CN1199421A - Method and chemical compounds for modifying polymers - Google Patents

Abstract

The invention relates to methods and compositions for improving the fluid, electrical or strength properties of a polymer by binding an effector moiety to the polymer via a protein. The invention particularly relates to improving the properties of paper by binding thereto a moiety capable of conferring a property such as improved wet strength, dry strength or sizing, via a protein such as a cellulase capable of binding to cellulose in the paper.

Description

The method and the compound of improvement polymkeric substance

1. technical field

The present invention relates to improve the method and the compound of polymer physics character.Especially, the present invention relates to improve the method and the compound of polymer physics character, wherein polymkeric substance and a kind ofly give fluid, electricity or intensive property that polymkeric substance improves, hereinafter referred to as the compound combination of " effector fragment ".

2. background of invention

Polymkeric substance is very general with the material that comprises polymkeric substance in daily life.Natural polymer comprises as protein (comprise Keratin sulfate, it is the main component in the hair), starch, pectin, guar gum, chitin, xylogen, agar, alginate and polysaccharide such as Mierocrystalline cellulose and hemicellulose (comprising xylan, seminose and pectinose).Common Cellulosed molded article has, as product based on lumber fibre or annual crop fiber (as hemp, straw, paddy rice, flax, jute), as paper with can be fiber, yarn, silk thread or multiple weaving and the cotton products of non-woven fabric or textile product form.Wood sugar is the principal constituent of xylan, the material that be also referred to as hemicellulose of xylan for existing in grass, cereal grass, straw, husk and timber.Starch is present in seed, fruit, leaf, the bulb etc.

Available number of chemical and physical treatment method are improved polymkeric substance and are comprised the physical properties of the material of polymkeric substance.These chemistry and physical treatment method can be used for the improvement of polymer architecture itself or comprise the improvement of bulk property of the material of polymkeric substance.

For example, by in material, sneaking into treatment agent such as wet tenacity toughener, dry strength toughener or other improves the compound of material physical properties, can improve the bulk property of the material that comprises polymkeric substance.Typically, the mixing of these compounds and material can not make compound and polymkeric substance combine closely, and therefore can run into the waste of compound and the problem that compound oozes out from material, thereby cause the instability of material character.Equate that with the ionic charge of the material that comprises polymkeric substance also opposite charge balance rules can reduce oozing out of compound by a kind of ionic charge of compound that wherein makes.Yet in the system of reality, the change in electrical charge of two kinds of components is very big, needs carefully and frequently to control.The improved effect of compound also depends on the covalent attachment with polymkeric substance, so that obtain appropriate improved effect.In addition, the combination for some chemical substance of facilitation and material also needs promotor.

In addition, as being applied to compound the surface of material by dipping or print process.Equally typically, compound also not with the surface bonding of material, and also can run into the problem of compound from the diffusion of design application site.

Known have multiple non-covalent binding interactions, for example an effect of mutually combining between effect that mutually combines between antibody and antigen and vitamin H and avidin or streptavidin.Typically, the enzyme that can change enzyme substrates also depend on and enzyme substrates between non-covalent mutually combining do in order to work.

This fermentoid comprises the enzyme of degradation polymer, as proteolytic enzyme, M-Zyme, chitinase, lignoenzyme, agarase, algin enzyme, zytase, mannase, amylase, cellulase and hemicellulase.For example, cellulase and hemicellulase can be respectively on Mierocrystalline cellulose and the hemicellulose sugar or polysaccharide molecule under the cracking, amylase can be from glucose under the cracking on the starch.

Mierocrystalline cellulose and cellulase protein, particularly the interaction that is bonded between the cellulase protein on the cellulosic fibre by the mode with catalytic activity is described and summarizes (cellulase: Beguin, Annu.Rev.Microbiol., 44,219-248,1990; Cellulase and zytase: Gilbert and Hazelwood, Journal of General Microbiology, 139,187-194,1993).This fermentoid comprises the cellulase and the hemicellulase in the protein territory that wherein comprises the function uniqueness.Particularly, the territory of catalytic activity being arranged structurally is different with Mierocrystalline cellulose bonding territory.These territories have the sequence of evolution conservative, to all these proteinoid all quite similar (303-315, June 1991 for people such as Gilkes, Microbiological Reviews).

Can separate the bonding territory on this proteinoid with active bit field by proteolysis.Found that isolated bonding territory still keeps binding ability (204 (2), 223-227, August 1986 for people such as Van Tilbeurgh, FEBS Letters).Existing suggestion utilizes the roughened means of the Mierocrystalline cellulose bonding territory of cellulase as the cellulose carrier surface pattern, and suggestion utilizes the polished finish (International Patent Application WO 93/05226) of cellulase activity bit field as these surface patterns.

Indication (people such as Ohmiya has been carried out from gene level in a lot of bonding territories, MicrobialUtilisation of Renewal Resource, 8,162-181,1993), and produced novel fusion rotein (people such as Kilburn, disclosed International Patent Application WO 90/00609 by subclone; People such as Ong, Enzyme Microb.Technol, 13,59-65, January 1991; People such as Shoseyov, disclosed International Patent Application WO 94/24158).Some such fusion rotein is used as anchorin in some special applications.By combining of fusion rotein and the cellulose carrier material that is used for the protein purification method, these albumen are used as auxiliary agent (people such as Kilburn, United States Patent (USP) 5,137,819 of protein purification; People such as Greenwood, Biotechnologyand Bioengineering, 44,1295-1305,1994).Fusion rotein the ability on the cellulose carrier of being fixed on be proposed fixing means as a kind of ezyme bio-reactor (people such as Ong, Bio/Technology, 7,604-607, June 1989; People such as Le, Enzyme Microb.Technol., 16,496-500, June 1994), with as the method (International Patent Application WO 93/21331) that on cellulose carrier, is connected chemical " label ".

3. invention summary

The invention provides a kind of method of handling polymkeric substance, make at least a character that is selected from fluid, electricity and intensive property be improved, comprising connecting the effector fragment is combined in described polymkeric substance by the albumen that is used to obtain described improvement, described effector fragment is different from described albumen and connects, and described albumen connects and is different from described polymkeric substance, and described effector fragment and described albumen connect by the significant quantity that obtains described improvement and exists.

Will be appreciated that polymkeric substance can comprise polymerizable molecular or a kind of polymeric material that comprises polymerizable molecular.Effector fragment and albumen connect and refer to that respectively at least a effector fragment and at least a albumen connect in addition.Therefore the present invention includes a kind of method of handling polymkeric substance, make at least a character that is selected from fluid, electricity and intensive property be improved, comprising connecting at least a effector fragment is combined at least a described polymkeric substance by at least a albumen that is used to obtain described improvement, described at least a effector fragment is different from described at least a albumen and connects, described at least a albumen connects and is different from described at least a polymkeric substance, and described at least a effector fragment and described at least a albumen connect by the significant quantity that obtains described improvement and exists.

According to a further aspect in the invention, the invention provides a kind of method of handling polymkeric substance, make at least a character that is selected from fluid, electricity and intensive property be improved, comprising the effector fragment is contacted with described polymkeric substance with the protein that is used to obtain described improvement, described effector fragment is different from described protein, described protein is different from described polymkeric substance, and described effector fragment and described protein exist by the significant quantity that obtains described improvement.The present invention includes a kind of method of handling polymkeric substance, make at least a character that is selected from fluid, electricity and intensive property be improved, comprising at least a effector fragment is contacted with at least a described polymkeric substance with at least a protein that is used to obtain described improvement, described at least a effector fragment is different from described at least a protein, described at least a protein is different from described at least a polymkeric substance, and described at least a effector fragment and described at least a protein exist by the significant quantity that obtains described improvement.

According to a further aspect in the invention, the invention provides a kind of chemical substance composition, comprise:

A) a kind of effector fragment; With

B) a kind ofly can be attached to protein on the polymkeric substance to described effector fragment;

Wherein said effector fragment is different from described protein, and described composition can make at least a character that is selected from fluid, electricity and intensive property of described polymkeric substance be improved.

The present invention also provides the composition of the material that comprises a kind of polymkeric substance, and the effector fragment is combined on this polymkeric substance by the albumen connection, described effector fragment is different from described albumen and connects, wherein said effector fragment and described albumen connect by significant quantity and exist, and at least a character that is selected from fluid, electricity and intensive property of described polymkeric substance is improved.

According to a further aspect in the invention, the invention provides the method for the composition fiber of a kind of treatment paper or paper, make and be selected from fluid, at least a character of electricity and intensive property is improved, comprising connecting the composition fiber that at least a effector fragment is combined in described paper or paper by at least a albumen that is used to obtain described improvement, described at least a effector fragment is different from described at least a albumen and connects, described at least a albumen connects the composition fiber that is different from described paper or paper, and described at least a effector fragment and described at least a albumen connect by the significant quantity that obtains described improvement and exists.4. detailed Description Of The Invention

The invention provides by the effector fragment that can give required character and be combined on the polymkeric substance to improve polymkeric substance or to comprise the method and the compound of fluid, electricity and/or the intensive property of the material of polymkeric substance.

Term polymer comprises the material that contains polymkeric substance.The material that contains polymkeric substance can be made up of polymkeric substance fully and maybe can comprise polymkeric substance and be compounded with other composition.

Polymkeric substance can comprise any polymkeric substance with any monomeric unit number.Preferably, polymkeric substance comprises natural polymer or its chemically modified derivative.For example, natural polymer can comprise protein such as Keratin sulfate, or polysaccharide such as starch, pectin, guar gum, chitin, xylogen, agar, alginate.Preferably, polymkeric substance comprises polysaccharide.Polysaccharide can comprise any polysaccharide, as seminose, wood sugar, Mierocrystalline cellulose or hemicellulose, is preferably Mierocrystalline cellulose.Comprising cellulosic material comprises as lumber fibre or annual crop fiber (as hemp, straw, paddy rice, flax, jute) sill, as paper.This material also can comprise the cotton products of fiber, silk thread or weaving and non-woven fabric, cloth or cotton paper form in addition.Preferably, described material comprises paper.

Can adopt the present invention to improve polymkeric substance fluid, electricity or intensive property arbitrarily.The polymer property that is enhanced comprises that wet tenacity and dry strength, sizability, hydrophobicity, achromatophilia and didirtresistance, fluid permeability, oleophobic and hydrophobicity, electricity lead and resistance, electric capacity, pH and biological metal.

Protein of the present invention can comprise any protein that can combine with polymkeric substance.Preferably, protein be can combine with polymkeric substance, dissociation constant (Kd) is less than 1 * 10 ^-3M's.Term " protein " comprises peptide, oligopeptide and polypeptide herein, and residue of protein, contains proteinaceous substances, amino acid chain and contain the peptide bond molecule.Sometimes by the context requirement (as, when protein is bonded on other molecule), protein finger protein matter residue.Term " albumen connection " finger protein matter or residue of protein, the effector fragment connects with polymkeric substance by it.Protein can comprise natural protein, or its segment or the modified protein that makes of genetically altered gene that make with chemically modified or synthesis method or by the coding marking protein.Term " modified protein " comprises the proteinic chemical analog that can combine with polymkeric substance herein.The proteinic example that can combine with polymkeric substance is well known, comprises being selected from cellulase, hemicellulase, mannase, zytase, proteolytic enzyme, M-Zyme, chitinase, lignoenzyme, agarase, algin enzyme and diastatic enzyme.For example known have a multiple cellulase that Mierocrystalline cellulose works that is incorporated into.The example of these cellulases for those can be from bacterium such as Cellulomonas fimi and mould such as viride (Trichoderma viride), black aspergillus (Aspergillus niger), penicillium funiculosum (Penicillium funiculosum), Trichoderma reesei and Humicola insolens isolating enzyme, they can be buied from Sigma Chemical Sigma-Aldrich Company Ltd., Novo Nordisk A/S, BDH Ltd. or ICN Biomedicals Ltd..In addition, also available protein as disclosed recombinant DNA technology preparation in the International Patent Application WO 94/24158.Usually cellulase comprises cellulase in conjunction with territory and cellulase activity territory.The present invention can adopt whole cellulase or its can with Mierocrystalline cellulose bonded segment.Utilize proteolytic enzyme such as papain to handle whole cellulase and obtain cellulase in conjunction with the territory.The present invention can adopt outer fiber enzyme and inner cellulose enzyme.

Preferably, protein comprises the natural enzyme that can combine with polymkeric substance.More preferably, for paper its catalytic activity by inactivation.The catalytic activity of enzyme can by as connect that effector fragment or enzyme are crosslinked to come inactivation.Can adopt suitable protein cross agent such as dialdehyde for example glutaraldehyde to carry out enzyme crosslinked.Preferably, protein comprises the natural cellulose enzyme of inactivation.

The effector fragment can any mode easily be incorporated into can with protein that polymkeric substance combines on.For example, the effector fragment can be coupled to protein by the appropriate reaction functional group direct covalent bonds on effector fragment and the protein.The identification of appropriate reaction functional group and, if necessary, the chemically modified of they being done for the facilitation covalent bond is in this area routine techniques personnel's the ability level scope.The example that covalent linkage constitutes comprises by the carbodiimide of carboxyl or dimethyl formamide activation makes carboxyl and amido constitute amido linkage.

The effector fragment can be incorporated into the arbitrarily suitable part of polymkeric substance conjugated protein.The effector fragment can be incorporated into the protein N-terminal of polymkeric substance conjugated protein, and is for example amino by the N-end.In addition, it also can be incorporated into proteinic C-end, for example by C-end carboxyl.In addition,, for example introduce proteinic functional group in the gal4 amino acid chain or in its side chain or for combining, make the effector fragment be incorporated into protein with the effector fragment by the optional functional group that exists.For example, the effector fragment can connect by aryl or the heteroaryl in the carboxyl in the amide group in the amino in the hydroxyl in the sulfydryl in the Gelucystine, Serine or the Threonine, Methionin or the arginine, asparagine or the glutamine, aspartic acid or the L-glutamic acid or phenylalanine, tyrosine, tryptophane or Histidine, the or derivatives thereof.The effector fragment can be connected in protein by a kind of connection agent.Connect agent can comprise as can with the bifunctional molecule of proteins react site and the segmental reactive site reaction of effector so that protein and effector fragment connect.May be favourable at protein and this connection agent of the intersegmental introducing of effector sheet as a kind of spacer, thereby this makes and enough separates not three-dimensional influence activity separately on two material spaces.Providing aspect the suitable connection effector fragment and proteinic functional group, it also is favourable connecting agent.

In addition, perhaps also as the part that connects agent, non-covalent combination that can be by molecule is to being connected in the effector fragment on the protein.Such molecule is non-covalent to comprise vitamin H and avidin, streptavidin or neutralite in conjunction with right example.

Therefore, it is that effector fragment covalency is connected in streptavidin that a kind of possibility is arranged, and polymer scale hop protein covalency is connected in vitamin H.These compositions in conjunction with facilitation the streptavidin of each component and the combination of biotin moiety, thereby the effector fragment is connected with the polymkeric substance conjugated protein.Will be appreciated that effector fragment-streptavidin component can mix with albumen-vitamin H component before or after protein ingredient is incorporated into polymkeric substance.Will be appreciated that also but effector fragment covalency connects vitamin H, and the protein covalency connects avidin, streptavidin or neutralite

Will be appreciated that to have the effector of more than one types fragment to be connected in polymkeric substance.The effector fragment that can adopt two or more types is to strengthen effect separately or two or more effects are provided simultaneously.

Will be appreciated that common effector fragment can connect with the polymer scale hop protein before or after the polymer scale hop protein is incorporated into polymkeric substance.Method of the present invention can comprise the effector fragment is contacted with polymkeric substance with proteinic conjugate, maybe can comprise the conjugate of effector fragment with protein and polymkeric substance contacted.In addition, can be in single stage method original position finish connecting of effector fragment and proteinic connection and protein and polymkeric substance.

The present invention's precise nature that pairing effect thing fragment is not incorporated into that albumen connects and the albumen connection is incorporated into the mode of polymkeric substance defines.Can connect by chemical bond such as covalent linkage or by non-covalent physics mutual relationship, connection, contact, attraction or affinity.

The effector fragment can comprise any fragment that can give required physical properties.The effector fragment can comprise atom, molecule or a compound or its residue that can give required physical properties.The effector fragment can comprise the compound that can give required physical properties in one embodiment.For example, treatment agent can comprise wet tenacity toughener such as aldehyde for example glutaraldehyde or dialdehyde starch or its cationic derivative, polyamide resin, polyacrylamide amine copolymer oxalic dialdehyde, the albumen modified of polymkeric substance, synthetic latex, the formaldehyde of glyoxylyl polyacrylamide, polymine, polymeric amine epichlorohydrin polymkeric substance, polyamide-based amine epichlorohydrin polymer, urea formaldehyde and melamine formaldehyde (MF) or other be used to strengthen the polymkeric substance of paper wet strength; Dry strength toughener such as starch, negatively charged ion or cationic starch, polyacrylamide, both sexes, negatively charged ion or cationic polyacrylamide multipolymer, negatively charged ion or cation guar gum, Viscogum BE or its positively charged ion or anion modified thing, polyvinyl alcohol, carboxymethyl cellulose; Sizing agent such as sylvic acid comprise sylvic acid, and the adduction sylvic acid comprises saponified fumaric acid glue rosin adducts, and rosin acid derivative comprises Yatall MA, and lipid acid comprises tetradecanoic acid, palmitinic acid or stearic acid; Other hydrophobizing agent comprises the derivative of alkenyl succinic anhydride (ASA) or 2-oxetanone (2-oxetanone) compound such as alkyl or alkynyl ketene dimer or polymer (AKD) or ASA or AKD, natural gum, adduction natural gum, timber or toll oil rosin, chain length is the straight chain of about 4 to 30 carbon atoms or branch is saturated or unsaturated carboxylic acid, the alkyl ketene dimer that makes by carboxylic acid, chain length is the alkyl succinic anhydride of about 4 to 30 carbon atoms, perfluoro or part fluorocarboxylic acid or its deutero-alkyl ketene dimer, perfluoro or part fluoro-alkyl succinyl oxide; Refuse to dye or stain control agent; Oleophobic or hydrophobizing agent such as fluoric compound comprise the fluoro derivatives of fluoro lipid acid or ASA or AKD; Softening agent comprises for example expansin (people such as McQueen-Mason, Proc.Natl.Acad.Sci.USA, 91,6574-6578 (July 1995)) of tensio-active agent, sanitising agent, fatty amide or zymin as the treatment agent that can destroy the fiber hydrogen bond; The reagent such as the metal of electroconductibility are provided; Inflexible reagent can be provided; The reagent of absorptive character can be provided; Hydrophilic reagent can be provided; Can improve the reagent of density; Metalating agent; Can improve the preparation of pH, as buffer reagent (for example being used to strengthen the ability of antiacid degraded).

In another embodiment, the effector fragment can comprise that linking agent or skeleton form agent or its residue, itself can be used for improving the physical properties of polymkeric substance, thereby or can be used for improving the character that proteinic character is improved polymkeric substance, or can be used for introducing the preparation that other improves polymer physics character.The example that preferred crosslinked skeleton forms agent comprises dialdehyde, as glutaraldehyde.For example dialdehyde such as glutaraldehyde can make the cellulase derived protein form skeleton.Cellulase/glutaraldehyde skeleton makes paper that the wet tenacity and the dry strength of improvement be arranged, to the paper applying glue and/or can introduce other reagent such as TiO ₂Or CaCO ₃

The detailed summary that is used for the compound of papermaking is made (Paper Chemistry, Chapman Hall New York, 1991) by people such as Robert, its whole contents at this with for referencial use.This reference has been summarized retention aid, wet strength additives, dry strength additive, sizing agent and filler especially.

Predicate " paper " is meant any bonded sheet or Web materials herein, comprise the network that Mierocrystalline cellulose is woven into, wherein comprise come from fibre of plant and randomly sneak into multiple proportioning derive from plant, mineral, animal or synthetic fiber, and the inorganic materials particulate of randomly sneaking into multiple proportioning, as oxide compound, carbonate and the vitriol of metallic element.Term " paper " comprises that the scraps of paper or net weight are greater than 200g/m ²Cardboard.

Cellulosic plant origin comprises timber, straw, bagasse, Stipa capillata, bamboo, kanaff fibre, grass, jute, ramie, hemp, cotton, flax.Thick plant-sourced Mierocrystalline cellulose is processed into paper pulp, utilize mechanical means or chemical process then or use both means by this paper pulp papermaking.Preparation and purification process classification according to paper pulp, the paper pulp of cellulose can be divided into mechanical pulp, chemical machinery and thermochemimechanical pulp, semi-chemical pulp, high yield dissolving pulp, full dissolving pulp (referring to " paper pulp and paper; chemistry and chemical technology (Pulp and paper; Chemistryand Chemical Technology) ", the third edition, the 1st volume, the 164th, 165 page, edit ISBN 0-471-03175-5 (V.1) by James P.Cassay).

The effector fragment can or contain the material manufacturing of polymkeric substance and is added in the polymkeric substance in the process segment at any suitable polymkeric substance.

If the effector fragment is applied to paper, wet pulp skeleton process or any stage interpolation in the process of extruding and volume skeleton formation paper be added or formed to the effector fragment can at paper pulp in the stage.In addition, also can be added to the effector fragment on the Manufactured paper product, for example, paper be dipped into contain in order in the bath of the reagent of joint efficiency thing fragment or utilize suitable spraying, smear, brush, coating or print process be connected in the effector fragment on the paper that has been shaped.

If the effector fragment is applied to cotton products, the effector fragment can be added in any cotton goods process segment.It can be connected on cotton goods, silk thread, yarn or weaving or chemically woven fabrics or the fabric.For example, material be dipped into contain in order in the bath of the reagent of joint efficiency thing fragment or utilize suitable spraying, smear, brush, coating or print process connect the effector fragment.

When making polymkeric substance or containing the material of polymkeric substance, by selecting to connect effector segmental opportunity, may command effector fragment is distributed on the surface that is confined to material in the polymer materials or basically.

Be used to improve the situation of material monolithic character for the effector fragment, guarantee that effector fragment uniform distribution in material is favourable.Therefore, the manufacturing stage connects the effector fragment in early days.For example when the effector fragment was used to improve the bulk property of paper, in paper-making process, the effector fragment should add in the stage at paper pulp.

Be used to improve the situation of material surface character for the effector fragment, can fully be limited to the effector fragment on the material surface level, its benefit of following is to have reduced the segmental consumption of required effector.Therefore, preferably, the effector fragment should add at the late stage of making.For example when the effector fragment was used to improve the surface properties of paper, in paper-making process, the effector fragment should be applied to the surface of paper.

Depend on the purposes needs, one of paper or two plane surfaces are coated with the effector fragment.For example use two surfaces of the effector fragment treatment paper that comprises wet strength agent, and remaining its one or more edges are not handled, this facilitation the preparation of sandwich structure, the ply of paper that has poor wet strength properties and good fluid absorbent in this structure is sandwiched in two layers of ply of paper with good wet tenacity performance.Such structure can be passed through the wicking action transmit fluid in its middle layer, and is particularly useful in the manufacturing of immersion type diagnosis test paper.

A special aspect of the present invention is can the reverse mode to improve polymkeric substance or to contain the ability of the physical properties of polymer materials.The method that the conventional processing polymkeric substance is given special physical properties usually is irreversible.And conventional treatment method usually makes polymkeric substance be unsuitable for regeneration.If the regeneration for paper is handled with conventional wet tenacity toughener, be paper slurrying again more difficult or impossible.The invention provides the method that makes the release of effector fragment, material regeneration.For example at the polymkeric substance first line of a couplet the segmental proteinic protease treatment of effector is arranged, can discharge the effector fragment from the material that contains polymkeric substance by cleavable; In addition, connect agent by the selectivity cleavable and can be coupled to protein to the effector fragment; The starch that available starches enzymatic lysis linking agent such as aldehyde replace.

Another benefit of the present invention is that required physical properties is imparted on the material basically immediately.In the facture of giving paper wet strength of routine, the thermal treatment that needs several weeks is with aging.

Below will be with scheming and embodiment description the present invention for referencial use.In the drawings:

The influence of the wet tenacity that the plain enzyme concn of Fig. 1 display fibers is given the cellulase of glutaraldehyde cross-linking;

Fig. 2 shows the influence of the wet tenacity that glutaraldehyde concentration is given the cellulase of glutaraldehyde cross-linking;

Fig. 3 shows the influence of the wet tenacity that pH gives the cellulase of glutaraldehyde cross-linking;

The influence of the wet tenacity that Fig. 4 displays temperature is given the cellulase of glutaraldehyde cross-linking;

Fig. 5 shows the influence of the wet tenacity that the incubation time gives the cellulase of glutaraldehyde cross-linking;

Fig. 6 shows the influence of the wet tenacity that the preincubation time gives the cellulase of glutaraldehyde cross-linking;

The cellulase that Fig. 7 shows glutaraldehyde cross-linking is to the influence of the wet tenacity of the paper made by different wood pulps.

Will be appreciated that following is embodiment only, and can revise details in the scope of the invention.

Experiment

Principle and operation that the effector fragment connects

On behalf of characterization, the rules of following definition use cellulase as biological bridging agent the effector fragment to be connected in cellulosic technology.The preparation of original material

Use the phosphate buffered saline (PBS) (1/3PBS) of 1/3 concentration.Below be the prescription of 1/3PBS:

200 liters of deionized waters or demineralized water (DEMI water)

197g AMSP (NaH ₂PO ₄)

767g disodium hydrogen phosphate,anhydrous (Na ₂HPO ₄)

389g sodium-chlor (NaCl)

Anhydrous material not necessarily recomputates described weight but should deduct " crystal water " for moisture salt.

The cellulase that is adopted derives from mould, can the aqueous solution or the lyophilized powder form obtain.Penicillium funiculosum

Come from penicillium funiculosum cellulase (Dorset U.K.) can brown powder obtain for Sigma Aldrich Co.Ltd., Poole, should below 0 ℃ the storage.

When being used as the additive of handsheet, at first in 1/3 PBS, this cellulase is made 20% total solid solution.In the big beaker of the shallow end, put into the zymin that 200g does.Then to wherein slowly adding 800g 1/3 PBS.Slowly stir the mixture with glass rod.Owing to can make enzyme denaturation, vigorous stirring solution is not with dispersed powders.The all available glass rod of any zymin piece is broken gently.If one day before use preparation enzyme solution needs it 4 ℃ of storages down.Trichoderma?reesei

The cellulase that comes from Trichoderma reesei can be from Sigma Aldrich Co.Ltd., and Poole, Dorset, U.K. press powder type maybe can be from Novo Nordisk A/S, and Bagsvaerd, Denmark press aqueous solution form and obtain.When using powder, also adopt method and the operation for preparing the Penicillium funiculosum aqueous solution herein.

In total protein content in the enzyme solution is (adding 10 parts of dried egg whites as per 100 parts of dried fibers) cellulase solution to be added in the raw material.With Coomassie Brilliant Blue G250 dyestuff (Sedmak and Grassberg (Analytical Biochemistry, 79,544-552 (1997)) make protein staining, with UV absorb (=620nm) measure the total protein content of the cellulase solution of making.1, the test of cellulase binding fiber

Mierocrystalline cellulose sample (between 25-500mg, often being 100mg typically), for example Microcrystalline Cellulose (Avicel, SigmaCell) or unsized paper slurry be weighed in a series of test tube/flasks.

Cellulase solution (typically, in the 3ml damping fluid, every ml contains 200-600mg albumen) is added in each test tube.At first add proteic accurate concentration (Analytical Biochemistry, 79,544-552 (1977)) in beginning with the method for testing measurement institute of Sedmak and Grassberg development in conjunction with test.

Temperature required following jolting test tube (typically between 4 ℃-30 ℃, being generally room temperature) for some time (typically being 1-90min, usually between 5-15min).Then sampling (0.5-1ml) is tested.

Use desk-top microcentrifuge in 1ml Eppendorf pipe centrifugal 5 minutes of sample, keep the concentration (unconjugated cellulase) that supernatant liquor is used for measuring the supernatant liquor residual protein.

Deduct the cellulose protease amount that the supernatant liquor protein concentration represents to be incorporated into Mierocrystalline cellulose group with initial protein concentration.

Adopt in test bovine serum albumin (BSA) in contrast.

The result accounts for institute with the proteic amount of cellulose-binding and adds proteic per-cent or the proteic amount of cellulose-binding and account for albumen/cellulosic per-cent (%w/w) expression.2, use chemoluminescence method to manifest the biotinylation of preparation 1. cellulases that the segmental connection of effector is used for the cellulose protease of ECL detection architecture

Preparation biotin amido group N-hydroxy-succinamide ester (B _CapNHS) N, dinethylformamide (DMF) solution (1mg/ml).The solution (77mg/ml) of preparation cellulase in distilled water.

The 1ml cellulase solution is added to 1ml B _CapIn the NHS solution, following mixture room temperature incubation of jolting 2.5 hours.Then reaction solution 500ml 1/3 PBS damping fluid (PBS, pH7.5:Na ₂HPO ₄, 11.5g; NaH ₂PO ₄, 2.96g; NaCl, 5.84g with distilled water diluting to 1L) fully dialysis 1 hour.2. combine [surface that cellulase is applied to the scraps of paper] of biotinylated cellulase and the scraps of paper

Under the jolting in 4 ℃ in the culture dish of the shallow end a unsized paper sheet, be generally 2cm ²And the protein concentration scope in 1/3 PBS is 0.05 to 100 μ g albumen ml ^-1Biotinylated cellulase (10ml) incubation 45 minutes to 2 hours together.Also use and contain polysorbas20 (0.1%vv ^-1) the experiment of PBS.3. combining and the preparations of the scraps of paper subsequently [cellulase is applied in the paper substrate body] of biotinylated cellulase and paper pulp

Paper pulp and containing polysorbas20 (0.1%vv ^-1) 1/3 PBS in biotinylation cellulase room temperature incubation 45 minutes together under jolting.Use the papermaking filter screen by paper pulp-biotinylation cellulase system scraps of paper.The scraps of paper are taken off from filter screen, roll and dried overnight.4.HRP the streptavidin of mark combines with the biotinylation cellulase

Recommendation (Amersham Led., Amersham, U.K. by manufacturers; Whitehaead, people such as T.P., Clin.Chem.26,1531-1546,1997) carry out the combination of streptavidin of HRP mark and the ECL of biotinylation cellulase detects.

Under the jolting in 4 ℃ or room temperature paper and milk powder (4%wv in PBS ^-1) incubation 45 minutes to be to shelter the non-feature combination of HRP streptavidin conjugate together.Then be used in and contain polysorbas20 (0.1%vv ^-1) 1/3 PBS in 0.5% (wv ^-1) milk powder cleaned the scraps of paper 3 * 3 minutes.After each the cleaning, discard solution and change to new soln.

Use is containing polysorbas20 (0.1%vv ^-1) 1/3 PBS in 0.5% (wv ^-1) milk powder makes horseradish peroxidase (HRP)-streptavidin conjugate solution of 1: 1000.Add an amount of volume (2 to 10ml) solution and cover the scraps of paper, then under jolting in 4 ℃ of incubations 45 minutes.

Be used in and contain polysorbas20 (0.1%vv ^-1) 1/3 PBS in 0.5% (wv ^-1) milk powder cleaned the scraps of paper 3 * 5 minutes.After each the cleaning, discard solution and change to new soln.Then cleaned the scraps of paper 3 * 5 minutes with 1/3 PBS.After each the cleaning, discard solution and change to new soln.

The Mierocrystalline cellulose that then is combined with cellulase-vitamin H-HRP-streptavidin conjugate can develop with the ECL method, or uses the OPD standard measure.3, enhanced chemiluminescence (ECL) method

This method needs to carry out in photographic laboratory.

(consumption is about 0.13ml/cm to the mixing of Amersham ECL detection reagent 1+2 equal-volume ²Paper).Then excessive damping fluid is removed and added the complete covering paper of detection reagent surface from paper.

Do not stir the at room temperature lucky incubation of a paper 1 minute.Then detection reagent is removed also folder is blotted unnecessary reagent in two toilet papers.Then the paper that blots is transferred on a slice adhibit quality film and twines and guarantee to remove any gas bag.

Paper is put into a bellows, the delay minimization between making incubation paper and exposing paper to the last film.This film is carefully placed the top of paper, guarantees that film is not moved this film exposure 15 seconds in the exposure.Then removing first tunic exists side by side and promptly changes second tunic of the paper that will expose 1 minute subsequently.

Then immediately film development with display result.Available if desired 1 to 60 minute other diaphragm that exposes.4, the effector of detection by quantitative cellulose-binding segmental (OPD) method

1 OPD sheet (60mg; O-phenylenediamine dihydrochloride, Sigma Chemicals UK) is dissolved in 150ml 0.06M phosphate-citrate salts damping fluid (0.2M Na ₂HPO ₄, 121.5ml; 0.1M citric acid 121.5ml with distilled water diluting to 500ml, and pH transferred to 5.0) the preparation substrate buffer solution, the final OPD concentration of gained is 0.4mg ml ^-1Notice that this reagent is photosensitivity.Just add the fresh 30%H of 10 μ l in every before use 25ml substrate buffering salt ₂O ₂

The pattern that contains the biotinylation cellulase is placed 50ml Falcon pipe.The substrate buffer solution that 25ml is prepared is added in the test tube, and jolting at room temperature 30 seconds to 20 minutes, is generally 5 to 15 minutes, then adds 1ml 3M H ₂SO ₄Termination reaction.Then in 492nm place measurement absorbancy, for calculating is in the concentration of the biotinylation cellulase on the paper or in the paper, with reference to OD ₄₉₁Typical curve to the biotinylation cellulase concentration.5, use carbodiimide chain attachment paper effector fragment and enzyme peptide

Carbodiimide and carboxyl reaction form activated carboxyl.Follow amino attack activated carboxyl and constitute peptide bond.This chemical reaction is used for linking to each other with amino on the peptide containing the paper effector fragment of free carboxy.

The chemistry that is used for the carbodiimide that the effector fragment with paper is connected with cellulase is based on ordinary method (people J.Biol.Chem such as Hoare, 242 (10), 2447-2453,1967).

In following method, with sylvic acid coupling cellulase.

Cellulase (21mg ml ^-1) be dissolved in the distilled water, sylvic acid (100mg) is dissolved in 25ml 10% (vv ^-1) in the methyl alcohol.In 1.0ml sylvic acid solution, add 0.5ml 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide-HCl (WS-CDI; 63mg ml ^-1), and pH is transferred to 4.5 ± 0.5 with HCl (0.1N).Then at room temperature stir the mixture (5min).Add the 2ml cellulase solution, and mixture is at room temperature stirred (16hr).

Add sodium acetate and unnecessary sylvic acid (0.1M; PH5.0) termination reaction is removed WS-CDI by complete dialysis in phosphate buffered saline buffer.

Then by described method with link coupled cellulase with the sylvic acid bridging on Mierocrystalline cellulose.6, the cellulase with glutaraldehyde cross-linking proves wet tensile character

Carried out the cellulase of glutaraldehyde cross-linking is applied to experiment in the unsized paper slurry, and proved that it can give scraps of paper wet strength properties.

Prepare the unsized paper slurry by the following method: the 10g unsized paper is cut into 1cm ²Square and in family expenses herbal medicine mill (CH100, Kenwood Ltd.UK), it was macerated 3 minutes with 100ml distilled water.

Take out 2.15g unsized paper slurry (10%wv ^-1), wherein contain the 0.2g Mierocrystalline cellulose, and carry out following interpolation: 1,10ml 1/3 concentration phosphate buffered saline (PBS) (PBS), pH7.0 is as a reference.2,10ml contains T.reesei cellulase (2mg ml ^-1) 1/3 PBS3,10ml contain glutaraldehyde (25 μ l ml ^-1) 1/3 PBS4a, 10ml contain T.reesei cellulase (2mg ml ^-1) and glutaraldehyde (25 μ l ml ^-1) 1/3PBS, wherein before adding paper pulp under room temperature common incubation 1 hour.4b, 10ml contain T.reesei cellulase (2mg ml ^-1) and glutaraldehyde (25 μ l ml ^-1) 1/3PBS, directly add in the paper pulp.

The equal room temperature incubation 1 hour in horizontal shaking table of all samples before making the scraps of paper.

For the preparation test scraps of paper, make volume increase to 100ml with distilled water, utilize the papermaking instrument of lab design to prepare the scraps of paper (6cm in the following manner ²): with pulp suspension (0.2%WV ^-1) pour in the plastics strumbox that thin nylon filter screen is housed.Adding vacuum makes paper pulp constitute the scraps of paper that supported by screen cloth several seconds.Filter screen is taken out from instrument, and the scraps of paper are sandwiched in second nylon screen and with thieving paper and absorb water.Carefully from becoming the paper screen cloth to take off, flatten the scraps of paper then dry with roll-in.

Measure wet tenacity by following method; A, the paper stability in water

Each is measured the sample (1.5cm of the scraps of paper ²) place the Universal bottle and add 25ml distilled water respectively.The sign of jolting test tube and the forfeiture of periodic test pattern integrity.

The results are shown in Table 1.

Table 1 pattern is in the detection of water stability

Sample number	Repeat samples number	Situation in water after the jolting	Initial destruction	Total destruction
					????1	????1 ????2	Disintegrate	?????- ?????-	?????- ?????-
????2	????1 ????2	Disintegrate	?????- ?????-	?????- ?????-
					????3	????1 ????2	Disintegrate	?????- ?????-	?????- ?????-
????4a	????1 ????2	Complete	???＜18h ???＜18h	????36h ????36h
					????4b	????1 ????2	Complete	???＞8d ???＞8d	????＞8d ????＞8d

In table 1, "-" refers to B, the paper intensity of inapplicable experiment

Get and respectively test scraps of paper sample (4cm * 1cm) also 25 μ l distilled water are dripped to the middle part of paper to guarantee uniform distribution.Paper is suspended in two clips with well, and on bottom clips, a container is installed.Adding water and weighing in container makes paper tear the weight of required water.

The results are shown in Table 2, proves that the pattern with the preparation of glutaraldehyde cross-linking cellulase has maximum wet tensile.

The mensuration of table 2 paper intensity

Sample number	Repeat samples number	Add weight (g)
			????1	??????1 ??????2	?????27.43 ?????43.48
????2	??????1 ??????2??	?????＜22.00 ?????＜22.00
			????3	??????1 ??????2	?????＜22.00 ?????34.63
????4a	??????1 ??????2	?????66.34 ?????49.96
			????4b	??????1 ??????2	?????＞77.33 ?????64.20

Compare the wet tenacity of testing pattern again with BSA, to estimate the specificity of bridging protein effect.Press following preparation pattern: 1,10ml 1/3 concentration phosphate buffered saline (PBS) (PBS), pH7.02,10ml contain T.reesei cellulase (2mg ml ^-1) 1/3 PBS3,10ml contain glutaraldehyde (25 μ l ml ^-1) 1/3 PBS4,10ml contain BSA (2mg ml ^-11/3 PBS) 5,10ml contains T.reesei cellulase (2mg ml ^-1) and glutaraldehyde (25 μ l ml ^-1) 1/3PBS, before scraps of paper preparations, be added in the paper pulp and room temperature incubation 1 hour on horizontal shaking table.6,10ml contains BSA (2mg ml ^-1) and glutaraldehyde (25 μ l ml ^-1) 1/3 PBS, before scraps of paper preparations, be added in the paper pulp and room temperature incubation 1 hour on horizontal shaking table.

Pattern and 25ml water are added in the 50ml Universal bottle, and disintegrate fully until pattern with the mixing tank vortex with the laboratory.The results are shown in Table 3.

The proof of paper stability when table 3 mixes at the water mesoscale eddies

Sample number	Repeat samples number	Disintegrate fully the required time (second)
			????1	??????1 ??????2	??????????＜5 ??????????＜5
????2	??????1 ??????2	??????????＜5 ??????????＜5
			????3	??????1 ??????2	??????????＜5?? ??????????＜5
????4	??????1 ??????2	??????????＜5 ??????????＜5
			????5	??????1 ??????2	??????????＞1020 ??????????＞1080
????6	??????1 ??????2	????????????10 ????????????10

Compared with the control, though use the pattern of crosslinked BSA (sample 6) preparation to show that wet tensile increases, it is littler 100 times than glutaraldehyde cross-linking cellulase.

Top condition for glutaraldehyde/cellulose treatment method of determining to improve wet tenacity changes following parameters successively.Be decided to be for this work control parameters: cellulase (2mg ml ^-1), glutaraldehyde (0.6%vv ^-1) be added in the paper pulp respectively; At 25 ℃ of following pulp suspensions in damping fluid (pH7.0).Before scraps of paper preparations with mixture incubation 60 minutes.Change each parameter below successively: cellulase (0.5 to 8mg ml ^-1); (0.1 to 2.5vv for glutaraldehyde ^-1); PH (5.0 to 10.0); 25 ℃, 37 ℃ and 45 ℃ of temperature; The incubation time (5 to 120 minutes); The preincubation time of cellulase and glutaraldehyde (15 to 60 minutes).

All scraps of paper dried overnight at room temperature before wet tensile is measured.The results are shown among Fig. 1 to 6.7, with dissimilar paper pulp proof glutaraldehyde cross-linking cellulase (GCC)-wet tensile performances

GCC-wet tenacity composition is applied on the paper with dissimilar pulp preparation: ground wood pulp (GWP), thermochemimechanical pulp (CTMP), hardwood pulp (HWP), softwood pulp (SWP) and unsized paper slurry (W-LP; 70%HW:30%SW).Paper pulp prepares according to a conventional method, certainly, should will spend the night in GWP and the CTMP paper pulp immersion water before mixing with the promotion fiber dispersion.

Paper pulp PBS damping fluid (10ml); Or PBS damping fluid (10ml)+cellulase (20ml)+glutaraldehyde (0.6%vv ^-1) handle.Then before the wet tensile test, pulp sample is made the test scraps of paper (6cm by described method ²).The results are shown in Table 4 and use diagrammatic representation in Fig. 7.

The result shows that the wet tensile of all tested paper pulp all is improved.The final strength of the scraps of paper of use GWP or CTMP preparation is bigger than what prepare with HWP, SWP and W-LP.But the wet tensile that the GCC composition produces in HWP and SWP sample has the increase of bigger per-cent.

Table 4: the wet tensile of the scraps of paper that different paper pulp are made

Paper pulp	Treatment process	Vortex mixed (second) 1	Wet tensile (g)
				Ground wood pulp	Damping fluid cellulase glutaraldehyde cellulase+glutaraldehyde	135 120 315 ＞1200	105.4 45.0 94.5 249
CTMP	Damping fluid cellulase glutaraldehyde cellulase+glutaraldehyde	480 420 420 ＞1200	78.5 88 71.5 242.5
				HWP	Damping fluid cellulase glutaraldehyde cellulase+glutaraldehyde	＜5 ＜10 ＜10 ＞300	＜15.3 ＜15.3 ＜15.3 119
SWP	Damping fluid cellulase glutaraldehyde cellulase+glutaraldehyde	＜5 ＜5 ＜5 ＞300	＜15.3 ＜15.3 ＜15.3 207
				W-LP	Damping fluid cellulase glutaraldehyde cellulase+glutaraldehyde	＜5 ND ND ＞300	＜15.3 ND ND 193

Required time 8, the glutaraldehyde cross-linking cellulase of completely destroy gives wet tensile reversible proof

For proving the reversibility of the wet tensile of giving by the glutaraldehyde cross-linking cellulase, with following Sigma Chemical-Aldrich Company Ltd., Fancy Road, Poole, Dorset, the following protein enzyme solution of enzyme preparation of BH177NH supply: ficin (4 μ l ml in pH 6.5 PBS damping fluids ^-1Solution); Papain (5 μ lml in pH 6.5 PBS damping fluids ^-1Solution); Proteinase K (the 2.8mg ml in pH 8.0 PBS damping fluids ^-1Solution); Alpha-chymotrypsin (the 1.0mg ml in pH 8.0 PBS damping fluids ^-1Solution).

Take the scraps of paper that glutaraldehyde cross-linking cellulase enhanced unsized paper slurry makes (1.5 * 1.5cm) and press the listed treatment process incubation of table 5.

Table 5 wet tenacity reversibility treatment process

Sample number	Treatment process
			1	Ficin (10ml)+PBS damping fluid (pH 6.5)
2	Papain (10ml)+10mlPBS damping fluid (pH 6.5)
		3	Proteinase K (1ml)+19mlPBS damping fluid (pH 8.0)
4	Alpha-chymotrypsin (1ml)+19mlPBS damping fluid (pH 8.0)
		5	Ficin (10ml)+papain (10ml)
6	Proteinase K (1ml)+alpha-chymotrypsin (1ml)+18mlPBS damping fluid (pH 8.0)
		7	Ficin (10ml)+papain (10ml)+Proteinase K (1ml)+alpha-chymotrypsin (1ml)
8	0.2M phosphoric acid buffer pH 6.5 (20ml)
		9	0.2M phosphoric acid buffer pH 8.0 (20ml)

Sample in horizontal shaking table in 70rpm, 30 ℃ of following incubations.Test sample when 4 hours and 20 hours, whether vortex mixed was observed paper in 10 seconds and is kept perfectly after 20 hours.Repeated experiments, the results are shown in Table 6.

The paper destructive is measured in the table 6 different treatment method

Treatment process	The incubation time (h) 4 20		After 10 seconds vortex mixed in addition
				Ficin	X X	XX XX	XXXX XXXX
Papain	XX XX	XXXX XXXX	--- ---
				Proteinase K	XX XX	XX XX	XXXX XXXX
Alpha-chymotrypsin	0 0	XX XX	XXXX XXXX
				Papain+ficin	XX XX	XXXX XXXX	--- ---
Alpha-chymotrypsin+Proteinase K	X X	XX XX	XXXX XXXX
				Whole 4 kinds of proteolytic enzyme	XX XX	XXXX XXX	--- XXXX
Contrast phosphoric acid buffer (pH 6.5)	0 0	0 0	0 0
				Contrast phosphoric acid buffer (pH 8.0)	0 0	0 0	0 0

Qualitative observation is represented with arbitrary scale 0 to XXXX, wherein 0 represents paper not visible damaged, and XXXX represents the paper completely destroy; The paper integrity is promptly lost before----representative test.9, determine that protease treatment is to being strengthened the influence of the recyclability of paper by glutaraldehyde cross-linking cellulase wet tenacity

The paper that the paper pulp (0.2g) that adopts glutaraldehyde cross-linking cellulase wet tenacity to strengthen unsized paper slurry (0.2g) or do not handle with any wet tenacity toughener is made carries out a series of processing.

Handle the square that 1 scraps of paper made from glutaraldehyde cross-linking cellulase wet tenacity enhanced paper pulp (0.2g) is cut into 1cm * 1cm, and place a culture dish with the 20ml 0.2m phosphate buffered saline buffer (pH 8.0) that contains Proteinase K (14mg).Under the jolting (60rpm) in 37 ℃ sample incubation 2 hours.Then the scraps of paper are taken out and are dipped in the phosphate buffered saline buffer (pH 8.0), then put in the Universal bottle that contains 20ml fresh phosphoric salt buffer (pH 8.0).The sample vortex mixed to macerate paper.Anyly residue in fiber in the culture dish all 6 when initial incubation, centrifugal collection under the 000rpm is washed and is added in the sample of macerating in the Universal bottle with phosphate buffered saline buffer (pH 8.0).Add 2ml T.reesei cellulase (10mg ml ^-1) and 0.5ml glutaraldehyde solution (25%), sample 25 ℃ of following incubations 60 minutes.Then with the new scraps of paper of this specimen preparation.

Handle 2 placing a Universal bottle by the unsized paper sheet (0.2g) and the 10ml 1/3 concentration PBS that do not add the pulp preparation that any wet tenacity toughener only makes with PBS.To macerate paper, gained paper pulp is in order to prepare the new scraps of paper the sample vortex mixed.

Handling 3 scraps of paper (0.4g) and the 30ml 1/3 concentration PBS that made by glutaraldehyde cross-linking cellulase wet tenacity enhanced paper pulp places a mixing tank to macerate.Take out gained paper pulp and make the new scraps of paper.

Handle 4 scraps of paper made from glutaraldehyde cross-linking cellulase wet tenacity enhanced paper pulp (0.2g) and be cut into 1cm ²Sheet place a mixing tank to macerate with 30ml 1/3 concentration PBS then.Add 2mg T.reesei cellulase (10mg ml ^-1) and 0.5ml glutaraldehyde solution (25%), sample 25 ℃ of following incubations 60 minutes.Then with the new scraps of paper of this specimen preparation.

Using vortex mixer 1cm ²Sample in water, destroy to test before its integrity, each scraps of paper is dried overnight at room temperature.The test result of paper is listed in the table 7.

Table 7: the integrity of recycled writing paper

Method	Vortex mixed (time, second)	The description of paper situation
			1	300	Paper is broken into three
2	5	Paper disintegrates fully
			3	50	A lot of small shredss are arranged
4	345	Duck eye is arranged in paper

The result shows that the paper pulp that contains GCC keeps some wet tensile character when making the new scraps of paper; The paper pulp made from protease treatment is opposite with physical damage, generates the better paper of intensity when regeneration, can make the regeneration scraps of paper reach best wet tensile character and continue to add GCC.10, the proof that the wet tenacity of paper, dry strength and sizability improve after glutaraldehyde and the cellulose treatment

Do experiment to determine the influence of cellulase (protein aglucon) and glutaraldehyde (effector fragment) to wet tenacity, dry strength and the sizability of paper.In experiment, adopt following material and general purpose discipline: cellulase

Adopt water base Trichoderma reesei cellulase preparation (" Cellulast 1.5L ", by Novo Nordisk Bioindustry S.A.92017 Nanterre Cedex, France provides).Glutaraldehyde

In the following example, adopt glutaraldehyde 25% aqueous solution, can (Poole, Dorset U.K.) buy from Merck Ltd.Feedstock production:

Unless specialize, the batching of employing is the leaf wood of ECF bleaching and the mixture (ratio is 70: 30 HW/SW) of softwood pulp.With 1/3PBS and not filled preparation raw material.Process is as follows:

The softwood pulp of the hardwood pulp of 280g bleaching and 120g bleaching is added among 18 liters of 1/3PBS.Vigorous stirring makes fiber dispersion.Then raw material is transferred in the hollander, and making beating is to (needing 30 to 35 minutes usually) till all degree of beating value is 25oSR.Then adding 1/3PBS again transfers to 2% to the raw material ultimate density.Adding/the incubation of additive

Cellulase solution and glutaraldehyde solution are added in this heavy-gravity (concentration 2%) raw material.Two liters of raw materials (containing the 40g fiber) are placed a metal tin, and raw material is slowly moved with alap speed stirring.Vigorous stirring should be avoided otherwise enzyme denaturation can be caused in incubation period.Raw material is in room temperature (20 to 25 ℃).At first in raw material, add cellulase solution (avoid any solution to splash or splash), add enzyme after one minute, add glutaraldehyde water solution again.

The incubation time of additive is 15 minutes, has added from enzyme and has started at.In the incubation process movement velocity of raw material may transfiguration easily/faster.If change more obviously then need reduce agitator speed as much as possible.

After 15 minutes incubation period are finished, the heavy-gravity raw material is added in the blender loader.Blender loader

Only with DEMI water heavy-gravity raw material in the blender loader rare to concentration be 0.25%.Adopt stirring velocity mixing raw material commonly used in the blender loader.The formation of handsheet

In white water box, charge into the DEMI water that forms for handsheet.Handsheet is formed lead pack in the mould, one liter of raw material from blender loader is added in the sheet frame box, add the water from white water box simultaneously.Content in the sheet frame box stirs (lifting up and down five times) with the porous agitator.When lift for the 5th time finish after, place agitator water surface to help to be still in the motion of sheet frame box water.Then water pump is got back to the feasible base that just wets that forms in the plain boiled water box.

The severe degree that depends on stirring some foams can occur in the sheet frame box.Wet originally base forms the back foam and can exist, and also may be very many.Continue to take out several seconds to remove the air in the base if remove the back, then can make a part of lather collapse with pump at water.The compacting of handsheet and drying

Wet base and handsheet lead are removed from mould and suppress.The scraps of paper water content that is pressed into should be 70%.Follow in the electrically heated rotary drum dryer the scraps of paper drying that is pressed into.The surface temperature of doing the baking machine is between 60 ℃ to 105 ℃, and the rotating speed of drying machine will make the scraps of paper and its hot surface that are pressed into reach 35 to 180 seconds duration of contact.The final water content of the scraps of paper should (typically be 5%) between 4 to 7%.

If the water content of the compacting back scraps of paper is less than 70%, next the scraps of paper may be bonded at the rotary drum dryer surface when adopting described condition.This may be owing to inhomogeneous exerting pressure on the scraps of paper amplitude caused.Should avoid in the operation that this thing happens.

When the surface temperature of rotary drum dryer less than 105 ℃ but be 70 ℃ or when higher, then need longer duration of contact so that the final water-content of handsheet is 5%.

If the surface temperature of rotary drum dryer is lower than 70 ℃, then needs further to increase duration of contact or increase and be pressed in original pressure on the wet base to remove more water or both all adopt.Might reduce to the water content of the scraps of paper that are pressed into less than 60%.Test

Publish according to " Tappi Test Methods " TAPPI, Technology Park Atlanta, PO BOX 105113, Atlanta GA 30348, USA, listed method is carried out the aging and test of paper among the ISBN 0-89852-200-5 (the 1st and 2 volume).Method T456om-87 has defined the wet tension rupture strength of paper and cardboard; T4940m-81 has defined the tension rupture strength of paper and cardboard; HST (Hercules sizing test) is defined as the applying glue experiment of paper with ink resistance T530pm-83; With T441om-84 definition Cobb test.

Carry out a series of cellulose concentration, glutaraldehyde concentration, time of drying and temperature, digestion time and temperature and do the experiment that changes separately.The results are shown in down in the tabulation, wherein:

" weather aging " refer to define among the T402om-83 at 23 ℃ ± 1 ℃, 50.0 ± 2% times storages of relative humidity specified time;

" oven ageing " refers to handle 30 minutes down at 80 ℃;

" standard drying conditions " refers at 105 ℃ following dry 35 seconds.

Test wet and dry tensile strength respectively with method T456om-87 and T4940m-81, wet tensile is represented with per-cent with the ratio of dry tensile strength.These data are listed in the table, and wherein this is worth that high wet strength is good more more.Measure sizing efficiency with HST (Hercules sizing test) (TAPPI method T530om-83), data are unit record with the second.The big more sizing efficiency of this numerical value is good more.The HST value of gained is preferably more than 20 seconds, more preferably greater than 120 seconds, more preferably greater than 200 seconds.Also available Cobb test (TAPPI method T441om-90).Data are with the grams/m of unit ²Record." saturated fully " refers to that paper does not have applying glue at all.The Cobb value is low more, and sizing efficiency is good more.The Cobb value of gained is preferably less than 30g/m ², more preferably less than 21g/m ²The wet tenacity of the cellulase under the standard conditions in the exsiccant handsheet/glutaraldehyde system and sizability " the % wet tenacity after the weather aging in 24 hours "

		Protein on the fiber
					????0％	????5％	????10％
		Glutaraldehyde on the fiber	????10％	????0.26				????1.63	????2.64
????20％	????0.80				????3.44	????6.24
			????40％	????0.97			????5.00	????8.86

Contrast :-0.25%Kymene SLX=4.57% " the % wet tenacitys after 2 all weather agings "

		Protein on the fiber
					????0％	????5％	????10％
		Glutaraldehyde on the fiber	????10％	????0.94				????1.97	????3.01
????20％	????0.99				????3.57	????6.06
			????40％	????1.05			????5.56	????10.30

Contrast :-0.25%Kymene SLX=9.01% " HST (second) after the weather aging in 24 hours "

		Protein on the fiber
					????0％	????5％	????10％
		Glutaraldehyde on the fiber	????10％	????1				????51	????81
????20％	????1				????89	????128
			????40％	????1			????108	????159

Contrast :-0.25%Kymene SLX=1s " HST (second) after 2 all weather agings "

		Protein on the fiber
					????0％	????5％	????10％
		Glutaraldehyde on the fiber	????10％	????1				????66	????125
????20％	????1				????86	????163
			????40％	????1			????120	????132

Contrast :-0.25%Kymene SLX=1s " HST after the oven ageing (second) "

		Protein on the fiber
					????0％	????5％	????10％
		Glutaraldehyde on the fiber	????10％	????1				????60	????108
????20％	????1				????101	????165
			????40％	????1			????108	????149

Contrast :-0.25%Kymene SLX=1s " Cobb (gsm) after the weather aging in 24 hours "

		Protein on the fiber
					????0％	????5％	????10％
		Glutaraldehyde on the fiber	????10％	Saturated fully				????39.0	????25.3
????20％	Saturated fully				????24.7	????22.3
			????40％	Saturated fully			????24.4	????21.6

Contrast :-0.25%Kymene SLX=saturated fully " Cobb (gsm) after 2 all weather agings "

		Protein on the fiber
					????0％	????5％	????10％
		Glutaraldehyde on the fiber	????10％	Saturated fully				????35.3	????21.1
????20％	Saturated fully				????24.1	????20.4
			????40％	Saturated fully			????23.1	????20.3

Contrast :-0.25%Kymene SLX=saturated fully " Cobb after the oven ageing (gsm) "

		Protein on the fiber
					????0％	????5％	????10％
		Glutaraldehyde on the fiber	????10％	Saturated fully				????35.0	????22.8
????20％	Saturated fully				????29.2	????20.7
			????40％	Saturated fully			????24.5	????20.3

Contrast: the wet tenacity of the cellulase/glutaraldehyde system under the complete saturated contingent condition of-0.25%Kymene SLX=in the exsiccant handsheet and sizability " the % wet tenacity after the weather aging in 24 hours "

		Protein/glutaraldehyde on the fiber
				????5％/20％	???10％/40％
		Drying conditions (surface temperature/duration of contact)	23 ℃/spend the night			?????0.65	?????4.13
????60℃/120s	?????2.00			?????5.65
			????70℃/180s		?????2.54	?????6.50
????105℃/35s	?????2.86			?????6.97

Contrast :-0.25%Kymene SLX (105 ℃/35s)=6.03% " the % wet tenacity after the oven ageing "

		Protein/glutaraldehyde on the fiber
				???5％/20％	???10％/40％
		Drying conditions (surface temperature/duration of contact)	23 ℃/spend the night			?????2.14	?????5.66
????60℃/120s	?????3.04			?????6.11
			????70℃/180s		?????3.25	?????7.28
????105℃/35s	?????2.99			?????8.82

Contrast :-0.25%Kymene SLX (105 ℃/35s)=11.38% " HST (second) after the weather aging in 24 hours "

		Protein/glutaraldehyde on the fiber
				??5％/20％	??10％/40％
		Drying conditions (surface temperature/duration of contact)	23 ℃/spend the night			?????3	??????3
????60℃/120s	?????51			??????39
			????70℃/180s		?????154	??????184
????105℃/35s	?????83			??????208

Contrast :-0.25%Kymene SLX (105 ℃/35s)=1s " HST (second) after 2 all weather agings "

		Protein/glutaraldehyde on the fiber
				???5％/20％	???10％/40％???
		Drying conditions (surface temperature/duration of contact)	23 ℃/spend the night			??????3	??????2
????60℃/120s	??????55			??????56
			????70℃/180s		??????158	??????221
????105℃/35s	??????82			??????231

Contrast :-0.25%Kymene SLX (105 ℃/35s)=1s " HST after the oven ageing (second) "

		Protein/glutaraldehyde on the fiber
				??5％/20％	???10％/40％
		Drying conditions (surface temperature/duration of contact)	23 ℃/spend the night			??????2	??????2
????60℃/120s	??????56			??????42
			????70℃/180s		??????134	??????193
????105℃/35s	??????96			??????157

Contrast :-0.25%Kymene SLX (105 ℃/35s)=1s " Cobb (gsm) after the weather aging in 24 hours "

		Protein/glutaraldehyde on the fiber
				????5％/20％	????10％/40％
		Drying conditions (surface temperature/duration of contact)	23 ℃/spend the night			Saturated fully	Saturated fully
????60℃/120s	??????26.8			??????26.9
			????70℃/180s		??????21.9	??????19.6
????105℃/35s	??????28.0			??????21.1

Contrast :-0.25%Kymene SLX (105 ℃/35s)=fully saturated " Cobb (gsm) after 2 all weather agings "

		Protein/glutaraldehyde on the fiber
				????5％/20％	????10％/40％
		Drying conditions (surface temperature/duration of contact)	23 ℃/spend the night			Saturated fully	Saturated fully
????60℃/120s	??????25.6			??????25.8
			????70℃/180s		??????20.4	??????20.3
????105℃/35s	??????22.7			??????19.3

Contrast :-0.25%Kymene SLX (105 ℃/35s)=fully saturated " Cobb after the oven ageing (gsm) "

		Protein/glutaraldehyde on the fiber
				????5％/20％	????10％/40％
		Drying conditions (surface temperature/duration of contact)	23 ℃/spend the night			Saturated fully	Saturated fully
????60℃/120s	??????26.3			??????26.3
			????70℃/180s		??????21.5	??????20.6
????105℃/35s	??????22.9			??????20.6

Contrast :-0.25%Kymene SLX (105 ℃/35s)=fully saturated under two stage aging conditions wet/dry strength and sizability " the % wet tenacity after the weather aging in 48 hours " of the cellulase in the exsiccant handsheet/glutaraldehyde system

Drying conditions (surface temperature/duration of contact)			The % wet tenacity
				Experiment numbers	Drying conditions A	Drying conditions B
??????1	????40℃/60s	????70℃/180s					?????4.66
			??????a	????55℃/60s	????70℃/180s	?????3.73
??????b	????40℃/180s	????70℃/180s					?????5.29
			??????ab	????55℃/180s	????70℃/180s	?????2.61
??????c	????40℃/60s	????105℃/35s					?????5.57
			??????ac	????55℃/60s	????105℃/35s	?????5.58
??????bc	????40℃/180s	????105℃/35s					?????6.21
			??????abc	????55℃/180s	????105℃/35s	?????5.01

Contrast :-0.25%Kymene SLX (105 ℃/35s)=8.48% " the % wet tenacity after the oven ageing "

Drying conditions (surface temperature/duration of contact)			The % wet tenacity
				Experiment numbers	Drying conditions A	Drying conditions B
??????1	????40℃/60s	????70℃/180s					?????5.38
			??????a	????55℃/60s	????70℃/180s	?????4.56
??????b	????40℃/180s	????70℃/180s					?????5.82
			??????ab	????55℃/180s	????70℃/180s	?????2.84
??????c	????40℃/60s	????105℃/35s					?????5.17
			??????ac	????55℃/60s	????105℃/35s	?????5.49
??????bc	????40℃/180s	????105℃/35s					?????6.03
			??????abc	????55℃/180s	????105℃/35s	?????5.53

Contrast :-0.25%Kymene SLX (105 ℃/35s)=12.53% " dry strength after the weather aging in 48 hours "

Drying conditions (surface temperature/duration of contact)			Dry strength/kNm -1
				Experiment numbers	Drying conditions A	Drying conditions B
??????1	????40℃/60s	????70℃/180s					?????5.40
			??????a	????55℃/60s	????70℃/180s	?????5.12
??????b	????40℃/180s	????70℃/180s					?????5.43
			??????ab	????55℃/180s	????70℃/180s	?????4.77
??????c	????40℃/60s	????105℃/35s					?????5.09
			??????ac	????55℃/60s	????105℃/35s	?????5.50
??????bc	????40℃/180s	????105℃/35s					?????5.13
			??????abc	????55℃/180s	????105℃/35s	?????5.27

Contrast :-blank (105 ℃/35s)=4.28kNm ^-10.25%Kymene SLX (105 ℃/35s)=4.41kNm ^-1" dry strength after the oven ageing "

Drying conditions (surface temperature/duration of contact)			Dry strength/kNm -1
				Experiment numbers	Drying conditions A	Drying conditions B
??????1	????40℃/60s	????70℃/180s					?????5.24
			??????a	????55℃/60s	????70℃/180s	?????4.65
??????b	????40℃/180s	????70℃/180s					?????5.11
			??????ab	????55℃/180s	????70℃/180s	?????4.75
??????c	????40℃/60s	????105℃/35s					?????5.09
			??????ac	????55℃/60s	????105℃/35s	?????5.18
??????bc	????40℃/180s	????105℃/35s					?????5.46
			??????abc	????55℃/180s	????105℃/35s	?????4.78

Contrast :-blank (105 ℃/35s)=3.93kNm ^-10.25%Kymene SLX (105 ℃/35s)=4.64kNm ^-1" HST after the weather aging in 48 hours "

Drying conditions (surface temperature/duration of contact)			HST/ second
				Experiment numbers	Drying conditions A	Drying conditions B
??????1	????40℃/60s	????70℃/180s					????277
			??????a	????55℃/60s	????70℃/180s	????216
??????b	????40℃/180s	????70℃/180s					????243
			??????ab	????55℃/180s	????70℃/180s	????169
??????c	????40℃/60s	????105℃/35s					????258
			??????ac	????55℃/60s	????105℃/35s	????274
??????bc	????40℃/180s	????105℃/35s					????310
			??????abc	????55℃/180s	????105℃/35s	????195

Contrast :-0.25%Kymene SLX (105 ℃/35s)=1s " HST after 2 all weather agings "

Drying conditions (surface temperature/duration of contact)			HST/ second
				Experiment numbers	Drying conditions A	Drying conditions B
??????1	????40℃/60s	????70℃/180s					????304
			??????a	????55℃/60s	????70℃/180s	????241
??????b	????40℃/180s	????70℃/180s					????190
			??????ab	????55℃/180s	????70℃/180s	????178
??????c	????40℃/60s	????105℃/35s					????239
			??????ac	????55℃/60s	????105℃/35s	????251
??????bc	????40℃/180s	????105℃/35s					????290
			??????abc	????55℃/180s	????105℃/35s	????171

Contrast :-0.25%Kymene SLX (105 ℃/35s)=1s " HST after the oven ageing "

Drying conditions (surface temperature/duration of contact)			HST/ second
				Experiment numbers	Drying conditions A	Drying conditions B
??????1	????40℃/60s	????70℃/180s					????314
			??????a	????55℃/60s	????70℃/180s	????205
??????b	????40℃/180s	????70℃/180s					????242
			??????ab	????55℃/180s	????70℃/180s	????149
??????c	????40℃/60s	????105℃/35s					????212
			??????ac	????55℃/60s	????105℃/35s	????275
??????bc	????40℃/180s	????105℃/35s					????308
			??????abc	????55℃/180s	????105℃/35s	????210

Contrast :-0.25%Kymene SLX (105 ℃/35s)=1s " Cobb after the weather aging in 48 hours "

Drying conditions (surface temperature/duration of contact)			???Cobb/gsm
				Experiment numbers	Drying conditions A	Drying conditions B
??????1	????40℃/60s	????70℃/180s					?????21.7
			??????a	????55℃/60s	????70℃/180s	?????21.9
??????b	????40℃/180s	????70℃/180s					?????22.0
			??????ab	????55℃/180s	????70℃/180s	?????25.1
??????c	????40℃/60s	????105℃/35s					?????20.7
			??????ac	????55℃/60s	????105℃/35s	?????20.1
??????bc	????40℃/180s	????105℃/35s					?????22.1
			??????abc	????55℃/180s	????105℃/35s	?????25.9

Contrast :-0.25%Kymene SLX (105 ℃/35s)=fully saturated " Cobb after 2 all weather agings "

Drying conditions (surface temperature/duration of contact)			???Cobb/gsm
				Experiment numbers	Drying conditions A	Drying conditions B
??????1	????40℃/60s	????70℃/180s					?????20.8
			??????a	????55℃/60s	????70℃/180s	?????19.2
??????b	????40℃/180s	????70℃/180s					?????28.2
			??????ab	????55℃/180s	????70℃/180s	?????21.2
??????c	????40℃/60s	????105℃/35s					?????22.1
			??????ac	????55℃/60s	????105℃/35s	?????20.6
??????bc	????40℃/180s	????105℃/35s					?????21.7
			??????abc	????55℃/180s	????105℃/35s	?????21.9

Contrast :-0.25%Kymene SLX (105 ℃/35s)=fully saturated " Cobb after the oven ageing "

Drying conditions (surface temperature/duration of contact)			???Cobb/gsm
				Experiment numbers	Drying conditions A	Drying conditions B
??????1	????40℃/60s	????70℃/180s					?????21.7
			??????a	????55℃/60s	????70℃/180s	?????20.4
??????b	????40℃/180s	????70℃/180s					?????22.3
			??????ab	????55℃/180s	????70℃/180s	?????22.3
??????c	????40℃/60s	????105℃/35s					?????20.5
			??????ac	????55℃/60s	????105℃/35s	?????20.4
??????bc	????40℃/180s	????105℃/35s					?????20.9
			??????abc	????55℃/180s	????105℃/35s	?????22.3

Contrast :-0.25%Kymene SLX (105 ℃/35s)=the dry rules of complete saturated difference are to the contrast " different dry rules are to the influence (oven ageing data) of wet tenacity " of the cellulase/wet tenacity of glutaraldehyde system and the influence of sizability

Additive	Drying conditions (surface temperature/duration of contact)		The % wet tenacity
				Drying conditions A	Drying conditions B
	5% protein/20% glutaraldehyde	????105℃/35s				????????-	?????2.99
5% protein/20% glutaraldehyde			????70℃/180s	????????-	?????3.25
	5% protein/20% glutaraldehyde	????40℃/60s				????105℃/35s	?????5.17
5% protein/20% glutaraldehyde			????55℃/180s	????70℃/180s	?????5.49
	???0.25％Kymene?SLX	????105℃/35s				????????-	?????12.53

" different dry rules are to the influence (oven ageing data) of HST "

Additive	Drying conditions (surface temperature/duration of contact)		HST/ second
				Drying conditions A	Drying conditions B
	5% protein/20% glutaraldehyde	????105℃/35s				????????-	????96
5% protein/20% glutaraldehyde			????70℃/180s	????????-	????134
	5% protein/20% glutaraldehyde	????40℃/60s				????105℃/35s	????212
5% protein/20% glutaraldehyde			????55℃/180s	????70℃/180s	????149
	??0.25％Kymene?SLX	????105℃/35s				????????-	????1

" different dry rules are to the influence (oven ageing data) of Cobb "

Additive	Drying conditions (surface temperature/duration of contact)		???Cobb/gsm
				Drying conditions A	Drying conditions B
	5% protein/20% glutaraldehyde	????105℃/35s				???????-	?????22.9
5% protein/20% glutaraldehyde			????70℃/180s	???????-	?????21.5
	5% protein/20% glutaraldehyde	????40℃/60s				????105℃/35s	?????20.5
5% protein/20% glutaraldehyde			????55℃/180s	????70℃/180s	?????22.3
	??0.25％Kymene?SLX	????105℃/35s				???????-	Saturated fully

In a word, the cellulase of described digital proof paper/glutaraldehyde is handled and is made wet tenacity, dry strength and the sizability of paper be improved.11, the proof of the biological metalization of paper

The biological metalization of paper is proved to be.This technology is based on the avidity of streptavidin to vitamin H.Biotin labeling is connected in cellulase, and then cellulase links to each other with the streptavidin of gold grain mark again.

Containing polysorbas20 (0.1%vv in the greenhouse handle under the jolting ^-1) 1/3 PBS damping fluid (pH7.4) in paper pulp incubation 45 minutes.Make the scraps of paper (6cm ²), roll and dried overnight at room temperature.

The pattern (1.5cm that contains the biotinylation cellulase ²) and do not have the biotinylation cellulase in the same old way under jolting, the room temperature with 5% (wv in 10mM PBS pH 7.4 ^-1) BSA incubation 30 minutes together.

Pressing manufacturer's recommendation uses the streptavidin conjugate of Auroprobe BLplus mark and promotor (Amersham Ltd., Amersham U.K.) is used for connecting and development gold grain (people such as Fostel, Chromosoma, 90,254, (1984); People such as Hutchinson, J.CellBiol., 95,609, (1982)).Accelerator solution is produced orange/brown with the silver bag by gold grain, proves the existence of metal.The contrast scraps of paper that do not contain the biotinylation cellulase do not show orange/brown, thereby not by metal bag quilt.12, the capacitance measurement of biological metal paper

The capacitance characteristic of the electric capacity of biological metal paper with contrast scraps of paper comparison change paper with the cellulase that determines whether golden mark.

With the cellulase of cellulase, golden mark, strengthen the cellulase of golden mark and do not have the contrast pulp preparation scraps of paper of cellulase.The scraps of paper are clipped between two metal sheets that are connected in capacitance meter.Metal sheet is placed on guarantees between metal sheet to keep constant and reproducible distance in the shelf.

Utilize following formula to calculate electric capacity (C) $C=\frac{\mathrm{\ϵo\ϵrA}}{d}$ The distance between d=two plates wherein

The A=area

ε o=constant

ε r=relative permittivity

The gained measuring result shows that the existence of the cellulase of golden mark has increased the electric capacity of the scraps of paper.Measuring result is listed in the table 7.

Table 7: capacitance measurement

Sample	Electric capacity (pF)
		Machine is proofreaied and correct (contrast)	10.00
The paper that does not have cellulase	10.97
		Paper+cellulase	10.65
The cellulase of paper+golden mark	13.86

13, amylase is in conjunction with the proof of starch

With two kinds of amylase of HPLC indication: derive from the α-Dian Fenmei (X-A type crude preparation by using) of aspergillus oryzae (Aspergillus oryzae) and derive from black aspergillar starch glucoside sugar (from Sigma Aldrich Co.Ltd., Poole, Dorset, United Kingdom obtains).Determine the catalytic main peak of each preparation with starch glucose release test.In evaluation, multiple starch is compared every kind of proteic joint efficiency of mensuration with BSA.

32mg ml ^-1The α-Dian Fenmei solution of (dry weight) is allocated among the 0.1M PBS (pH 7.0).Among the sample introduction 100 μ l to HPLC, adopt Bio-Sil SEL gel permeation column, leacheate is the 0.1M phosphate buffered saline buffer, and flow velocity is 1ml min ^-1The reducing sugar of collecting elutriant (1ml) and from starch suspension, discharging with standard microtitration test (glucose) detection.

With glucose and the cellobiose in the following quantitative test test samples.Test at room temperature and carry out on the microtiter plate.Reagent composition: 0.1M phosphate buffered saline buffer pH7.0 (250Eu/ml) the 60 μ l 0.1M phosphate buffered saline buffer pH7.0 of 0.1M phosphate buffered saline buffer pH7.0 (800Eu/ml) the 10 μ l notatins of amino pyrine reagent (the 0.1M phosphate buffered saline buffer pH 7.0 of 19.7mM4-aminophenazone) the 10 μ l peroxidases of 10 μ l phenol reagent (the 0.1M phosphate buffered saline buffer pH7.0 of 0.128M phenol), 10 μ l

These reagent compositions are mixed and be added in the hole of microtiter plate.Add 100 μ l samples and then add excessive substrate (starch) again.Redness occurs and show have amylase to exist.

Also available identical way is made the HPLC curve of amyloglucosidase.With the blue technical measurement amyloglucosidase of Coomassie for containing about 262mg ml ^-1Proteic liquid preparation.Be diluted to the diluent of original content 0.007 with 0.1MPBS (pH7.0), get 100 μ l HPLC sample introductions and under 230nm 0.1AUS, monitor.The reducing sugar of collecting the 1ml elutriant and from starch suspension, discharging by described method detection.

Estimate α-Dian Fenmei and amyloglucosidase ability in suspension in conjunction with conventional starch.Starch (0.2g; Roquette) be added among the 9ml 0.1M PBS (pH7.0) and add 1ml α-Dian Fenmei solution (Coomassie indigo plant is determined as 9.5mg ml ^-1).Incubation is 20 minutes on shaking table.

Sample under rotating speed 13000rpm centrifugal 5 minutes is got 100 μ l sample feedings to the HPLC post.Peak height and T=0 sample in conjunction with α-Dian Fenmei contrasted in 20 minutes.Thereby calculate enzyme in conjunction with percentage.Also measure the combination rate of amyloglucosidase with cationic starch.BSA is used as contrast and is used for identical method.The ultimate density of employed BSA is 0.2% (WV ^-1) in 0.1M PBS.

In being listed in the table below in conjunction with result of experiment.The starch binding curve

Enzyme	Substrate	The % combination rate
			α-Dian Fenmei	Starch	????32
Amyloglucosidase	Starch	????27
			Amyloglucosidase	Cationic starch	????45
????????BSA	Starch	????7
			????????BSA	Cationic starch	????6

The result shows that α-Dian Fenmei and the equal specificity of starch glucoside sugar are incorporated on starch and the cationic starch, therefore is suitable for and does the protein connection of effector fragment in conjunction with starch.

Claims (36)

1, a kind of method of handling polymkeric substance, make at least a character that is selected from fluid, electricity and intensive property be improved, it comprises connecting by the albumen that is used to obtain described improvement the effector fragment is combined in described polymkeric substance, described effector fragment is different from described albumen and connects, described albumen connects and is different from described polymkeric substance, and described effector fragment and described albumen connect by the significant quantity that obtains described improvement and exists.

2, the method for processing polymkeric substance as claimed in claim 1 can make the fluid dipped type of described polymkeric substance improve.

3, the method for processing polymkeric substance as claimed in claim 1 can make the sizability of described polymkeric substance be improved.

4, the method for processing polymkeric substance as claimed in claim 1 can make the conductivity of described polymkeric substance be improved.

5, the method for processing polymkeric substance as claimed in claim 1 can make the metallicity of described polymkeric substance be improved.

6, the method for processing polymkeric substance as claimed in claim 1 can make the wet tenacity performance of described polymkeric substance be improved.

7, the method for processing polymkeric substance as claimed in claim 1 can make the dry strength performance of described polymkeric substance be improved.

8, any method of claim as described above, wherein said polymkeric substance is a polysaccharide.

9, method as claimed in claim 8, wherein said polymkeric substance are Mierocrystalline cellulose.

10, any method of claim as described above, wherein said polymkeric substance is the composition fiber of paper or paper.

11, any method of claim as described above, wherein said albumen connects and comprises natural enzyme or its fragment.

12, method as claimed in claim 1, wherein said albumen connect and comprise and be selected from cellulase, hemicellulase, mannase, zytase, proteolytic enzyme, M-Zyme, chitinase, lignoenzyme, agarase, algin enzyme and diastatic enzyme or its fragment.

13, any method of claim as described above, wherein said albumen connects and comprises polysaccharidase and fragment thereof.

14, as the method for claim 13, wherein said albumen connects and comprises cellulase and fragment thereof.

15, as the method for claim 14, wherein said albumen connects the cellulose binding domain that comprises cellulase.

16, any method of claim as described above, wherein said effector fragment is connected in described albumen by connecting agent.

17, as the method for claim 16, wherein said connection agent comprise non-altogether bond close right.

18, as the method for one of claim 1 to 16, wherein said effector fragment is covalently bonded in described albumen and connects.

19, any method of claim as described above, wherein said effector fragment can be from the described polymkeric substance optionally cracking get off.

20, a kind of chemical substance composition comprises:

A) effector fragment; With

B) can be attached to protein on the polymkeric substance to described effector fragment;

Wherein said effector fragment is different from described protein, and described composition can make at least a character that is selected from fluid, electricity and intensive property of described polymkeric substance be improved.

21, a kind of material compositions, it comprises by the albumen connection combination segmental polymkeric substance of effector thereon, described effector fragment is different from described albumen and connects, wherein said effector fragment and described albumen connect by significant quantity and exist, and at least a character that is selected from fluid, electricity and intensive property of described polymkeric substance is improved.

22, a kind of method of handling polymkeric substance, make at least a character that is selected from fluid, electricity and intensive property be improved, contact comprising the albumen that makes polymkeric substance and effector fragment and be used to obtain described improvement, described effector fragment is different from described albumen and connects, and be different from described polymkeric substance, described albumen is different from described polymkeric substance, and described effector fragment and described albumen exist by the significant quantity that obtains described improvement.

23,, comprise the step that described effector fragment and described proteinic conjugate are contacted with described polymkeric substance as the method for claim 22.

24,, comprise the step that described effector fragment is contacted with the conjugate of described protein and described polymkeric substance as the method for claim 22.

25, a kind of method of handling polymkeric substance, make at least a character that is selected from fluid, electricity and intensive property be improved, comprising connecting at least a effector fragment is combined at least a described polymkeric substance by at least a albumen that is used to obtain described improvement, described at least a effector fragment is different from described at least a albumen and connects, described at least a albumen connects and is different from described at least a polymkeric substance, and described at least a effector fragment and described at least a albumen connect by the significant quantity that obtains described improvement and exists.

26, a kind of method of handling polymkeric substance, make and be selected from fluid, at least a character of electricity and intensive property is improved, comprising at least a effector fragment is contacted with at least a described polymkeric substance with at least a protein that is used to obtain described improvement, described at least a effector fragment is different from described at least a protein, and be different from described at least a polymkeric substance, described at least a protein is different from described at least a polymkeric substance, and described at least a effector fragment and described at least a protein exist by the significant quantity that obtains described improvement.

27, the method of the composition fiber of a kind of treatment paper or paper, make and be selected from fluid, at least a character of electricity and intensive property is improved, comprising connecting the composition fiber that at least a effector fragment is combined in described paper or paper by at least a albumen that is used to obtain described improvement, described at least a effector fragment is different from described at least a albumen and connects, described at least a albumen connects the composition fiber that is different from described paper or paper, and described at least a effector fragment and described at least a albumen connect by the significant quantity that obtains described improvement and exists.

28, the method for the composition fiber of a kind of treatment paper or paper, at least a character that is selected from fluid, electricity and intensive property is improved, comprising connecting the composition fiber that the effector fragment is combined in described paper or paper by the albumen that is used to obtain described improvement, described effector fragment is different from described albumen and connects, described albumen connects the composition fiber that is different from described paper or paper, and described effector fragment and described albumen connect by the significant quantity that obtains described improvement and exists.

29, as the method for claim 28, wherein said effector fragment can be given the wet tenacity that described paper improves.

30, as the method for claim 28, wherein said effector fragment can be given the dry strength that described paper improves.

31, as the method for claim 28, wherein said effector fragment can be given the sizability that described paper improves.

32, as the method for claim 28, wherein said effector fragment is a linking agent.

33, as the method for claim 32, wherein said effector fragment is a dialdehyde crosslinking agent.

34, as the method for claim 33, wherein said effector fragment is a glutaraldehyde.

35, as the method for one of claim 28 to 34, it is cellulase that wherein said albumen connects.

36, effector fragment and proteolytic enzyme are in a kind of purposes of handling in the method that at least a character that is selected from fluid, electricity and intensive property that polymkeric substance makes described polymkeric substance is improved.

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