CN103695388B - α-amylase and Aspergillus niger strain expressing α-amylase - Google Patents

Abstract

The object of the present invention is to provide an α-amylase and an Aspergillus niger strain expressing α-amylase, the amino acid sequence of the α-amylase is SEQ? ID? NO: 1, one of its encoding mRNA sequences is SEQ? ID? NO:2. The mutant strain Aspergillus niger 1-B5 obtained by the ultraviolet mutagenesis method of the present invention can efficiently express the α-amylase AclaP11 derived from Aspergillus clavus, and the strain number is CGMCC? No. 8443. The enzyme activity of Aspergillus niger 1-B5 was 1.97 times that of the starting strain, and the optimum pH of the expressed α-amylase AclaP11 was 6.0, and the optimum temperature was 50°C. Compared with the starting strain, the enzymatic properties did not change . In addition, the Aspergillus niger 1-B5 described in the present invention is a food safety bacterium (GRAS), so the α-amylase secreted and expressed by it can also be used in the field of food additives, further expanding the market space of the α-amylase.

Description

α-amylase and Aspergillus niger strain expressing α-amylase

technical field

The invention belongs to the technical field of separation and expression of amylase, and in particular relates to an α-amylase derived from Aspergillus clavatus and a filamentous fungal host Aspergillus niger cell for recombinantly expressing the α-amylase.

Background technique

α-amylases (E.C.3.2.1.1) are capable of catalyzing the endohydrolysis of 1,4-α-glycosidic linkages in oligo- and polysaccharides, acting in a random fashion on starch, glycogen and related poly- and oligosaccharides, releasing α- Configuration of the reducing group. At present, α-amylase is widely used in a variety of industrial applications, such as bakery, wine, corn steep liquor and ethanol production, and vinegar fermentation and other fields.

The sources of α-amylase are very extensive and can be isolated from animals, plants and microorganisms. Among them, amylase derived from microorganisms has the characteristics of abundant sources, diverse performances and easy industrial production, which can meet the needs of various industrial applications and is the most widely used in industry. In the process of starch treatment in modern industry, the hydrolysis method of microbial amylase has completely replaced the traditional chemical hydrolysis method. Although there are a variety of microorganisms that can produce α-amylase, including filamentous fungi, yeast, bacteria, and actinomycetes. However, at present, α-amylases that can meet the needs of industrial applications are mainly derived from bacteria and filamentous fungi. Bacterial α-amylases are usually derived from Bacillus, and fungal α-amylases are usually derived from Aspergillus. The α-amylase produced by fungi is called fungal α-amylase.

In the literature reported so far, fungal α-amylases can be roughly divided into three types according to enzymatic properties or action conditions: (1) Neutral fungal α-amylases: different from bacterial α-amylases, fungal α-amylases There are relatively few sources of fungal α-amylase. The temperature and pH of most fungal α-amylases are relatively mild. Start to inactivate. At present, the α-amylase derived from Aspergillus oryzae (variety) which is the most commercially produced and most widely used belongs to this enzyme. (2) Heat-resistant or acid-resistant fungal α-amylase: This type of enzyme has a good thermal stability when the pH is between 2.5 and 4.5, and the action temperature exceeds 60°C. Compared with neutral fungal α-amylases, heat-resistant or acid-resistant fungal α-amylases can simplify the liquefaction and saccharification processes, reduce the chance of bacterial contamination in the deep processing of starch such as sugar production, and reduce the corresponding production costs. These enzymes have already been produced and used in industry, and have great potential for development and utilization. (3) Fungi with raw amylase activity: α-amylase, in addition to the ability to hydrolyze soluble starch or other gelatinized starch, also has the ability to hydrolyze raw starch. It is used in the simultaneous saccharification and fermentation (SSF) of raw meal alcohol industry , used in conjunction with glucoamylase, can greatly increase the utilization rate and efficiency of starch, and effectively increase the alcohol yield.

A variety of α-amylase genes have been reported to be expressed in different hosts, and the heterologous expression of fungal α-amylase genes is mainly concentrated in eukaryotic expression systems. Compared with prokaryotic expression systems (such as E. coli expression systems or Bacillus expression systems, etc.), eukaryotic expression systems have great advantages in expressing heterologous proteins of eukaryotic origin, such as post-translational processing modifications (such as glycosylation , formation of disulfide bonds), recognition of introns, cutting out of signal peptides, correct folding and secretion of peptide chains, etc. In addition, there are a variety of fungal expression systems listed as GRAS (generally recognized as safe) strains by the US FDA, which makes it easier to obtain the recognition and approval of relevant agencies for the production and application of some heterologous proteins for food or medicine.

At present, a large number of α-amylases from the genus Aspergillus (Aspergillus.sp) have been reported and used in industrial production. These amylases have excellent enzymatic properties, but the expression level of the production strains is generally low, which seriously restricts the development of amylases. wide application.

Contents of the invention

The purpose of the present invention is to provide an α-amylase and a bacterial strain expressing α-amylase. In the present invention, the α-amylase gene of Aspergillus clavus is transformed into Aspergillus niger to construct an engineering strain expressing α-amylase recombinantly; and by carrying out ultraviolet mutagenesis on the engineering bacterium, an Aspergillus niger mutant strain with high α-amylase production is obtained , to make up for the deficiencies of the prior art.

The present invention firstly provides an α-amylase, the amino acid sequence of which is SEQ ID NO: 1, and a coding mRNA sequence of which is SEQ ID NO: 2.

In a second aspect, the present invention relates to an enzyme composition comprising an alpha-amylase as described above.

Another aspect of the present invention relates to Aspergillus strains for expressing the above-mentioned α-amylase;

One of the α-amylase-expressing Aspergillus niger 1-B5 (Aspergillus Niger1-B5) has been preserved on November 7, 2013 at the China Microbial Strains of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing The General Microorganism Center (CGMCC) of the Preservation Committee, the strain number is CGMCCNo.8443.

The mutant strain Aspergillus niger 1-B5 obtained by the ultraviolet mutagenesis method of the present invention can efficiently express the α-amylase AclaP11 derived from Aspergillus clavus, and its enzyme activity is 1.97 times that of the starting strain; the α-amylase expressed by the mutant strain Aspergillus niger 1-B5 The optimum action pH of the enzyme AclaP11 was 6.0, and the optimum action temperature was 50°C. Compared with the starting strain, the enzymatic properties did not change. In addition, the Aspergillus niger 1-B5 described in the present invention is a food safety bacterium (GRAS), so the α-amylase secreted and expressed by it can also be used in the field of food additives, further expanding the market space of the α-amylase.

Description of drawings

Fig. 1: the pGm plasmid map that the present invention uses;

Figure 2: Comparison of mycelium morphology between the mutant Aspergillus niger 1-B5 and the starting strain;

Figure 3: Comparison of colony morphology between the mutant Aspergillus niger 1-B5 and the starting strain;

Figure 4: The pH-residual activity curve of the α-amylase AclaP11 expressed by Aspergillus niger 1-B5;

Figure 5: Temperature-residual activity curve of the alpha amylase AclaP11 expressed by Aspergillus niger 1-B5.

Detailed ways

The present invention uses conventional techniques and methods commonly used in the fields of genetic engineering and molecular biology, such as those described in reference books such as MOLECULARCLONING: ALABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and CURRENT PROTOCOLS IN MOLECULARBIOLOGY (Ausubel, 2003). However, this does not mean that the present invention is limited to the specific methods, experimental schemes and reagents recorded in the examples, those skilled in the art can choose the disclosed technology to implement the schemes described in the examples of the present invention.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. DICTIONARYOFMICROBIOLOGYANDMOLECULARBIOLOGY, 3nd Ed. (Singleton et al., 2006) and COLLINSDICTIONARYBIOLOGY (Hale et al., 2003) provide the general explanation of many terms used in the present invention for the skilled person, specifically as follows:

As used herein, the term "starch" refers to any substance composed of complex polysaccharide carbohydrates of plants, including amylose and _amylopectin having ( _{C6H10O5 ) x} , where X can be any number.

As used herein, the term "alpha-amylase" refers to an enzyme that catalyzes the hydrolysis of 1,4-alpha-glycosidic linkages. These enzymes have also been described as enzymes that accomplish exo- or endo-hydrolysis of 1,4-α-D-glycosidic linkages in polysaccharides containing 1,4-α-linked D-glucose units. Another term used to describe these enzymes is "glycogenase".

As used herein, the term "recombinant" when used in reference to a cell, nucleic acid, protein or vector means that the cell, nucleic acid, protein or vector has been modified by introducing a heterologous nucleic acid or protein or by altering a native nucleic acid or protein , or the cells are derived from cells so modified. Thus, for example, a recombinant cell expresses genes that are not found in the native (non-recombinant) form of the cell, or expresses native genes that are abnormally, underexpressed, or not expressed at all.

As used herein, the terms "protein" and "polypeptide" are used interchangeably herein. The conventional one-letter or three-letter codes for amino acid residues are used herein.

As used herein, the term "signal sequence" refers to an amino acid sequence bound to the N-terminal portion of a protein, which promotes secretion of the mature form of the protein outside the cell. The definition of a signal sequence is a functional definition. The mature form of the extracellular protein lacks a signal sequence, which is cleaved during secretion.

As used herein, the term "gene" refers to a segment of DNA involved in the production of a polypeptide, including regions preceding and following the coding region, as well as intervening sequences (introns) between each coding segment (exons).

As used herein, the term "nucleic acid" includes DNA, RNA, single- or double-stranded, and chemical modifications thereof.

Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acids are written left to right in amino to carboxy orientation.

As used herein, the terms "nucleic acid" and "polynucleotide" are used interchangeably herein.

As used herein, the term "vector" refers to a polynucleotide sequence designed to introduce a nucleic acid into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phagemids, cassettes, and the like.

As used herein, the term "expression vector" means a DNA construct comprising a DNA sequence operably linked to suitable control sequences capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effectuate transcription, an optional operator sequence to control transcription, a sequence encoding a suitable ribosome binding site on the mRNA, an enhancer, and sequences controlling the termination of transcription and translation.

As used herein, the term "promoter" refers to a regulatory sequence involved in binding RNA polymerase to initiate transcription of a gene. Promoters can be inducible or constitutive.

As used herein, when describing proteins or the genes that encode them, the term applied to the gene is generally italicized (eg a gene encoding AclaP11 amylase could be indicated as AclaP11). Terms used for proteins are generally not italicized (for example, the amylase encoded by the AclaP11 gene may be indicated as AclaP11).

A polynucleotide or polypeptide having a certain percentage of sequence identity to another sequence means that, when aligned, that percentage of bases or amino acid residues are the same when comparing the two sequences. The alignment and percent homology or identity can be determined using any suitable software program known in the art, such as BLAST (Altschule et al. Basic local alignment search tool. Journal of Molecular Biology, 1990, 215(3):403-410). Since the genetic code is degenerate, more than one codon can be used to encode a specific amino acid, and the present invention includes polynucleotides encoding a specific amino acid sequence.

As used herein, the term "host strain" or "host cell" refers to a suitable host for an expression vector or DNA construct comprising an alpha-amylase-encoding polynucleotide of the present invention. In particular, the host strain is preferably a filamentous fungal cell. The host cell may be a wild-type filamentous fungal host cell or a genetically modified host cell. The term "host strain" or "host cell" refers to a nuclear protoplast produced by a cell of a filamentous fungal strain.

As used herein, the term "filamentous fungi" refers to all filamentous forms of organisms of the subdivision Fungi (see INTRODUCTORY MYCOLOGY, 4th Ed. (Alexopoulos, 2007) and AINSWORTHANDBISBYDICTIONARYOFTHEFUNGI, 10th Ed. (Kirketal., 2008)). These fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose, and other complex polysaccharides. The filamentous fungi of the present invention are morphologically, physiologically and genetically distinct from yeast. Vegetative growth of filamentous fungi is accomplished by elongation of the hyphae, and carbon metabolism is obligately aerobic. In the present invention, the filamentous fungal parental cells can be, but not limited to, Aspergillus sp. (such as A.clavatus, A.fumigatus, A.awamori) , Aspergillus flavus (A.flavus), Aspergillus terreus (A.terreus) and Aspergillus oryzae (A.oryzae)), Penicillium sp. (such as Penicillium chrysogenum (P.chrysogenum)), Xinsa Neosartoryasp. (e.g. N. fischeri), Gliocladium sp. (e.g. G. roseum), Trichoderma sp. Trichoderma sp. (eg T. reesei, T. viride, T. koningii, T. harzianum), Humicola Humicolasp. (such as H.insolens and H.grisea), Chrysosporiumsp., Fusariumsp., Cells of Neurospora sp., Hypocreas sp. and Emericella sp.

As used herein, the term "Aspergillus" or "Aspergillus sp." refers to any fungal genus previously or currently classified as Aspergillus.

As used herein, the term "culturing" refers to growing a population of microbial cells in a liquid or solid medium under appropriate conditions.

As used herein, the term "heterologous" in reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in the host cell. The term is intended to include proteins encoded by naturally occurring genes, mutant genes and/or synthetic genes.

As used herein, the term "endogenous" in reference to a polynucleotide or protein refers to a polynucleotide or protein naturally occurring in a host cell.

As used herein, the terms "transformed," "stably transformed," and "transgenic" with reference to a cell mean that the cell contains a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or As episomal plasmids that are maintained for multiple generations.

In the context of the insertion of a nucleic acid sequence into a cell, the term "introduction" means "transfection", "transformation" or "transduction", including the integration of a nucleic acid sequence into a eukaryotic or prokaryotic cell, where the nucleic acid sequence can Integrated into the genome of the cell (eg, chromosomal, plasmid, plastid, or mitochondrial DNA), converted to a spontaneous replicon, or expressed transiently (eg, transfected mRNA).

As used herein, the term "enzyme activity unit" refers to the amount of enzyme that produces a given amount of product per given time under specified conditions. In some embodiments, the term "amylase activity unit" (CU) is defined as under given temperature and pH conditions, in the presence of excess thermostable α-glucosidase (α-glucosidase), each Amount of enzyme required to release 1 micromol of p-nitrophenol from a non-reducing-endblocked p-nitrophenylmaltoheptaoside (BPNPG7) substrate in min.

"CGMCC" refers to China General Microorganism Culture Collection Management Center, Beijing 100101, China,

"ATCC" means the American Type Culture Collection, Manassas, VA20108, USA,

"NRRL" Northern Agricultural Research Institute Culture Collection, Peoria, IL61604, USA,

Recombinantly expressed enzymes and host cells:

In some embodiments of the invention, the microorganism is genetically engineered to express a heterologous alpha-amylase, preferably the host cell is a filamentous fungal cell.

Some preferred fungal host cells include A. nidulans, A. awamori, A. oryzae, A. aculeatus, A. niger, A. japonicus, T. reesei, A. viride Mold, Fusarium oxysporum (F. oxysporum) and Fusarium solani rot (F. solani).

In some embodiments, the filamentous fungal host cell is a strain of Aspergillus sp. Useful Aspergillus host strains include but are not limited to Aspergillus nidulans (Yeltone et al., Proc. Natl. Acad. Sci. USA, 1984, 81:1470-1474; 45; Johnstone et al. EMBOJ., 1985, 4:1307-1311); Aspergillus niger (Kelly et al., EMBOJ., 1985, 4:475-479); Aspergillus awamori (NRRL3112, UVK143f (USP5364770), ATCC22342, ATCC44733 and ATCC14331) and Aspergillus oryzae (ATCC11490). In a further embodiment, the filamentous fungal host cell is a strain of Aspergillus niger.

According to the present invention, a DNA construct comprising a nucleic acid encoding an alpha-amylase, such as AclaP11, is constructed for introduction into a host cell. In some embodiments, the DNA construct is introduced into the host cell via an expression vector that includes regulatory sequences operably linked to an alpha-amylase (such as AclaP11 ) coding sequence.

In some embodiments, a nucleic acid encoding an alpha amylase (AclaP11) is operably linked to a suitable promoter that is transcriptionally active in a fungal host cell. The promoter may be derived from a gene encoding a protein homologous or heterologous to the host cell. Preferably, the promoter is useful in an Aspergillus host. Non-limiting examples of useful promoters include those derived from genes encoding Aspergillus awamori and Aspergillus niger glucoamylase (glaA) (Nunberg et al., Mol. Cell Biol., 1984, 4:2306-2315; USP5364770; USP6590078; Gwynne et al., Nat .Biotechnol.,1987,5:713-719; Boeletal.,EMBOJ.,1984,3:1581-1585), Aspergillus niger α-amylase, Aspergillus oryzae TAKA amylase and Aspergillus niger neutral α-amylase gene promoter . In a further embodiment, the promoter is the promoter of the gene encoding Aspergillus niger glucoamylase.

In some embodiments, the coding sequence for alpha amylase (AclaP11) is operably linked to a signal sequence. The DNA encoding the signal sequence is preferably naturally associated with the gene to be expressed. In a further embodiment, the signal sequence linked to the alpha-amylase coding sequence is encoded by the gene encoding the alpha-amylase (eg, the signal sequence for AclaP11 is encoded by the AclaP11 gene).

In some embodiments, the expression vector also includes a termination sequence. Non-limiting examples of useful terminators include those encoding the Aspergillus niger, A. tubingensis, and A. awamori glucoamylase genes (Nunberg et al., Mol. Cell Biol., 1984, 4:2306-2315 and Boe et al., EMBO J., 1984, 3:1581-1585). In a further embodiment, the termination sequence is the terminator of the gene encoding Aspergillus tubingensis glucoamylase.

In some embodiments, the expression vector includes a selectable marker. Examples of preferred selectable markers include, but are not limited to, markers that confer resistance to antimicrobial agents (eg, hygromycin, bleomycin, chloramphenicol, and phleomycin). Genes that confer a metabolic advantage, such as nutritional selectable markers, are also useful in the present invention, including those markers known in the art such as amdS, argB, and pry4. In a further embodiment, the selectable marker is the Aspergillus nidulans amdS gene, which encodes an acetamidase that enables the transformed cells to grow on acetamide as a nitrogen source. The use of the A. nidulans amdS gene as a selectable marker is described in Kelly et al., EMBO J., 1985, 4:475-479 and Penttila et al., Gene, 1987, 61:155-164.

The expression vector comprising a DNA construct carrying a polynucleotide encoding an alpha-amylase may be any vector capable of self-replicating in a given fungal host organism or integrating into the host DNA. In some embodiments, the expression vector is a plasmid. In a further embodiment, the promoter in the expression vector is the promoter of the gene encoding Aspergillus niger glucoamylase, the terminator is the terminator of the gene encoding Aspergillus tubingensis glucoamylase, and the selectable marker is the Aspergillus nidulans amdS gene.

Methods for inserting polynucleotides encoding alpha amylases such as AclaP11 into expression vectors are well known in the art. This is generally accomplished by cleavage and ligation reactions at convenient restriction sites. In some embodiments, the restriction enzyme sites used are AflII and NotI.

Transformation, expression and culture of host cells:

Introduction of DNA constructs or vectors into host cells includes various techniques such as transformation, electroporation, nuclear microinjection, transduction, transfection (e.g. lipofection-mediated transfection and DEAE-Dextrin-mediated transfection), Precipitation of DNA with calcium phosphate baths, high-velocity bombardment with DNA-coated microprojectiles, and protoplast fusion. General transformation techniques are known in the art. For transformation of Aspergillus strains, reference is made to Yeltone et al., Proc. Natl. Acad. Sci. USA, 1984, 81:1470-1474 and EP238023.

Preferably, a genetically stable transformant is constructed with a vector system whereby a nucleic acid encoding an α-amylase (such as AclaP11) is stably integrated into the chromosome of the host strain, and then the transformant can be purified by known techniques.

In one non-limiting example, stable transformants containing the amdS marker differed from non-stable transformants by their faster growth rate on acetamide-containing solid media and the formation of smooth, rather than rough-edged, round colonies. Differentiate. Additionally, in some cases, further stability testing can be performed by growing transformants on solid non-selective medium (i.e. medium lacking acetamide), harvesting spores from this medium, and determining subsequent germination and The percentage of these spores grown on selective media containing acetamide was determined. Alternatively, other methods known in the art can be used to select transformants.

In some embodiments, preparation of Aspergillus sp. for transformation includes preparation of protoplasts from fungal mycelium (see Campbell et al., Curr. Genet., 16:53-56). Mycelium is obtained from germinated vegetative spores. The mycelium is treated with an enzyme that digests the cell wall, producing protoplasts. The protoplasts are then protected by an osmotic stabilizer present in the suspension medium. These stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate and the like. Usually the concentration of these stabilizers varies between 0.8 and 1.2M.

The uptake of DNA by host strains can be determined by the concentration of calcium ions. Generally, 10-10 mM CaCl ₂ can be used in community uptake solutions. In addition to the requirement for calcium ions in the uptake solution, other compounds that are generally included are buffer systems such as TE buffer (10 mM Tris (pH 7.4) and 1 mM EDTA) or 10 mM MOPS (pH 6.0) buffer and polyethylene glycol (PEG ).

Typically a suspension containing host cells or protoplasts such as Aspergillus sp. protoplasts or cells that have been permeabilized at a density of about ¹⁰⁷ /mL is used in transformation. In some embodiments, a volume of approximately 100 μL of an appropriate solution of these protoplasts or cells (eg, 1.2M sorbitol and 50 mM CaCl ₂ ) is mixed with the desired DNA. In some embodiments, a high concentration of PEG is added to the uptake solution. 0.1-1 volume of 25% PEG4000 can be added to the protoplast suspension. However, preferably about 0.25 volume is added to the protoplast suspension. Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride, and the like can also be added to the uptake solution and aid in conversion. Similar methods can be used for other fungal host cells (cf. USP6022725 and 6268328).

Typically, the mixture is then incubated at about 0°C for 10-30 min. Additional PEG is added to the mix to further enhance uptake of the desired gene or DNA sequence. In some embodiments, 5 to 15 volumes of the transformation mixture are added with 25% PEG4000. However, more or less volume may be suitable. 25% PEG4000 is preferably about 10 times the volume of the transformation mixture. After addition of PEG, the transformation mixture can be incubated at room temperature or on ice before adding sorbitol and CaCl2 solutions. The protoplast suspension was then further added to the molten aliquot of growth medium. When the growth medium includes growth selection conditions such as acetamide or antibiotics, it only allows the growth of transformants.

In general, cells are cultured in standard media containing physiological salts and nutrients (see Pourquie et al., BIOCHEMISTRYANDGENETICSOFCELLULOSEDEGRADATION, Academic Press, 1988, 71-86 and Ilmenetal., Appl.Environ.Microbiol., 1997,63:1298- 1306). Common commercially prepared media (such as Yeast Malt Extract (YM) broth, Luria Bertani (LB) broth and Sabouraud Dextrose (SD) broth) can also be used in the present invention.

Culture conditions are also standard (eg, incubation of cultures in a suitable medium in a shaker incubator at approximately 30° C. until the desired expression level of an alpha-amylase such as AclaP11 is achieved). Cultivation conditions for a given filamentous fungus are known in the art and can be found in the scientific literature and/or from sources of the fungus such as the American Type Culture Collection (ATCC) and the Fungal Genetics Collection (FGSC).

Analysis of enzyme expression and determination of enzyme activity:

To assess the expression of alpha amylases such as AclaP11, analysis can be performed at the protein level or at the nucleic acid level. Analytical methods that can be used include Northern blots, dot blots (for DNA or RNA analysis), Southern blots, autoradiography, RT-PCR (reverse transcriptase polymerase chain sequence) in situ hybridization. In addition, gene expression can be performed by immunological methods, such as immunohistochemical staining of cells, tissue sections, or immunoassays of tissue culture media. Evaluated eg by Western blot or ELISA. Such immunoassays can be used to qualitatively and quantitatively assess the expression of alpha amylases such as AclaP11. The details of such methods are known to those of skill in the art, and many reagents for performing such methods are available commercially. In some embodiments, the expression of an alpha amylase, such as AclaP11, is analyzed by SDS-PAGE.

The activity of α-amylases such as AclaP11 can be measured by the DNS method described in Miller, Anal. Chem., 1959, 31:426-428. Enzyme activity can also be determined by measuring the ability to liquefy soluble starch per unit time (refer to GB8275-2009 and GB/T24401-2009). In some embodiments, the activity of an alpha-amylase (such as AclaP11) is determined by using an alpha-amylase activity assay kit (Megazyme).

The α-amylase and recombinant strains of the present invention will be described below in conjunction with specific examples.

In the following Disclosure and Experimental sections, the following abbreviations apply:

AclaP11 (α-amylase having the amino acid sequence described in SEQ ID NO: 1), ° C (degrees Celsius), rpm (rotational speed per minute), dlH2O (deionized water, Milli-Q filter), kD (kilodalton), g (gram), mg (milligram), μg (microgram), cm (centimeter), μm (micrometer), L (liter), mL (milliliter), μL (microliter), M (molar concentration), mM (milliliters) molar concentration), CU (enzyme activity unit), min (minute), h (hour), d (day), Tris (trishydroxymethylaminomethane), SDS (sodium dodecyl sulfate), PAGE (polypropylene Amide gel electrophoresis), MOPS (morpholine propanesulfonic acid).

Cloning of Example 1 α-amylase gene AclaP11

Genomic DNA was extracted from overnight cultures of Aspergillus clavus CGMCC3.5289 using the Fungal Genomic DNA Extraction Kit (Omega) following the manufacturer's instructions.

Design PCR primers according to the α-amylase gene sequence of XM_001271888 on the NCBI, and the sequence of the forward primer AclaP11-F for cloning the AclaP11 gene in Aspergillus clavus is as follows:

5'-ACGGC CTTAAG AAGATGCCTCGGATTTGGTCCTC

(the sequence shown in the underline is AflII restriction site),

The reverse primer AclaP11-R sequence is:

5'-ATT- GCGGCCGCAGCTCAAGCACAGATCTTGCTTC

(The underlined sequence is the NotI restriction site).

The gene was amplified from A. clavus genomic DNA using Phusion DNA polymerase (Thermoscientific).

Amplification conditions:

Step 1: Keep at 98°C for 3 minutes.

The second step: hold at 98°C for 10s, then hold at 72°C for 75s, and repeat this step 30 times.

Step 3: Keep at 72°C for 10 minutes.

The above PCR product was purified using a gel purification kit, and the purified PCR product was digested with restriction enzymes AflII and NotI (Fermentas); meanwhile, the plasmid pGm (genetics) was digested with restriction enzymes AflII and NotI The map is shown in Figure 1) for enzyme digestion. The digested product was purified using a gel purification kit, and the above two digested products were ligated with T4 DNA ligase (Fermentas). The ligation product was transformed into Trans5α E. coli (Transgen) and selected with ampicillin. To ensure accuracy, several clones were sequenced (Invitrogen).

Plasmids were purified from correctly sequenced E. coli clones using a plasmid midiprep kit (Axygen) following the manufacturer's instructions. The resulting plasmid was named pGm-AclaP11, and the sequencing results showed that the nucleotide sequence encoding the α-amylase amplified by PCR of the present invention was SEQ ID NO: 2, and its translated amino acid (protein) sequence was SEQ ID NO: 1.

Embodiment 2: PEG-mediated protoplast fusion transforms Aspergillus niger

Take the Aspergillus niger G1 spore suspension in the center of the CMA plate (9cm petri dish), wait for the colony to cover the whole petri dish, cut 1/4 size of the culture based on 200mL CMA liquid medium, culture at 30°C, 200rpm for 14-16h .

Collect the mycelium with a sterile Miracloth filter cloth, and wash it once with solution A, transfer the washed mycelium to 40mL protoplastization solution under aseptic conditions, and incubate at 30°C and 200rpm for 1-2h, The progress of protoplastization was detected by microscopic observation.

Filter the above-mentioned warm bath liquid with a sterile Miracloth filter cloth, and the obtained filtrate is the protoplast solution. The protoplast solution was divided into two 50mL sterile disposable centrifuge tubes, and the volume of each tube was adjusted to 45mL with solution B, centrifuged at 4000rpm for 8min to obtain the precipitate and discard the supernatant. Wash the pellet two more times with 20 mL of Solution B. Resuspend the pellet in 10 mL of Solution B and count the protoplasts with a hemocytometer. The protoplasts were centrifuged again and the supernatant was discarded. According to the counting results of the hemocytometer, an appropriate amount of solution B was added to resuspend the pellet, so that the number of protoplasts was 1×10 ⁷ /mL.

On ice, add 100 μL of the above protoplast solution into pre-chilled sterile 15 mL centrifuge tubes, 1 tube for each transformation reaction. Add 10 μg of plasmid. Add 12.5 μL solution C, mix gently and then place on ice for 20 min.

The MMSA top agar tube was melted and kept at 55°C. Remove the above-mentioned 15mL centrifuge tube from ice, add 1mL solution C and 2mL solution B to the tube, and mix each tube gently, and the resulting mixture is the protoplast mixture. 1 mL of the above protoplast mixture was added to each of the 3 top agar test tubes, and immediately poured onto the MMSA plate, and the plate was incubated at 30°C for 7-10 days.

Solution A: 2.5mL of 1MK ₂ HPO ₄ , 2.5mL of 1MKH ₂ PO ₄ , 48.156g of MgSO ₄ , add dlH ₂ O to a final volume of 500mL, and filter to sterilize with a 0.22μm microporous membrane.

Solution B: 5 mL of 1 MTris (pH 7.5), 2.77 g of CaCl ₂ , 109.32 g of sorbitol, added dlH ₂ O to a final volume of 500 mL, and sterilized by filtration through a 0.22 μm microporous membrane.

Solution C: 250g PEG4000, 2.77g CaCl ₂ , 5mL 1MTris (pH 7.5), add dlH ₂ O to a final volume of 500mL, filter and sterilize with a 0.22μm microporous membrane.

Protoplastization solution: 0.6 g of lysing enzyme (Lysing Enzyme from Trichodermaharzianum, Sigma) was dissolved in 40 mL of solution A, and sterilized by filtration with a 0.22 μm microporous membrane.

MMSA plates: 0.59g acetamide (Sigma), 3.4g CsCl (Sigma), 0.52g KCl, 1.52g _KH2PO4 , _218.5g sorbitol, 1mL trace elements (see below), 20g agar, add _dlH2O to final volume 972.5mL, add 25mL of 40% glucose and 2.5mL of 20% MgSO ₄ sterilized by filtration through a 0.22μm microporous membrane after autoclaving.

MMSA top agar tube: 0.59 g acetamide (Sigma), 3.4 g CsCl (Sigma), 0.52 g KCl, 1.52 g KH ₂ PO ₄ , 218.5 g sorbitol, 1 ml trace elements (see below), 10 g low melting point agarose, add dlH ₂ O to a final volume of 972.5mL. After high-pressure steam sterilization, when the culture medium is not solidified, add 25mL of 40% glucose and 2.5mL of 20% MgSO ₄ sterilized by filtration with a 0.22μm microporous membrane, and then immediately dispense in Bacteria test tubes, 10mL per tube.

Trace elements: add _{1gFeSO4 · 7H2O} , _{8.8gZnSO4 · 7H2O} , _{0.4gCuSO4 · 5H2O} , _{0.15gMnSO4 ·} 4H2O, _0.1gNa2B4O7 ·10H in 250mLdlH2O ₂ O, 50 mg (NH ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O, 0.2 mL of concentrated HCl, after complete dissolution, dilute to 1 L with dlH ₂ O, and filter to sterilize with a 0.22 μm microporous membrane.

CMA plate: 20g glucose, 20g malt extract, 1g peptone, 15g agar, add dlH ₂ O to a final volume of 1000mL, autoclaved.

CMA liquid medium: 20g glucose, 20g malt extract, 1g peptone, add dlH ₂ O to a final volume of 1000mL, and sterilize by autoclaving.

After colonies grew on the plate, 25 colonies were picked, and genomic DNA was extracted from the overnight culture using the Fungal Genomic DNA Extraction Kit (Omega) according to the manufacturer's instructions. Perform PCR verification according to the experimental procedure described in Example 1, and sequence the obtained PCR products. Inoculate the spore suspension of the obtained positive clone and Aspergillus niger G1 (control group) into 25mL of TSB fermentation medium, culture at 30°C and 200rpm for 5 days, filter the obtained fermentation broth with 8 layers of gauze, and filter the filtrate at 14000×g Centrifuge 10min under condition, collect supernatant; Supernatant is carried out electrophoresis detection on the SDS-PAGE gel that concentration is 8%; To 55kD place (theoretical molecular weight of α-amylase of the present invention is 55kD) have obvious For the positive clones of the protein band, the amylase activity in the supernatant was measured by the α-amylase activity assay kit (Megazyme), and the positive clone with the highest enzyme activity was selected and named Aspergillus niger 1-B (Aspergillus niger1-B), Its enzyme activity is about 603.2CU/mL, and as a control group, the enzyme activity of the host cell Aspergillus niger G1 fermentation supernatant is only 46CU/mL, indicating that the engineering strain Aspergillus niger 1-B constructed by the present invention can efficiently express heterologously Alpha-amylase from Aspergillus clavus.

Embodiment 3: the ultraviolet mutagenesis and screening of starting bacterial strain Aspergillus niger 1-B

Aspergillus niger 1-B was used as the starting strain for ultraviolet mutagenesis and screening.

Inoculate the starting strain Aspergillus niger 1-B on CMA solid medium, culture at 30°C for 5 days until black spores grow, wash the spores with sterile water, dilute to the spore concentration level of 10 ⁶ , and put the spore suspension under the ultraviolet lamp Under the irradiation for 4 minutes, the irradiation distance is 22 cm, and the power of the ultraviolet lamp is 30w. The resulting spore suspension was diluted 100 times, coated on a CMA solid medium plate, and after 48 to 72 hours, a small and dense colony with a small colony radius was selected, streaked and inoculated on a CMA solid medium plate, and after it grew black spores, use After washing with sterile water, inoculate in TSB fermentation medium, measure enzyme activity after 5 days of fermentation, and select strain candidates with high enzyme activity. This is the first round of ultraviolet mutagenesis.

Using the same operation as above, the mutant strain obtained by the first round of ultraviolet mutagenesis was subjected to the second round of ultraviolet mutagenesis, and the hyphal form and colony form of a mutant strain in the obtained bacterial strain were significantly changed (as shown in Figure 2 and Figure 3).

The spore suspensions of the starting strain Aspergillus niger 1-B and the mutant strain were respectively inoculated in 25 mL of TSB fermentation medium, and cultured at 30° C. and 200 rpm for 5 days. The obtained fermentation broth was filtered with 8 layers of gauze, the filtrate was centrifuged at 14000×g for 10 min, and the supernatant was collected. According to the manufacturer's instructions, the enzyme activity of amylase in the supernatant was measured with α-amylase activity assay kit (Megazyme) (due to the long fermentation period and shake flask fermentation, environmental factors cannot be regulated in real time during the fermentation process, and the enzyme There will be differences between live data batches; but if it is the same batch of fermentation samples, the comparative relationship between the data between different samples has been tested and is credible and repeatable). The results showed that at pH 5.4 and 40°C, the enzyme activity of the fermentation supernatant of the starting strain was only 603.2CU/mL, while the enzyme activity of the mutant strain was as high as 1188.2CU/mL, which was 1.97 times that of the starting strain. The applicant named the mutant strain Aspergillus niger 1-B5 (Aspergillus niger1-B5), and it has been preserved in the General Microorganism Center (CGMCC) of China General Microorganism Culture Collection Management Committee (CGMCC) on November 7, 2013, and the strain number is CGMCCNo. 8443.

Embodiment 4: Analysis of the enzymatic properties of α-amylase

Be respectively 2.4,3.0,3.6,4.2,4.8,5.4,6.0,6.6,7.2,7.8 with the disodium hydrogen phosphate-citric acid buffer solution described in embodiment 3 starting bacterial strain Aspergillus niger 1-B and mutant strain The fermentation supernatant of 1-B5 was diluted to a suitable concentration, and the enzyme activity of the supernatant was measured with the α-amylase activity assay kit (Megazyme) at 40 °C according to the manufacturer's instructions; 100% based on the highest enzyme activity , calculate the residual activity, and make a pH-residual activity curve. The results are shown in Fig. 4, the optimal pH of the α-amylase AclaP11 expressed by the mutant strain Aspergillus niger 1-B5 is 6.0, which is consistent with the starting strain.

The fermentation supernatant of the starting strain Aspergillus niger 1-B and the mutant strain 1-B5 described in Example 3 was diluted to an appropriate concentration with disodium hydrogen phosphate-citric acid buffer solution of pH 6.0, and was determined by α-amylase activity The kit (Megazyme) measured the enzyme activity of the supernatant at 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C, and calculated the residual activity based on the highest enzyme activity as 100%. - Residual activity curve. The results are shown in Figure 5, the optimum action temperature of the α-amylase AclaP11 expressed by the mutant strain Aspergillus niger 1-B5 is 50°C, which is consistent with the starting strain.

The above results show that, compared with the starting strain Aspergillus niger 1-B, the enzymatic properties of the α-amylase recombinantly expressed by the mutant strain Aspergillus niger 1-B5 obtained in the present invention have not changed.

Example 5: Application example of α-amylase AclaP11

Bread is made using a single-fermentation process. The bread flour is selected from Pengtai's bread base flour (without any additives); Angel brand bread yeast; Galen brand shortening. By studying the relationship between the change of bread hardness and the amount of α-amylase added, it was found that α-amylase AclaP11 has a certain anti-aging effect. The research found that when adding 0.1-0.5ml/100g flour, α-amylase AclaP11 can significantly increase the specific volume of bread, reduce the hardness of bread crust and bread core, improve bread texture, and improve the baking quality of bread. α-amylase can improve the volume of bread, improve the taste of bread, make bread and other products larger and softer, which is of great significance to improve the quality of bread.

Claims (1)

1. an Aspergillus strain, described Aspergillus strain is aspergillus niger, and its deposit number is CGMCCNo.8443.