CN103642778B - A kind of β-endo-glucanase enzyme mutant and application thereof - Google Patents
A kind of β-endo-glucanase enzyme mutant and application thereof Download PDFInfo
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- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
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Abstract
The invention provides a kind of β-endo-glucanase enzyme mutant, the 56th amino acids of the β-endoglucanase of SEQ ID NO:1 that to be aminoacid sequence be becomes Ile from Val, and the 212nd amino acids becomes Asn from Asp.The optimum temperature of described mutant is 65 DEG C, and the enzyme of more than 80% can be kept within the scope of 60-70 DEG C to live; Described mutant processes 5min under 75 DEG C of conditions, the enzyme of more than 40% still can be kept to live, process 2min under 80 DEG C of conditions, and the enzyme of more than 50% can be kept to live, and compared with wild-type, the thermotolerance of mutant is significantly improved.The high efficiency recombinant expressed mutant protein of Trichodermareesei engineering bacteria energy of the present invention, the work of 20L ferment tank enzyme, up to 7120U/mL, improves 50.5% than wild-type.
Description
Technical field
The invention belongs to functional gene renovation technique field, specifically a kind of β-endo-glucanase enzyme mutant and application thereof.
Background technology
Under the background of our times crisis in food and energy dilemma, the new energy technologies such as cellulosic ethanol are more paid attention to.As one of the abundantest renewable resources, the Mierocrystalline cellulose produced by photosynthesis every year can reach 100,000,000,000 tons.Development cellulose degradation technology, is converted into the focus that the energy or industrial raw material are global concerns by agriculture and forestry organic waste material.For the production of cellulosic ethanol, the saccharification of lignocellulose is a step of most critical, accounted for the over half of whole cellulosic ethanol production cost, and the saccharification of lignocellulose depends on cellulose degrading enzyme.Current, reduce and use enzyme cost, raising Reducing sugar and cellulose conversion rate become the major objective of research.
β-endoglucanase is an important composition composition in cellulase system, it acts on the noncrystalline domain of cellulosic molecule inside, random hydrolysis β-Isosorbide-5-Nitrae-glycosidic link, by the brachymemma of long chain cellulose molecule, produce in a large number with the small molecules Mierocrystalline cellulose of non-reducing end.And in lignocellulose saccharification process, substrate generally will through High Temperature Pre process, and the reaction that cellulase participates in is all generally under higher temperature condition, so it is significant for the application that moves towards the industrialization of the saccharification of lignocellulose to find resistant to elevated temperatures β-endoglucanase.
Orthogenesis is a kind of albumen remodeling method conventional in protein engineering, can transform target protein under to protein structure and the unclear situation of function information, high-throughput screening method is used to find out the mutant of improved performance, carry out sudden change and the screening of many wheels, the performance of protein can be improved further.This Reconstruc-tion policy of orthogenesis shows huge advantage for the thermotolerance improving protein.
Summary of the invention
The object of this invention is to provide a kind of β-endo-glucanase enzyme mutant and application thereof.The present invention is by carrying out protein engineering transformation to the β-endoglucanase deriving from Penicillium decumbens (Penicillium decumbens), obtain mutant protein, compared with wild-type, the thermotolerance of mutant is significantly improved, thus is conducive to realizing its widespread use in saccharification of cellulose hydrolysis.
Applicant utilizes directed evolution technologies to carry out molecular modification to Penicillium decumbens β-endoglucanase, through a large amount of screenings, finds that these two mutational sites of V56I and D212N can cause the change of β-endoglucanase thermotolerance, thus facilitates the present invention.
One aspect of the present invention provides a kind of β-endo-glucanase enzyme mutant, and the 56th amino acids of the β-endoglucanase of SEQ IDNO:1 that to be aminoacid sequence be becomes Ile from Val, and the 212nd amino acids becomes Asn from Asp.
The aminoacid sequence of above-mentioned β-endo-glucanase enzyme mutant is SEQ ID NO:3, and the nucleotide sequence of its a kind of encoding gene is SEQ ID NO:4.
The present invention provides the expression vector carrying β-endo-glucanase enzyme mutant gene that encoding sequence is SEQ ID NO:4 on the other hand.
The present invention also provides Trichodermareesei (Trichoderma reesei) engineering strain carrying above-mentioned expression vector.
The present invention obtains a kind of β-endo-glucanase enzyme mutant by directed evolution technologies, and builds the Trichodermareesei engineering strain obtaining this mutant recombinant expressed.The optimum temperature of described mutant is 65 DEG C, and the enzyme of more than 80% can be kept within the scope of 60-70 DEG C to live; Described mutant processes 5min under 75 DEG C of conditions, the enzyme of more than 40% still can be kept to live, process 2min under 80 DEG C of conditions, and the enzyme of more than 50% can be kept to live, and compared with wild-type, the thermotolerance of mutant is significantly improved.The high efficiency recombinant expressed mutant protein of Trichodermareesei engineering bacteria energy of the present invention, the work of 20L ferment tank enzyme, up to 7120U/mL, improves 50.5% than wild-type.β-endo-glucanase enzyme mutant that the present invention obtains can significantly improve the conversion coefficient of lignocellulose, and hydrolysis result is significantly better than wild-type under the high temperature conditions, be more suitable for Mierocrystalline cellulose and turn sugared commercial production conditions, can significantly reduction enzyme cost, thus reduction production cost, be conducive to β-endo-glucanase enzyme mutant and turn widespread use in sugared industry at Mierocrystalline cellulose.
Accompanying drawing explanation
Fig. 1: β-endo-glucanase enzyme mutant GluM15 and wild-type Glu98 optimum temperature comparison diagram;
The thermotolerance comparison diagram of Fig. 2: β-endo-glucanase enzyme mutant GluM15 and wild-type Glu98.
Embodiment
Following examples set forth content of the present invention to illustrate better, and those skill in the art related can understand better by embodiment and grasp the present invention.But protection of the present invention and right are not limited to provided case.
The synthesis of embodiment 1 β-endo-glucanase enzyme mutant gene and amplification
In order to improve β-endoglucanase (the called after Glu98 deriving from Penicillium decumbens (Penicillium decumbens), its aminoacid sequence is SEQ ID NO:1, gene order is SEQ ID NO:2) thermotolerance, applicant uses directed evolution technologies to obtain the mutant of this more enzyme of number, then screens mutant.
PCR primer Glu98-F, Glu98-R are as follows in design:
Glu98-F1:GGC
gAATTCgAGCTGGCTCAGTTCTGTGACCAATATGG(underscore is restriction enzyme EcoRI recognition site)
Glu98-R1:ATA
gCGGCCGCcTAGTACACGCTCGCAGACCAGTGG(underscore is restriction enzyme NotI recognition site)
Use the gene order of β-endoglucanase Glu98 as template, by above-mentioned primer GeneMorph II random mutation PCR kit (Stratagene), pcr amplification is carried out to object fragment, reclaim test kit with glue and reclaim PCR primer, use EcoRI and NotI to reclaiming the good PCR primer of purifying and pET21a carrier carries out double digestion, after double digestion two kind of a fragment Ligase is connected, after order-checking, select goal gene to check order correct expression vector, be converted in e. coli bl21 (DE3), the LB coated containing Amp is dull and stereotyped, be inverted at 37 DEG C and cultivate, after son to be transformed grows, choose into 96 orifice plates one by one with toothpick, the LB+Amp substratum that 150ul contains 0.1mM IPTG is added in each hole, 37 DEG C of 220rpm cultivate about 6h, supernatant is abandoned after centrifugal, with damping fluid, thalline is resuspended, multigelation broken wall, obtain the Bacillus coli cells lysate containing β-endoglucanase.
30uL cell pyrolysis liquid correspondence is added two new 96 orifice plates, one in 70 DEG C of process 10min, another respective panels 4 DEG C refrigeration, two 96 orifice plates all add 30uL glucanase substrate, after 37 DEG C of reaction 30min, the reducing sugar generated is measured, with the amount of the reducing sugar generated to measure muton through pyroprocessing with without the enzyme activity after pyroprocessing by DNS method.The selection result shows, and the thermotolerance of most mutant considerable change does not occur, and decline has appearred in the thermotolerance of fractional mutant, and fractional mutant even loses enzyme activity, only has the mutant of only a few to show tolerance to hot conditions.After in this approach mutant being screened in a large number, screen the mutant that a strain thermotolerance is improved significantly.The goal gene of mutant is checked order, determines the amino acid sites of sudden change.Final applicant obtains mutation combination V56I and D212N that can improve β-endoglucanase thermotolerance.
Sequencing result shows, the β-endo-glucanase enzyme mutant containing V56I and D212N two point mutation that the present invention obtains, and its aminoacid sequence is SEQ ID NO:3, and coding nucleotide sequence is SEQ ID NO:4.
By β-endo-glucanase enzyme mutant gene called after GluM15, primer Glu98-F2, Glu98-R2 that design two ends introduce BamHI and XbaI enzyme cutting site are as follows:
Glu98-F2:GGC
gGATCCgAGCTGGCTCAGTTCTGTGACCAATATGG(underscore is restriction enzyme BamHI recognition site)
Glu98-R2:ATA
tCTAGAcTAGTACACGCTCGCAGACCAGTGG(underscore is restriction enzyme XbaI recognition site)
Use above-mentioned primer, carry out pcr amplification using the mutant obtained as template, obtain the β-endo-glucanase enzyme mutant gene GluM15 of two ends with BamHI and XbaI enzyme cutting site.PCR reaction conditions is: 94 DEG C of sex change 5min; 94 DEG C of sex change 30s, 56 DEG C of renaturation 30s, 72 DEG C extend 1min, 30 circulations, and 72 DEG C extend 10min.
Obtained the gene fragment of wild-type beta-endoglucanase Glu98 by above-mentioned same PCR method amplification, its coding nucleotide sequence is SEQ ID NO:2.
The structure of embodiment 2 Trichodermareesei engineering strain
The β obtained by above-mentioned clone-endo-glucanase enzyme mutant gene GluM15, is connected with trichoderma reesei expression carrier pPST by BamHI with XbaI site, builds and obtains expression vector pPST-GluM15.The expression vector pPST-Glu98 of wild-type is built according to same method.
Inoculate Trichodermareesei (Trichoderma reesei) mycelia on PDA flat board to growing spore; Cutting the length of side with vaccinating lancet is 3cm square bacterium block, is inoculated in 100mL YEG(containing 1% glucose, 0.5% yeast powder) in liquid nutrient medium, 30 DEG C, 220rpm cultivates 12-16h; Use multi-layer filter paper collecting by filtration mycelia; By the mycelia aseptic water washing collected, put into the triangular flask 30 DEG C of slow circumvolves filling 20mL lyase liquid (Sigma L1412) and jolt about 1.5-2 hour; Observe the generation situation of protoplastis, when there being a large amount of protoplastis to generate, enzymolysis solution being fallen in three layers of sterilizing lens wiping paper and filtering, collecting filtrate, the centrifugal 10min of 3000rpm; Abandon supernatant, with STC solution (20% sucrose, 50mM Tris-Cl, 50mM CaCl
2) wash twice after, add appropriate STC solution resuspended, make free protoplast concentration about 10
7individual/mL.
Get 5 μ g pPST-GluM15DNA to join in 100 μ L protoplastiss, add 25 μ L25%PEG and mix gently, ice bath 20min; Then slowly add 1mL25%PEG several times, mix gently, room temperature adds 2mLSTC solution after leaving standstill 5min, and protoplastis is added to the upper strata regeneration culture medium (0.1%MgSO containing 100 μ g/mL Totomycin
4, 1%KH
2pO4,0.6% (NH
4)
2sO
4, 1% glucose, 18.3% sorbyl alcohol, 0.7% agarose) in, cover containing 100 μ g/mL Totomycin subfoundation culture medium flat plate (2% glucose, 0.5% (NH4) after mixing gently
2sO
4, 1.5%KH
2pO
4, 0.06%MgSO
4, 0.06%CaCl
2, 2% agar), 28 DEG C of dark culturing a couple of days grow to transformant.
Extracting transformant genomic dna is template, utilizes primer HyB-F and HyB-R(HyB-F:ACAAAGATCGTTATGTTTATCGGCACT; And HyB-R:AGAAGAAGATGTTGGCGACCTCGTATT) increase hygromycin gene checking transformant.Primer Glu98-F and Glu98-R described in embodiment 1 is utilized to carry out pcr amplification goal gene checking transformant.Pcr amplification condition is 94 DEG C of sex change 5min; 94 DEG C of sex change 30s, 56 DEG C of renaturation 30s, 72 DEG C extend 1min, 30 circulations, and 72 DEG C extend 10min.PCR fragment size is observed by agarose gel electrophoresis.Through above-mentioned two PCR reaction checking positive transformant.To a wherein strain positive transformant called after Trichodermareesei GluM15(Trichoderma reesei GluM15).
Above-mentioned same method for transformation is adopted to be transformed in Trichodermareesei by the expression vector pPST-Glu98 of wild-type, build the Trichodermareesei engineering bacteria obtaining recombinant expressed wild-type beta-endoglucanase Glu98, called after Trichodermareesei Glu98(Trichoderma reesei Glu98).
Embodiment 3 transformant fermentation checking
Above-mentioned two strain engineering bacteria Trichodermareesei GluM15 and Trichodermareesei Glu98 are inoculated in MM fermention medium (1.5% glucose, 1.7% lactose, 2.5% corn steep liquor, 0.44% (NH respectively
4)
2sO
4, 0.09%MgSO
4, 2%KH
2pO
4, 0.04%CaCl
2, 0.018% tween-80,0.018% trace element, 0.018% polypropylene glycol-2000), cultivate 48 hours for 28 DEG C, then cultivate 48 hours for 25 DEG C, get fermented liquid supernatant respectively, measure its enzyme and live.Result shows, and the fermenting enzyme of wild-type beta-endoglucanase Glu98 is lived as 670U/mL, and the fermenting enzyme work of mutant GluM15 is up to 1032U/mL, improves 54% than wild-type.
The thermotolerance experiment of the recombinant expressed β-endoglucanase of embodiment 4
Respectively at 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C, under pH5.5 condition, β-endoglucanase enzyme activity determination is carried out to fermented supernatant fluid described in embodiment 3, live as 100% with the highest enzyme, calculate relative enzyme to live, do temperature-enzyme curve alive relatively.As shown in Figure 1, the optimum temperature of wild-type beta-endoglucanase Glu98 is 45 DEG C to result; And the optimum temperature of mutant GluM15 is 65 DEG C, and the enzyme of more than 80% can be kept within the scope of 60-70 DEG C to live.
The acetic acid-sodium acetate buffer solution of fermented supernatant fluid pH5.5 described in embodiment 3 is diluted to about 20U/mL, process 2min and 5min respectively under 75 DEG C and 80 DEG C of conditions after, measures remnant enzyme activity, live as 100% with the enzyme of untreated samples, calculate relative enzyme and live.As shown in Figure 2, wild-type beta-endoglucanase Glu98 processes 2min to result under 75 DEG C of conditions, and enzyme is lived only surplus less than 20%, lives be almost 0 to enzyme when 80 DEG C; And mutant GluM15 processes 2min and 5min respectively under 75 DEG C of conditions, the enzyme of 70% and 50% still can be kept to live, under 80 DEG C of conditions, process 2min, the enzyme of about 40% still can be kept to live.
In sum, compared with wild-type, the thermotolerance of β-endo-glucanase enzyme mutant that the present invention obtains is significantly improved, and is conducive to it and turns widespread use in sugar at Mierocrystalline cellulose.
Embodiment 5 fermentation checking is prepared with β-endo-glucanase enzyme product
Above-mentioned two strain engineering bacteria Trichodermareesei GluM15 and Trichodermareesei Glu98 are inoculated in shake-flask seed substratum (glucose 10-30g/L respectively, potato 100-200g/L), temperature 28-30 DEG C, rotating speed 200rpm, incubation time 23-25h, (substratum is: glucose 20-50g/L fermented liquid to be accessed secondary seed medium, ammonium sulfate 10-30g/L, magnesium sulfate 5-10g/L, potassium primary phosphate 15-30g/L), 30 DEG C, rotating speed 200rpm, after cultivating 48h, (fermention medium is: glucose 30-50g/L seed liquor to be proceeded to 20L fermentor tank, lactose 2.0-10g/L, corn steep liquor 20-50g/L, ammonium sulfate 10-30g/L, magnesium sulfate 5-10g/L, potassium primary phosphate 15-30g/L), liquid amount is 12.5L, inoculum size is 500mL, rotating speed 300rpm, ventilation is 0.5m3/h, leavening temperature is 30 DEG C, pH value 3.50.When glucose concn is lower than 5g/L, dissolved oxygen starts to add lactose after rising.Fermentation time is about 150h.After fermentation ends, filtered the filtrate obtaining clarification by flame filter press, be β-endoglucanase liquid product.
Carry out enzyme to above-mentioned β-endoglucanase liquid product respectively to live and determining the protein quantity, result shows, under 20L ferment tank condition, the fermenting enzyme of wild-type beta-endoglucanase body is lived as 4730U/mL, and the fermenting enzyme work of mutant is up to 7120U/mL, lives than wild-type enzyme and improve 50.5%.
The application of embodiment 6 β-endoglucanase in lignocellulose saccharification
The invention provides a kind of method of lignocellulose saccharification, its step comprises:
1) taking the xylose residue (lignocellulose-containing 60-70%) of a certain amount of over dry, is 1:10(m/v according to solid-to-liquid ratio) add the citric acid-sodium citrate damping fluid that pH is 4.5 ~ 5.5, mixing;
2) in step 1), β described in the embodiment of the present invention 5-endoglucanase liquid product is added according to the ratio of 5-10mg albumen/g substrate (described substrate is the xylose residue of above-mentioned over dry);
3) after shaking enzymolysis 24-48h at 50 DEG C ~ 60 DEG C, sampling bio-sensing analysis-e/or determining glucose content;
4) conversion coefficient of lignocellulose is calculated: the glucose content (mg/g substrate) × 100% that content (mg/g substrate) ÷ of the glucose obtained after conversion coefficient (%)=enzymolysis is total.
Result shows: when wild-type beta described in the embodiment of the present invention 5-endoglucanase addition is respectively 5 and 9mg albumen/g substrate, the glucose yield of substrate is respectively 420 and 490mg/g substrate, and conversion coefficient is respectively 62% and 72%; When saltant type β-endoglucanase addition is respectively 5 and 9mg albumen/g substrate, the glucose yield of substrate is respectively 560 and 645mg/g substrate, and conversion coefficient, up to 82% and 95%, improves 20% and 23% respectively than the conversion coefficient of wild-type.
The above results shows, β-endo-glucanase enzyme mutant that the present invention obtains can significantly improve the conversion coefficient of lignocellulose, and hydrolysis result is significantly better than wild-type under the high temperature conditions, be more suitable for Mierocrystalline cellulose and turn sugared industrial process conditions, can significantly reduction enzyme cost, thus reduce production cost.
Claims (5)
1. β-endo-glucanase enzyme mutant, is characterized in that, the aminoacid sequence of described β-endo-glucanase enzyme mutant is SEQ ID NO:3.
2. a gene, is characterized in that, described gene is for β according to claim 1-endo-glucanase enzyme mutant of encoding.
3. gene as claimed in claim 2, it is characterized in that, the nucleotides sequence of described gene is classified as SEQID NO:4.
4. an expression vector, is characterized in that, described expression vector carries gene according to claim 3.
5. a Trichodermareesei, is characterized in that, described Trichodermareesei carries expression vector according to claim 4.
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| CN104450651B (en) * | 2014-12-09 | 2017-08-15 | 青岛蔚蓝生物集团有限公司 | A kind of β glucosides enzyme mutant and its application |
| CN105154415A (en) * | 2015-10-19 | 2015-12-16 | 中国农业科学院饲料研究所 | Mutant endoglucanase with improved pH stability and heat stability as well as coding gene and application thereof |
| CN110004130B (en) * | 2019-04-09 | 2021-03-30 | 江南大学 | Genetically engineered bacterium for improving thermal gel hydrolysis efficiency and application thereof |
| US11987824B2 (en) * | 2020-12-22 | 2024-05-21 | Fornia Biosolutions, Inc. | Additional endoglucanase variants and methods |
| CN114015677A (en) * | 2021-11-26 | 2022-02-08 | 中农华威生物制药(湖北)有限公司 | Cellulase for promoting release of traditional Chinese medicine feed additive in intestinal tract and production method thereof |
| CN114317495B (en) * | 2022-01-10 | 2024-07-30 | 鑫缘茧丝绸集团股份有限公司 | Glucanase mutant with improved thermal stability and application thereof |
| CN117511918B (en) * | 2023-11-16 | 2025-01-24 | 山东弥美生物科技股份有限公司 | A beta-1,3-glucanase mutant and its application |
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