CN106434735A - Method of increasing yield of lignocellulose substrate hydrolase - Google Patents
Method of increasing yield of lignocellulose substrate hydrolase Download PDFInfo
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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
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Abstract
The invention discloses provides a method of increasing heterologous expression quantity of hemicellulase, cellulase or lignocellulose hydrolysis assistance zymoprotein in pichia pastoris. An xylanase (XynC) gene from Aspergillus niger is subjected to codon optimization according to pichia pastoris codon preference characteristics, and an optimized nucleotide sequence (AxynC) is artificially synthesized, and constructs and recombines expression vectors pPIC9K-AxynC and pPICZ alpha A-AxynC. Recombined pichia pastoris of high yield xylanase is constructed by a two-step shock conversion method. That is to say, linearized pPIC9K-AxynC converts the pichia pastoris GS115 to obtain a strain with better enzyme producing ability. The high enzyme activity is subjected to second shock conversion as a host bacterium, and the linearized pPICZ alpha A-AxynC is shocked to convert the strain to obtain double-plasmid recombined pichia pastoris of the high yield xylanase. Tests show that the codon optimized xylanase gene can be expressed successfully in the pichia pastoris, the high yield xylanase recombinant bacterium constructed by a two-step shock conversion method can stably and efficiently produce the xylanase, the flask level enzyme activity reaches 410U/mL, and a protein content in a fermentated supernate reaches 0.37mg/mL.
Description
Technical field
The present invention relates to technical field of bioengineering.In detail, the present invention relates to a kind of improve hemicellulase, fiber
Auxiliary the pheron method of expression and its double matter in recombinant yeast pichia pastoris of plain enzyme or lignocellulose Matrix lysis correlation
The structure of grain recombinant yeast pichia pastoris.
Background technology
Hemicellulase, the cellulase auxiliary enzymes related with lignocellulose Matrix lysis are all class multicomponent enzyme eggs
White general name, can collective effect be hydrolyzed to bioavailable lignocellulose is difficult to beneficial to bioavailable Fructus Vitis viniferae
Sugar, is therefore considered as one of enzyme preparation for having a high potential.Hemicellulase and cellulase belong to glycoside hydrolase, can
The hemicellulase enzyme system of lignocellulose degradation includes to cut the β-D-1,4 inscribe wood that xylan backbone produces xylooligosaccharide at random
Dextranase, the circumscribed xylosidase of β-D-1,4 and side-chain hydrolysis enzyme (α-l-arabfuranglycosidase, phlorose aldehydic acid enzyme with
And acetyl xylan esterase etc.).Cellulase system at least includes three class components, respectively 1,4-BETA-D-glucancellobio-hydrolase (CBH), glucosan
Restriction endonuclease (EG) and beta-glucosidase (BG), wherein EG acts on cellulose noncrystalline domain, hydrolyzes β-Isosorbide-5-Nitrae glycosidic bond, can
Linear fibre element polymer is blocked a large amount of cellulose small molecules of generation；CBH hydrolyzes Isosorbide-5-Nitrae-β-D- glycosidic bond, acts on linear fibre
The plain polymer end of dimension, generates cellobiose molecule；Cellobiose hydrolysis is then become glucose by BG.Auxiliary enzymes mainly include
AA9, plant extension albumen Expansin and expansion factor Swollenin etc..Auxiliary enzymes directly do not participate in the water of lignocellulose
Solution, but auxilin is added during hemicellulase and hydrolyzing ligno-cellulose with cellulosic enzyme, its water can be significantly improved
Solution efficiency.Under cellulase, hemicellulase and the mutual synergism of auxiliary enzymes, can be high by most abundant biology on the earth
Polymer fibre element and hemicellulose, are converted into the glucose that microorganism easily can be utilized, and produce bio-fuel by fermenting,
In order to alleviate current energy shortage problem.
Hemicellulase and cellulase wide material sources, are mainly derived from educable environmental microorganism, such as aspergillus niger, inner
Family name's Trichoderma spp., white rot fungi, thermophilic bacteria of series bacillus and some anaerobism etc..However, these innately have necessarily from producing enzyme
Proportioning defect, with regard to the most commonly used cellulase-producing fungi trichoderma reesei of current commercial Application (Trichoderma reesei)
For, lack excision enzyme CBH II and beta-glucosidase BG in its cellulase system, cause its cellulase system enzymolysis efficiency
Lowly.In addition, the commercial fibres element enzyme preparation that either still manually makes from biological self-produced enzyme system, egg contained therein
White race class up to more than 80 is planted, and causes the specific enzyme activity of hemicellulase and cellulase core enzyme component to reduce, so as to cause wood
Matter cellulose hydrolysis efficiency is low.What is more important, cellulose in different lignocellulosic materials, hemicellulose and wooden
There were significant differences for the ratio of element, and pretreated lignocellulose also has the residual of hemicellulose, and these factors cause jointly
Lignocellulose hydrolysis efficiency is low, and enzyme dosage is big.So, the redesign of cellulase preparation and it is customized to lignocellulose
Effectively hydrolyzing, reduce cellulase cost and provide new Research Thinking.Then, using hemicellulase component, cellulose
Enzyme component and auxiliary enzymes are compounded, it is possible to reduce enzyme dosage, improve the hydrolysis efficiency of lignocellulose.Therefore, based on above-mentioned
Thinking, obtains hemicellulase, cellulase and some auxiliary enzymes list enzyme components very crucial.
In the last few years, heterogenous expression pheron gene obtained single enzyme component and caused the concern that numerous studies.Pichia
Pastoris can realize post translational modification effect, and such as glycosylation, disulfide formation, allow albumen correctly to be folded, and
Activated target protein is obtained, in addition, Pichia sp. does not produce the endogenic enzyme group related to ligocellulose degradation
Point, and extracellular protein composition uncomplicated, recombinant yeast pichia pastoris bacterium fermented supernatant fluid even can without purification directly as
Single enzyme preparation is used, and therefore, using Pichia pastoris as first-selected Host Strains, realizes the heterologous table of enzyme (or albumen)
Reach to obtain single enzyme components such as high concentration and high-purity hemicellulase, cellulase and auxiliary enzymes, it has also become study hotspot.
Data shows, improve one of the approach of expression of exogenous gene in Pichia sp. be exogenous gene is carried out close
Numeral optimizes, and by codon optimization, can not only realize the successful expression of exogenous gene, while the table of purpose product can also be improved
The amount of reaching.Additionally, purpose product high yield can be realized by screening high copy conversion bacterial strain.In addition, external structure multicopy purpose
The plasmid (such as patent CN 101955958A and CN 104046649A etc.) of gene, is increased restructuring and finishes red ferment by homologous recombination
The copy number of genes of interest in mother, so as to improve the yield of purpose product.Therefore, the present invention is using Pichia sp. as host
Bacterium, rationality, efficiently constructs the recombinant pichia yeast strain of high yield hemicellulase component xylanase, obtains high enzyme activity
Xylanase, be that industrial applications are laid a good foundation.
Content of the invention
For the not high present situation of existing xylanase generally existing wood enzyme activity both at home and abroad, using the teaching of the invention it is possible to provide commercial production xylan
The genetic engineering bacterium of enzyme is little.The present invention carries out excellent to the gene of an important component xylanase in hemicellulase
Change, the xylanase gene after optimization is transformed in Pichia sp., it is achieved that high efficient expression of the xylanase in Pichia sp..
The xylanase of expression has higher activity, for answering for lignocellulosic material degraded production fermentability monosaccharide
With being with a wide range of applications.
It is an object of the invention to provide the xylanase base of a kind of method for obtaining high enzyme activity xylanase and a kind of optimization
Cause, the sequence G/C content after optimization is reduced to 41.77% by 52%, and it is 76.83% to optimize context homology, and builds
PPIC9K-AxynC and pPICZ α A-AxynC expression vector, to obtain the expression transformant of stability and high efficiency.
The double-mass model of xylanase (XynC) gene and the high yield xylanase after the codon optimization that the present invention is provided
The comprising the following steps that of restructured Pichia pastoris in expression system constructing：
1st, the optimization of xylanase gene：
The present invention carries out codon optimization to a kind of xylan carbohydrase (XynC) gene for coming from aspergillus niger, its nucleotides sequence
Row are as shown in SEQ ID NO.1.
The present invention using Gene Designer (DNA2.0, Menlo Park) and is based on Pichia sp. codon preference
SEQ ID NO.1 is optimized, the nucleotide sequence after optimization, AxynC is named as, sequence is as shown in SEQ ID NO.2.
Xylanase molecule amount 35.5kDa that the present invention is provided, xylanase XynC aminoacid sequence before and after codon optimization
Row are unchanged, and its sequence is as shown in SEQ ID NO.3.
2nd, the structure of expression vector：
Using plasmid pPIC9K and pPICZ α A as carrier, the xylanase gene AxynC after optimization is inserted promoter
AOX1 downstream, it is Not I that 5 ' end restriction enzyme sites are EcoR I, 3 ' end restriction enzyme sites, builds pPIC9K-AxynC and pPICZ α A-
AxynC expression vector.
3rd, the structure of live high-enzyme strain and screening：
Using the electroporated method of two steps, the restructured Pichia pastoris in expression system of high yield xylanase is built, i.e., made with Sac I
For pPIC9K-AxynC expression vector linearisation sites, expression vector linearisation is realized, by electroporated Pichia sp., utilize
G418 resistance screening live high-enzyme strain.Second conversion is carried out as host using live high-enzyme strain, will Sac I enzyme linearizing
The electroporated above live high-enzyme strain of pPICZ α A-AxynC, using high flux primary dcreening operation, it is poly- that shaking flask enzyme activity shines method acquisition high yield wood again
The recombinant pichia yeast strain of carbohydrase.
4th, optimize condition of enzyme production：
Using BMMY as culture medium, Optimal Medium original ph, derivant (methanol) amount, cultivation temperature and dissolved oxygen
(shaking table revolution), realizes XynC stability and high efficiency producing enzyme.
Beneficial effect of the present invention：
1 present invention achieves xylanase (XynC) gene codon optimization, Host Strains are Pichia yeast, after optimization
Reduce G/C content, be reduced to 41.77% by 52%, using RNA secondary structure folding software RNAstructure to optimization after
Nucleotide sequence realize secondary structure folding, and adjust base sequence, make start codon end base in open loop structure, be beneficial to
Ribosome is combined and expression of the xylanase gene in Pichia sp..
2nd, the present invention is during restructured Pichia pastoris in expression system constructing, using the electroporated method of two steps (or double matter
Grain), copy number of the genes of interest in recombinant yeast pichia pastoris can be effectively improved, with the electroporated method of a step (or simple substance grain) phase
More loaded down with trivial details than, it is to avoid multiple screening, two steps electroporated methods can more rationality, more efficiently obtain high yield recombinant yeast pichia pastoris.
3rd, the present invention constructs the double-mass model that containing alcohol oxygen type starts pPIC9K-AxynC the and pPICZ α A-AxynC of AOX1
(or Double) recombinant bacterial strain, using methanol as derivant, controls methanol addition, increases substantially restructuring destination protein expression
Amount.
Description of the drawings
Fig. 1：The comparison diagram of xylanase (XynC) gene optimization context.
Fig. 2：The recombinant expression carrier pPIC9K-AxynC of structure and the process schematic of pPICZ α A-AxynC.
Fig. 3：Expression vector pPIC9K-AxynC and the detection of pPICZ α A-AxynC nucleic acid electrophoresis：
1st, No. 4 swimming lanes be using gained band after restricted enzyme Sac I single endonuclease digestion；
2nd, No. 3 swimming lanes be using gained band after restricted enzyme EcoR I, Not I double digestion.
Fig. 4：Double-mass model recombinant yeast pichia pastoris build and screening process.
Fig. 5：Double-mass model recombinant yeast pichia pastoris and simple substance grain live high-enzyme strain enzymatic productivity comparison diagram.
Fig. 6：The SDS-PAGE spectrum of recombined xylanase AxynC in double-mass model strain fermentation supernatant.
Fig. 7：Double-mass model bacterial strain enzyme activity change curve under the horizontal Liquid fermentation condition of shaking flask.
Specific embodiment
With reference to example, the method for the present invention is described further.Those skill in the art related can be by reality
Apply example to more fully understand and grasp the present invention.But, the protection of the present invention and right are not limited to provided case.
Embodiment 1：Xylanase (XynC) gene codon optimization
In NCBI (http://www.ncbi.nlm.nih.gov/) GenBank in retrieve aspergillus niger Aspergillus
Niger xylanase sequence (accession number：KJ601783), codon optimization is carried out according to following methods：
(1) software GeneDesigner 2.0 is adopted, according to codon of the Pichia sp. codon preference to XynC
(SEQ ID NO.1) is tentatively replaced；And calculate G/C content, adjusted further according to G/C content further so as to value between 40～
50%.After analysis and regulation in gene order mRNA cryptic splice site and codon abundance, intuitively can distinguish by the software
Knots modification before and after analysis codon.
(2) using RNAStructure 3.2, the start codon end of mRNA secondary structure is predicted and free energy enters
Row analysis, selects sequence of the relatively low and start codon end of free energy in open loop structure, finally obtains sequence SEQ after optimizing
ID NO.2, is named as AxynC.
(3) nucleotide sequence (shown in SEQ ID NO.2) after optimizing is obtained using full genome synthetic method.
Xylanase gene optimizes context and compares as shown in Figure 1.
Embodiment 2：Recombinant expression carrier pPIC9K-AxynC and the structure of pPICZ α A-AxynC
Expression vector pPIC9K, pPICZ α A EcoR I and Not I double digestion, reclaim digestion products.After purification, synthesize
Genes of interest adopt T with carrier pPIC9K, pPICZ α A respectively4DNA ligase is attached, and converts bacillus coli DH 5 alpha, profit
Identified with bacterium colony PCR and obtain purpose carrier pPIC9K-AxynC and pPICZ α A-AxynC.In LB culture medium, culture is containing purposeful
12～the 16h of escherichia coli of carrier, extracts purpose carrier according to plasmid extraction handbook.Expression vector pPIC9K-AxynC and
The structure of pPICZ α A-AxynC and checking are as shown in Figures 2 and 3.
Embodiment 3：The electroporated method of two steps builds the recombinant yeast pichia pastoris of high yield xylanase and the screening of bacterial strain
Linearisation, electroporated Pichia pastoris GS115 competent cell are carried out using Sac I to pPIC9K-AxynC.Turn
Change liquid spreading RDB solid medium, cultivate 3 days in 30 DEG C.Picking grow normal bacterium colony be transferred to containing variable concentrations (1,
2nd, 4 and 6mg/mL) on the YPD flat board of Geneticin G418, the bacterium colony of energy normal growth on picking high concentration flat board.Obtain above-mentioned
The bacterium colony for taking is inoculated in the 250mL triangular flask of 30mL BMMY culture medium respectively, 30 DEG C, and 220rpm is cultivated 4 days, is added per 24h
Methanol so as to final concentration of 0.5%, after culture terminates, is collected by centrifugation supernatant, determines enzyme activity with Zelkova schneideriana Hand.-Mazz. xylan as substrate,
Wherein enzyme activity highest is No. 16 bacterium (being named as P.pastoris GS115/pPIC9K-AxynC#16).With Sac I couple
PPICZ α A-AxynC carries out linearisation, No. 16 bacterium competence cells of electroporated recombinant yeast pichia pastoris.Conversional solution coating is containing rich
The YPDS solid medium of Lay mycin (100ug/mL), the single bacterium colony on picking flat board is inoculated into respectively to be trained equipped with 500ul BMMY
48 deep-well plates (5mL/ hole) of foster base carry out high flux screening, carry out preliminary screening by above-mentioned cultural method, to enzymatic productivity relatively
Good bacterial strain (4 plants) carries out shaking flask enzyme activity and shines again, and its vigor highest (is named as P.pastoris for No. 29 double-mass model bacterial strains
GS115/pPIC9K/pPICZ α A-AxynC#29), the concrete flow process that builds is as shown in accompanying drawing Fig. 4.No. 29 bacterium of double-mass model bacterial strain and 16
Number bacterium enzymatic productivity compares, and enzyme activity improves 34%, as illustrated in figure 5 of the drawings.The SDS-PAGE of No. 29 bacterium product xylanase is tested
Card is as shown in Figure 6.
Embodiment 4：Recombined xylanase (AxynC) determination of activity
Xylanase activity is determined by DNS method.Reaction system be 0.5% 480 μ L of Zelkova schneideriana Hand.-Mazz. xylan (with pH5.0's
The citrate buffer solution of 50mM is formulated) and the appropriate 20 μ L of enzyme night for diluting, in 50 DEG C of water bath heat preservation 10min, insulation terminates
0.5mL DNS solution terminating reaction is added afterwards immediately.Water-bath 7min colour developing in the boiling water.Be cooled to room temperature, draw 200 μ L in than
In color ware, 1.8mL deionized water is added, mix mensuration absorbance value at the 540nm.Control sample, with 20 μ L pH5.0
50mM citrate buffer solution replace enzyme liquid.Enzyme activity is defined：At 50 DEG C, in pH5.0 reaction system, the recombined xylanase water
Enzyme amount needed for solution Zelkova schneideriana Hand.-Mazz. xylan 1 μm of oL of release per minute is an enzyme activity unit U.In embodiment, all enzyme activity are surveyed
Fixed, each sample all takes 3 Duplicate Samples.
Embodiment 5：The preparation of high activity recombined xylanase (AxynC)
Single factor test optimization is carried out by fermentation condition, optimal conditions mainly include that the initial pH of fermentation medium, methanol are dense
Degree, cultivation temperature and shaking table revolution (i.e. dissolved oxygen).As a result show, the recombinant yeast pichia pastoris are cultivated as follows and can obtain high enzyme
Xylanase living：No. 29 bacterium single bacterium colonies of double-mass model bacterial strain are inoculated in triangular flask of the 250mL equipped with 30mL BMGY culture medium
In, 220rpm, 30 DEG C of culture 28h, make OD600Between 8～10.Bacterium solution is collected in aseptic 50mL centrifuge tube on super-clean bench,
5000 × g is centrifuged 5min, collects thalline.Supernatant is abandoned, thalline is all transferred to liquid amount for 50mL BMMY culture medium (just
Beginning pH5.5) 500mL triangular flask in, 250rpm, 28 DEG C, in culture medium add methanol so as to final concentration of 1.5%, cultivate to
240h.0.5mL sample is taken per 24h, determines Biomass (OD600), 5min being centrifuged in 4000 × g, collects supernatant.With Fagus sinensis Oliv.
Xylan determines enzyme activity as substrate and Braford method determines protein content.In optimum results such as accompanying drawing shown in Fig. 7, table is induced
Reach 240h, highest enzyme activity 410U/mL, protein content 0.37mg/mL, specific enzyme activity 1105U/mg.Note：Shaking flask induction producing enzyme is not optimised
Front experiment condition：Single bacterium colony is inoculated into BMMY culture medium (initial pH6.0), adds methanol final concentration of 0.5%, 220rpm, 30
DEG C culture 240h, remaining condition of culture is constant.
Claims (7)
1. a kind of using double-mass model restructured Pichia pastoris in expression system raising hemicellulase, cellulase or lignocellulose base
The method of the related auxiliary enzymes protein yield of matter hydrolysis, it is characterised in that by building high yield hemicellulase, cellulase or wood
Matter cellulose matrix hydrolyzes the related pichia yeast expression system for aiding in pheron to realize enzyme in extracellular efficient secretion.Concrete step
Rapid as follows：
1) codon optimization：The present invention is based on Pichia sp. codon preference using software Gene Designer, realizes half fine
The optimization of the plain enzyme of dimension, cellulase or lignocellulose Matrix lysis correlation auxiliary enzymes protein gene.
2) expression vector establishment：Using plasmid pPIC9K and pPICZ α A as carrier, by the hemicellulase after optimization or cellulose
Enzyme gene inserts promoter AOX1 downstream, and it is Not I, to build containing half that 5 ' end restriction enzyme sites are EcoR I, 3 ' end restriction enzyme sites
The recombinant expression carrier of cellulase, cellulase or auxiliary enzyme gene.
3) recombinant bacterial strain is obtained：Using the electroporated method of two steps, (fine containing half as pPIC9K recombinant expression carrier using Sac I
The plain enzyme of dimension, cellulose enzyme gene or auxiliary enzymes protein gene) linearisation sites, expression vector linearisation is realized, is converted by electricity
Pichia sp. is proceeded to, screens live high-enzyme strain.Subsequently, linearizing for Sac I pPICZ α A recombinant expression carrier is (fine containing half
The plain enzyme of dimension, cellulose enzyme gene or auxiliary enzymes protein gene) expression vector electroporated enter the above-mentioned restructuring for screening finish red ferment
In mother, the double-mass model bacterial strain of higher enzymatic productivity is obtained.
4) optimize induction condition of enzyme production：Using BMMY as culture medium, Optimal Medium original ph, derivant (methanol)
Amount, cultivation temperature and shaking table revolution (dissolved oxygen), realize hemicellulase, cellulose enzyme gene or auxiliary enzymes stability and high efficiency producing enzyme.
2. according to claim 1, its step 1) in codon optimization, be primarily referred to as encoding some with lignocellulose base
The optimization of the codon of matter hydrolysis relevant enzymes.These enzymes include：Cellulase (endoglucanase, exoglucanase
With beta-glucosidase etc.), hemicellulase (xylanase, xylosidase, arabinofuranosidase, glucuronate
Enzyme, acetyl xylan esterase and phenolic acid esterase etc.) and some auxilins (AA9, plant extension albumen Expansin, expansion
Factor S wollenin etc.).
3. according to claim 1, using double-mass model restructured Pichia pastoris in expression system, hemicellulase, cellulase are improved
Or double-mass model refers to two kinds of plasmids with different resistances in the method for auxiliary enzymes protein yield.
4. according to claim 1, using double-mass model restructured Pichia pastoris in expression system, hemicellulase, cellulase are improved
Or double-mass model refers to two kinds with of the same race or different hemicellulases, cellulase or auxiliary in the method for auxiliary enzymes protein yield
The plasmid of enzyme gene.
5. according to claim 1, using double-mass model restructured Pichia pastoris in expression system, hemicellulase, cellulase are improved
Or the method for auxiliary enzymes protein yield, the recombinant bacterial strain of its description is recombinant yeast pichia pastoris.
6. according to claim 1, using double-mass model restructured Pichia pastoris in expression system, hemicellulase, cellulase are improved
Or the method for auxiliary enzymes protein yield, its step 3) described in two step electric shock transformation methods, this method be applied to restructuring finish
Red saccharomycetic building process and convert for embodying carrier transforming sequence in no particular order.
7. according to claim 1, its step 4) described in recombinant pichia yeast strain can stability and high efficiency produce half fiber
Some auxilins of plain enzyme, cellulase or lignocellulose Matrix lysis correlation.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107699584A (en) * | 2017-09-30 | 2018-02-16 | 武汉轻工大学 | The preparation method of xylanase gene, recombinant expression carrier, recombinant strains, zytase and preparation method thereof and feed |
| CN107699507A (en) * | 2017-10-11 | 2018-02-16 | 广东海纳川生物科技股份有限公司 | A kind of Pichia pastoris for expressing recombinant plectasin |
| CN110029123A (en) * | 2019-04-03 | 2019-07-19 | 江西农业大学 | The method and application of Yeast expression carrier building and expression circumscribed-type cellulase |
| CN111500479A (en) * | 2020-04-29 | 2020-08-07 | 江南大学 | Construction and application of non-methanol-induced dual-promoter pichia pastoris engineering bacteria |
| CN111850027A (en) * | 2020-06-12 | 2020-10-30 | 天津科技大学 | Pichia pastoris engineering strain for heterologous expression of cellulase gene CBH II and application |
| CN113322270A (en) * | 2021-03-12 | 2021-08-31 | 上海国龙生物科技有限公司 | Preparation method and application of pichia pastoris for expressing mixed enzyme preparation |
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| CN107699584A (en) * | 2017-09-30 | 2018-02-16 | 武汉轻工大学 | The preparation method of xylanase gene, recombinant expression carrier, recombinant strains, zytase and preparation method thereof and feed |
| CN107699507A (en) * | 2017-10-11 | 2018-02-16 | 广东海纳川生物科技股份有限公司 | A kind of Pichia pastoris for expressing recombinant plectasin |
| CN110029123A (en) * | 2019-04-03 | 2019-07-19 | 江西农业大学 | The method and application of Yeast expression carrier building and expression circumscribed-type cellulase |
| CN111500479A (en) * | 2020-04-29 | 2020-08-07 | 江南大学 | Construction and application of non-methanol-induced dual-promoter pichia pastoris engineering bacteria |
| CN111500479B (en) * | 2020-04-29 | 2022-12-27 | 江南大学 | Construction and application of non-methanol-induced dual-promoter pichia pastoris engineering bacteria |
| CN111850027A (en) * | 2020-06-12 | 2020-10-30 | 天津科技大学 | Pichia pastoris engineering strain for heterologous expression of cellulase gene CBH II and application |
| CN113322270A (en) * | 2021-03-12 | 2021-08-31 | 上海国龙生物科技有限公司 | Preparation method and application of pichia pastoris for expressing mixed enzyme preparation |
| CN114561303A (en) * | 2022-02-14 | 2022-05-31 | 大连理工大学 | Trichoderma reesei engineering strain secreting high-performance cellulase and application thereof |
| CN114561303B (en) * | 2022-02-14 | 2023-11-17 | 大连理工大学 | Trichoderma reesei engineering strain secreting high-performance cellulase and application thereof |
| CN117924452A (en) * | 2024-03-21 | 2024-04-26 | 华南农业大学 | Application of recombinant corn expansin and synergistic cellulase thereof in degradation of lignocellulose |
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