CN116970591A - Thermophilic endo-cellulase mutant and preparation method thereof - Google Patents
Thermophilic endo-cellulase mutant and preparation method thereof Download PDFInfo
- Publication number
- CN116970591A CN116970591A CN202311206014.4A CN202311206014A CN116970591A CN 116970591 A CN116970591 A CN 116970591A CN 202311206014 A CN202311206014 A CN 202311206014A CN 116970591 A CN116970591 A CN 116970591A
- Authority
- CN
- China
- Prior art keywords
- endo
- cellulase
- mutant
- thermophilic
- enzyme
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108010084185 Cellulases Proteins 0.000 title claims abstract description 51
- 102000005575 Cellulases Human genes 0.000 title claims abstract description 51
- 238000002360 preparation method Methods 0.000 title abstract description 4
- 230000035772 mutation Effects 0.000 claims abstract description 16
- 241000499912 Trichoderma reesei Species 0.000 claims abstract description 6
- 150000001413 amino acids Chemical class 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 11
- 108020004707 nucleic acids Proteins 0.000 claims description 8
- 102000039446 nucleic acids Human genes 0.000 claims description 8
- 150000007523 nucleic acids Chemical class 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 7
- 239000013604 expression vector Substances 0.000 claims description 7
- 241000223259 Trichoderma Species 0.000 claims description 4
- 101150043930 cel5A gene Proteins 0.000 claims description 3
- 108091026890 Coding region Proteins 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 230000009465 prokaryotic expression Effects 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 abstract description 25
- 108090000790 Enzymes Proteins 0.000 abstract description 25
- 230000000694 effects Effects 0.000 abstract description 24
- 229940088598 enzyme Drugs 0.000 abstract description 17
- 230000009466 transformation Effects 0.000 abstract description 8
- 108091036078 conserved sequence Proteins 0.000 abstract description 4
- 238000000556 factor analysis Methods 0.000 abstract description 3
- 238000010801 machine learning Methods 0.000 abstract description 3
- 244000005700 microbiome Species 0.000 abstract description 2
- 102000003712 Complement factor B Human genes 0.000 description 9
- 108090000056 Complement factor B Proteins 0.000 description 9
- 238000000855 fermentation Methods 0.000 description 8
- 230000004151 fermentation Effects 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 7
- 239000002994 raw material Substances 0.000 description 6
- 239000002028 Biomass Substances 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108010059892 Cellulase Proteins 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 125000003275 alpha amino acid group Chemical group 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 229940106157 cellulase Drugs 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 235000000346 sugar Nutrition 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 description 2
- 229920001453 Arcel Polymers 0.000 description 2
- 229920002498 Beta-glucan Polymers 0.000 description 2
- 239000002154 agricultural waste Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000433 anti-nutritional effect Effects 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 235000010980 cellulose Nutrition 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 229940079919 digestives enzyme preparation Drugs 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 229920005610 lignin Polymers 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000010902 straw Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 101100016206 Cellvibrio japonicus (strain Ueda107) celC gene Proteins 0.000 description 1
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 229920002488 Hemicellulose Polymers 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 101100172078 Phanerodontia chrysosporium Eg5A gene Proteins 0.000 description 1
- 241000282849 Ruminantia Species 0.000 description 1
- 101100284150 Salipaludibacillus agaradhaerens cel5A gene Proteins 0.000 description 1
- 238000002441 X-ray diffraction Methods 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000013528 artificial neural network Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000013527 convolutional neural network Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000013135 deep learning Methods 0.000 description 1
- 238000013136 deep learning model Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 108010091371 endoglucanase 1 Proteins 0.000 description 1
- 108010091384 endoglucanase 2 Proteins 0.000 description 1
- 108010092450 endoglucanase Z Proteins 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000004293 potassium hydrogen sulphite Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 1
- 238000000547 structure data Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Classifications
-
- 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)
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/885—Trichoderma
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention belongs to the field of biotechnology and microorganisms, and particularly relates to a thermophilic endo-cellulase mutant and a preparation method thereof. According to the invention, through a new strategy of enzyme protein rational transformation, and by means of a cross strategy of machine learning and novel B factor analysis, potential mutation sites in a highly conserved sequence of thermophilic endo-cellulase are discovered in the thermophilic endo-cellulase, so that the endo-cellulase activity reaches 5800IU/Ml, and is improved by 85.2% compared with enzyme protein before transformation. According to the invention, the thermophilic endo-cellulase mutant is obtained through transformation, and the trichoderma reesei is prepared, so that the enzyme activity and the thermal stability are greatly improved, and the application value is high.
Description
Technical Field
The invention belongs to the field of biotechnology and microorganisms, and particularly relates to a thermophilic endo-cellulase mutant and a preparation method thereof.
Background
Lignocellulose is a very valuable and abundant natural resource on earth, which opens up many possibilities for the production of various value-added products as a renewable raw material. However, the structure of the biomass is intricate and complex, and the biomass consists of three major types of polymers, namely cellulose (35% -50%), hemicellulose (20% -30%) and lignin (20% -30%). The main technical bottleneck for realizing the resource utilization of the straw is how to degrade sugar in agricultural wastes with high efficiency and low cost. The development of the efficient low-cost straw sugar platform is a key for promoting the resource utilization of agricultural wastes, and is a necessary way for promoting the industrialization of biomass resources. However, the major hurdles of the biomass conversion commercialization process are the difficulty in effectively degrading the complex structure of lignocellulose and the high cost of cellulase production, so that the maximum value of the whole lignocellulose biorefinery is realized in order to fully exert the optimal potential of each lignin component, and therefore, the development of efficient and low-cost enzyme preparations becomes a research hotspot for various nationists.
According to Trichoderma reeseiT. reesei) Endo-cellulases (EG, EC 3.2.1.4) play a key role in the cleavage of internal beta-1, 4-glucosidic bonds during cellulose degradation, and play an important role in the degradation of lignocellulose-based degradation barriers by producing a large amount of glucose after catalytic attack on biopolymer chains in the amorphous region.
The endo-cellulase has wide application in various industries such as biofuel production, brewing, papermaking, feed production and the like, for example, anti-nutritional factors such as beta-glucan and the like in coarse feed interfere with digestion, absorption and utilization of other nutrients of the whole daily ration, prevent the action of endogenous digestive enzymes of ruminants and intracellular nutrients, reduce the nutritional value of traditional agricultural feed, and have low utilization rate of raw materials of the traditional agricultural feed, so that a large amount of raw materials of the traditional feed are wasted. The enzymolysis of cellulase is urgently needed in the pre-digestion of the raw material of the coarse fodder, and the anti-nutritional factors such as beta-glucan and the like are destroyed, which is helpful for the range expansion of the raw material of the fodder and the improvement of the digestion utilization rate of the raw material of the fodder.
The complex environment in large-scale industrialized applications, the greatly fluctuating temperature and pH ranges, and the tolerance to various harsh conditions in the treatment of cellulosic biomass, dictate that industrial cellulases require extremely strong environmental robustness. Enzyme activity and thermal stability of industrial enzyme preparations are key factors for evaluating the environmental robustness and excellent hydrolytic performance of enzymes, so that development of more efficient and robust cellulases has been a target of development. Directed evolution has been the primary strategy for improving cellulases; B-FIT is a popular method of predicting mutation sites, which can determine the flexibility of atoms, side chains and even the whole region. But this method relies on structure-related information to predict stability changes and cannot be applied in the absence of tertiary structure information. Particularly for highly conserved sequence active sites in thermophilic endocellulases, it is often difficult to rationally engineer, thereby limiting further improvement of industrial properties of thermophilic enzymes.
Disclosure of Invention
The invention develops a new strategy for enzyme protein rational transformation, and tests and applies the new strategy in thermophilic endo-cellulase by means of a cross strategy of machine learning and novel B factor analysis, so that potential mutation sites in a highly conserved sequence of thermophilic endo-cellulase are efficiently explored, and the technical bottleneck that enzyme activity and thermal stability are difficult to be improved is broken through.
The invention provides an endo-cellulase mutant, which is characterized in that the 299 th amino acid of the wild-type endo-cellulase cel5A is obtained by mutation from Y to C.
Further provided are nucleic acids encoding the endo-cellulase mutants. Preferably, it is obtained by mutation of TAC to TGC in the coding sequence of amino acid 299 of the wild-type endo-cellulase cel5A gene.
The invention also provides an expression vector containing the coding nucleic acid. Preferably, it is a prokaryotic expression vector.
The invention further provides recombinant host bacteria containing the coding nucleic acid or the expression vector.
In particular, it is trichoderma reeseiTrichoderma reesei) Preferably, it is Trichoderma reeseiTrichoderma reesei) A2H. Which can produce the endo-cellulase mutant with high yield. Wherein Trichoderma reesei A2H is described in Chinese patent ZL2023102126130And is disclosed (the application date is 2023, 03 and 07, the authorized bulletin date is 2023, 09 and 09, and the authorized bulletin number is CN 116083405B).
The present invention also provides a method for preparing an endo-cellulase mutant comprising the steps of culturing said recombinant host bacterium to produce said endo-cellulase mutant and collecting said endo-cellulase mutant.
Optionally, the method further comprises the step of purifying the endo-cellulase mutants.
The invention discloses a new strategy for enzyme protein rational transformation, which is used for testing and applying in thermophilic endo-cellulase by means of a cross strategy of machine learning and novel B factor analysis, so that potential mutation sites in a highly conserved sequence of thermophilic endo-cellulase are efficiently explored, the activity of endo-cellulase reaches 5800IU/Ml, and the activity of the endo-cellulase is improved by 85.2% compared with that of enzyme protein before transformation. And, after incubation at 70 ℃ for 30 min and 80 ℃ for 10 min, the modified endo-cellulase retains 93.83% and 91.48% of its initial activity and has extremely strong thermal stability. The transformation strategy successfully obtains the endo-cellulase mutant, so as to obtain the Trichoderma reesei strain with high yield of thermophilic endo-cellulase, further improve the industrial properties of enzyme protein, and break through the technical bottleneck that the activity and the thermal stability of enzyme are difficult to be improved.
Drawings
FIG. 1 is a graph showing the results of factor B calculation of amino acids in the range of 4 angstroms in an Arcel5A catalytic pore.
FIG. 2 is a graph showing the results of sorting transformants by fluorescence intensity by flow cytometry.
FIG. 3 is a graph showing the secretion amount of extracellular enzyme protein at different times of fermentation.
FIG. 4 is a diagram of an engineered strainT.reeseiEnzyme activity and initial strain after fermentation of A2H-Y299CT.reeseiA2H comparison result graph.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
Strains, media and culture conditions:
the cellulase fermentation medium contains 3.3% microcrystalline cellulose, 1.7% corn steep liquor, 0.5% (NH) 4 ) 2 SO 4 KH 0.6% 2 PO 4 0.1% MgSO 4 -7H 2 O, 0.25% CaCO 3 And 0.2% Tween-80. The initial pH of the fermentation medium was adjusted to 5.0 with 10M KOH. The spore suspension was then inoculated into 50 ml liquid medium in a 250 ml conical flask and incubated at 28℃for 5 days at 180 rpm.
Prediction of B factor values:
based on the first order sequence information, a deep learning method is adopted to select mutation sites. Factor B values are derived from X-ray diffraction crystal structure data of the protein, which reflect the degree of dispersion of the atomic electron shells. The factor B value of an amino acid reflects the flexibility of the amino acid at the current position. The B factor is higher for the flexible position than for the rigid position on average. Therefore, we use a deep learning model of neural network (CNN) and cyclic units (GRU) to predict the value of factor B. The model learns potential associations between amino acid sequences and factor B from a protein database, and orders predicted factor B values to select a range of mutation sites.
The enzyme activity detection method comprises the following steps:
the activity of the endocellulase was determined using carboxymethyl cellulose at a concentration of 0.8% (w/v). The reaction was carried out in 0.1M acetic acid-sodium acetate buffer (pH 5.5) at 37 ℃ for 30 minutes. The total amount of reducing sugars in the supernatant was determined by DNS method.
Embodiment one: endo-cellulase enzyme activity rational modification site excavation
Acidophilic fungiAcidomycespRichmondensisThe endo-cellulase cel5A gene (NCBI accession number: KXL 43823.1) belongs to glycoside hydrolase family 5 and is modeled using Swiss Model (https:// swissmodel. Expasy. Org /) and the PDB database (https:// www.rcsb.org /), the PDB template selected for modeling was 3QR3. Determination by analysis of the structure of ArCel5A27 amino acid residues within a radius of 4 angstroms from the catalytic site in the tunnel, including 27 amino acid residues such as K91, Y92, G93, N96, I97, A139, M178, I180, D183, I183, H184, T222, E228, K257, L259, I293, DD295, H297, Y299, M334, L335, T336, G366, W367 and T368 are critical to stability. It is speculated that substitution of higher factor B residues with lower factor B may decrease flexibility, increase rigidity, and thus increase the thermal stability of the protein. The predicted B factor values of the above amino acids were compared. In addition to Y299, the factor B value of the above conserved amino acid positions is relatively low. As shown in FIG. 1, the B factor value of Y299 was highest (15.63), indicating that it is the most flexible and thermally unstable site. Therefore, we considered that Y299 was a possible mutation site, and increasing the rigidity thereof could result in improvement of the thermal stability of ArCel 5A.
Embodiment two: high throughput screening of high enzyme activity transformants
Using error-prone PCR technology, using other amino acid residues to replace specific sites of Arcel5A, performing saturation mutation, amplifying the mutated PCR fragment, 22 amino acid modified FMDV 2A polypeptide sequence (VKQTLNFDLLKLAGDVESNPGP) and GFP fluorescent protein gene, cloning into pAN52-Ptef1-TtrPC-bar by using NEB Gibson assembly kit, and constructing plasmid Ptef1-Arcel5A-2A-GFP-TtrPC transformed Trichoderma reesei strainT.reeseiA2H. Since the transformants had GFP co-expression within the cells, the transformants were high-throughput screened by flow cytometry with fluorescence intensity (FIG. 2), and the primary screened transformants were high-throughput cultured and detected in 96-well plates for a second round of rescreening.
These transformants overexpressing ArCel5A showed different levels of GFP fluorescence signal. The initially selected transformants had high levels of GFP fluorescence signal. Subsequently, the independent transformants were cultured in 96-well plates and screened by endoglucanase activity. And after the secondary screening by enzyme activity analysis, the endo-cellulase activity of the positive transformant is 83.51 percent higher than that of the wild strain. By gene sequencing of positive transformant, it was found that the gene sequence of the 299 th position of ArCel5A of the transformant was mutated from TAC to TGC, the corresponding amino acid was mutated from Y to C, and the protein was secretedThe yields did not change much, indicating that ArCel5A-Y299C was able to fold normally and maintained a conformation very close to that of wild type ArCel 5A. The transformant with greatly improved endo-cellulase enzyme activity is named asT.reesei A2H-Y299C。
Embodiment III: activity and thermostability of mutant endocellulases
Trichoderma reesei transformantT.reeseiA2H-Y299C was successfully heterologously expressed in the fermentation supernatant by fed-batch fermentation, and SDS-PAGE protein maps showed (FIG. 3) that specific bands in ArCel5A-Y299C increased significantly as the fermentation proceeded. At 94h of fermentation, the endo-cellulase activity of ArCel5A reached 5800IU/mL, which was 85.2% higher than that of wild type ArCel5A (FIG. 4). The purification of ArCel5A-Y299C showed a clear single band of about 60 kDa in SDS-PAGE. As a thermophilic enzyme, arCel5A had excellent thermostability, and after incubation at 70℃for 30 min and 80℃for 10 min, the residual enzyme activities of the wild-type ArCel5A decreased to 76.85% and 71.22%, respectively. After rational engineering, the mutant ArCel5A-Y299C retained 93.83% and 91.48% of its original viability after exposure to the same temperature (table 1). Compared with the wild type, the thermal performance of the enzyme activity of the modified endo-cellulase is improved by 16.98-20.26%.
Table 1: comparison of thermal stability of ArCel5A to ArCel5A-Y299C
The experimental result shows that the 299 amino acid of the endo-cellulase plays an important role in the catalysis process, and particularly when the 299 amino acid is mutated from Y to C, the activity and the thermal stability of the enzyme can be greatly improved. To verify the rationality of the 299Y mutation to C, we analyzed the reason for the significant increase in mutant enzyme activity using two methods. First, we calculated the change in gibbs free energy (ΔΔg, kcal/mol) after 299Y saturation mutation using the I-mutant tool (https:// folding. Biofand. Org/I-mutant 2.0. Html). Gibbs free energy is an important indicator of the maximum work a thermodynamic system can perform at constant temperature and pressure (ΔΔg >0 represents an increase in stability and ΔΔg <0 represents a decrease in stability at ph=7.0, 50 ℃). Our calculation results are shown in table 2.
Table 2: mutation of amino acids Y299 Gibbs free energy Change in ArCel5A
The results are shown in table 2: only when the amino acid at position 299 was mutated from Y to C, the DeltaG value was the highest, indicating that the mutation of Y299C at this position was the most stable. The results of the Gibbs free energy calculation are consistent with the results of our experiments, which show that after the 299 th amino acid is mutated from Y to C, the structure of the enzyme becomes more stable, and the whole structure is more stable after the enzyme activity of the endo-cellulase is rationally modified. The stability is favorable for enzymatic reaction, so that the whole structure of the enzyme protein is more stable, the activity and the thermal stability of the enzyme are improved, and the reason that the thermal stability of the endo-cellulase is stronger after enzymatic transformation is explained.
Claims (10)
1. An endo-cellulase mutant, characterized in that it is obtained by mutation of Y into C at the 299 th amino acid of wild-type endo-cellulase cel 5A.
2. The nucleic acid encoding the endo-cellulase mutant of claim 1.
3. The coding nucleic acid according to claim 2, wherein the coding sequence of amino acid 299 of the wild-type endo-cellulase cel5A gene is obtained by mutation of TAC to TGC.
4. An expression vector comprising the nucleic acid encoding the nucleic acid of claim 2 or 3.
5. The expression vector of claim 4, which is a prokaryotic expression vector.
6. A recombinant host bacterium comprising the nucleic acid encoding claim 2 or 3, or the expression vector of claim 4 or 5.
7. The recombinant host bacterium according to claim 6, wherein the recombinant host bacterium is Trichoderma reeseiTrichoderma reesei）。
8. The recombinant host bacterium of claim 7, which is trichoderma reesei A2H.
9. A method of preparing an endo-cellulase mutant, characterized in that it comprises the steps of culturing the recombinant host bacterium according to any one of claims 6 to 8 to produce the endo-cellulase mutant, and collecting the endo-cellulase mutant.
10. The method of claim 9, further comprising the step of purifying the endo-cellulase mutant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311206014.4A CN116970591B (en) | 2023-09-19 | 2023-09-19 | Thermophilic endo-cellulase mutant and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311206014.4A CN116970591B (en) | 2023-09-19 | 2023-09-19 | Thermophilic endo-cellulase mutant and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116970591A true CN116970591A (en) | 2023-10-31 |
| CN116970591B CN116970591B (en) | 2023-12-05 |
Family
ID=88471605
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311206014.4A Active CN116970591B (en) | 2023-09-19 | 2023-09-19 | Thermophilic endo-cellulase mutant and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116970591B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070238155A1 (en) * | 1996-10-10 | 2007-10-11 | Gusakov Alexander V | Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose |
| US20150050701A1 (en) * | 2013-04-05 | 2015-02-19 | California Institute Of Technology | Cellulase compositions having improved thermostability and synergy |
| CN105441411A (en) * | 2014-08-15 | 2016-03-30 | 中国科学院青岛生物能源与过程研究所 | Endo cellulase Cel5A mutant with improved performances |
| CN107663520A (en) * | 2016-07-29 | 2018-02-06 | 东莞泛亚太生物科技有限公司 | Lift the cellulase of activity |
-
2023
- 2023-09-19 CN CN202311206014.4A patent/CN116970591B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070238155A1 (en) * | 1996-10-10 | 2007-10-11 | Gusakov Alexander V | Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose |
| US20150050701A1 (en) * | 2013-04-05 | 2015-02-19 | California Institute Of Technology | Cellulase compositions having improved thermostability and synergy |
| CN105441411A (en) * | 2014-08-15 | 2016-03-30 | 中国科学院青岛生物能源与过程研究所 | Endo cellulase Cel5A mutant with improved performances |
| CN107663520A (en) * | 2016-07-29 | 2018-02-06 | 东莞泛亚太生物科技有限公司 | Lift the cellulase of activity |
Non-Patent Citations (3)
| Title |
|---|
| ANTONIO DECHELLIS: "Supercharging 1 carbohydrate-binding module alone enhances 2 endocellulase thermostability, binding, and activity on cellulosic biomass", BIORXIV. * |
| 王培培: "荧光光谱法定量测定纤维素酶活性架构中单个氨基酸突变对底物结合力的影响", 生物化学与生物物理进展, vol. 43, no. 11 * |
| 白仁惠: "里氏木霉Cel5A 基因优化及其在毕赤酵母中的高效表达", 生物工程学报, vol. 32, no. 10 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116970591B (en) | 2023-12-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yamada et al. | Endowing non-cellulolytic microorganisms with cellulolytic activity aiming for consolidated bioprocessing | |
| Maleki et al. | A novel thermostable cellulase cocktail enhances lignocellulosic bioconversion and biorefining in a broad range of pH | |
| Amore et al. | Cloning and recombinant expression of a cellulase from the cellulolytic strain Streptomyces sp. G12 isolated from compost | |
| CN105802854B (en) | Cellulase high-yield strain and application thereof | |
| Qi et al. | Coordinated β-glucosidase activity with the cellulosome is effective for enhanced lignocellulose saccharification | |
| Liu et al. | Construction of consolidated bio-saccharification biocatalyst and process optimization for highly efficient lignocellulose solubilization | |
| JP2010536391A (en) | Enzymatic hydrolysis of lignocellulosic raw materials using auxiliary enzymes | |
| JP2013545491A (en) | Enhancement of ethanol production by xylose-utilizing zymomonas mobilis in biomass hydrolyzate medium | |
| US20110294184A1 (en) | Cellulolytic polypeptides and their use in micro-organisms for the production of solvents and fuels | |
| DK3000880T3 (en) | EXPRESSION OF RECOMBINANT BETA-XYLOSIDASE ENZYMERS | |
| Jain et al. | Functional expression of a thermostable endoglucanase from Thermoascus aurantiacus RCKK in Pichia pastoris X-33 and its characterization | |
| Datta | Recent strategies to overexpress and engineer cellulases for biomass degradation | |
| CN116970591B (en) | Thermophilic endo-cellulase mutant and preparation method thereof | |
| Rastegari | Molecular mechanism of cellulase production systems in penicillium | |
| CN113151264A (en) | Method for constructing high-yield cellulase strain by utilizing forward regulatory gene and application | |
| SUNG et al. | Omics applications to biofuel research | |
| CN116286751B (en) | Bifunctional cellulase mutant with improved catalytic efficiency and application thereof | |
| KR101539535B1 (en) | Fungal glycoside hydrolase 61 expressing microorganism and method for promoting cellulose degradation by using the same | |
| CN111549016B (en) | Extreme heat-resistant xylanase XYNA and mutant gene, application and preparation method thereof | |
| CN111621486B (en) | Heat-resistant xylanase XYNB with high enzyme activity at low temperature, mutant gene, application and gene sequence preparation method | |
| Mauriello et al. | Development of Improved Cellulase Variants for the Conversion of Spent Mushroom Substrate Supplemented with Wheat Straw by Directed Evolution | |
| Tongsuk et al. | CHARACTERIZATION OF LIGNOCELLULOSE-DEGRADING THERMOPHILIC ANAEROBIC BACTERIUM, CALDICELLULOSIRUPTOR SP. STRAIN EP2: ITS PROPERTIES AND GENOME | |
| US11254956B2 (en) | Myceliophthora thermophila host cell having improved cellulolytic activity and enzymatic compounds produced with same | |
| US9890370B2 (en) | Hyperthermostable endoglucanase | |
| CN105754973A (en) | Mutant W233D of beta-glucosidase and application of mutant W233D |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |