CN113388532B - Recombinant trichoderma reesei for producing asparaginase and construction method and application thereof - Google Patents
Recombinant trichoderma reesei for producing asparaginase and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant trichoderma reesei for producing asparaginase and a construction method and application thereof, and belongs to the technical field of biology. In the expression process of the recombinant trichoderma reesei, asparaginase protein is secreted to the outside of cells under the guiding action of a proper signal peptide, the produced asparaginase has high yield, high purity and high stability, the activity of the extracellular protease is low, asparaginase genes from trichoderma reesei and other microorganisms can be effectively produced, and the produced asparaginase can be applied to food related industries.
Description
Technical Field
The invention relates to the technical field of biology, in particular to recombinant trichoderma reesei for producing asparaginase and a construction method and application thereof.
Background
Trichoderma reesei (Trichoderma reesei or Hypocrea jecorina) is a mesophilic and saprophytic filamentous fungus. The initially isolated strain was identified as Trichoderma reesei QM6a. Starting from an original strain QM6a, a series of mutation breeding is carried out to screen out some cellulase high-yield strains, such as Rut-C30.
Trichoderma reesei is widely used because of its ability to naturally secrete large amounts of endogenous proteins to the extracellular space, while endogenous cellulases are fermented to yield up to 100g/L. Trichoderma reesei is also a common host for expression of homologous and heterologous proteins due to its diverse post-translational modifications. Cellulases and hemicellulases produced by trichoderma reesei are widely applied to various industries, such as cbh1, cbh2, egl1, egl2, xyn1, xyn2, xyn3 and the like. Promoters for cellulases and hemicellulases, characterized by inducibility and high expression, and partial promoters, such as cbh1 and cbh2 promoters, have been successfully used for the expression of heterologous proteins. However, in the process of expressing the heterologous protein, the endogenous protein is also expressed at the same time, and the expression amount of the endogenous protein is far larger than that of the heterologous protein. Therefore, the trichoderma reesei crude enzyme solution is mixed with a large amount of endogenous protein besides heterologous protein; when the crude enzymes are used for food processing, the quality of the food is seriously affected by a large amount of Trichoderma reesei endogenous protein mixed in the crude enzymes. These limit the use of heterologous protein expression in trichoderma reesei in the food industry. Therefore, there is a need for genetic engineering to improve the purity of heterologous proteins in crude enzyme solutions.
Disclosure of Invention
In order to solve the problem of low purity of heterologous protein expressed in the existing trichoderma reesei, the invention aims to provide the recombinant trichoderma reesei for producing the asparaginase and the construction method and the application thereof.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a recombinant trichoderma reesei for producing asparaginase, wherein the recombinant trichoderma reesei heterologously expresses the asparaginase with a nucleotide sequence shown in any one of SEQ ID No. 1-4; the recombinant trichoderma reesei is obtained by replacing original genes of cellulase CBH1, CBH2, EGL3 and xylanase XYN2 and XYN3 in trichoderma reesei with genes of asparaginase, and knocking out genes of cellulase, xylanase and protease required to be knocked out in trichoderma reesei.
The asparaginase is derived from eukaryotes, including filamentous fungi, and more preferably from the extreme thermophilic species Thermococcus kodakarensis, aspergillus niger and Aspergillus oryzae.
The recombinant Trichoderma reesei starting strain is Trichoderma reesei strain, preferably Trichoderma reesei QM6a, QM 9123, QM9414, QM 9136, NG14, NRRL 3653, NRRL 3652, NRRL 11236, sa28, DB746, ME-446, 4065MG4 or 4065MG5, more preferably Trichoderma reesei Rut-C30, RL-P37 or PC-3-7.
The invention also provides a group of plasmids for constructing the recombinant trichoderma reesei, which comprises a recombinant expression plasmid and a knock-out plasmid, wherein the recombinant expression plasmid sequentially comprises a promoter sequence of a cellulase or xylanase gene required to be replaced, a gene sequence coded by asparaginase, a recoverable resistance screening marker mediated by Cre-loxP and a terminator sequence of the cellulase or xylanase gene required to be replaced; the knockout plasmid sequentially comprises an upstream homology arm sequence of a cellulase, xylanase or protease gene to be knocked out, a recoverable resistance screening marker based on Cre-loxP mediation and a downstream homology arm sequence of the cellulase, xylanase or protease gene to be knocked out.
Further, the desired alternative cellulases or xylanases comprise cellulases CBH1, CBH2, EGL3 and xylanases XYN2, XYN3; the cellulase, xylanase or protease to be knocked out is knocked out by using enzymes coded by EGL1, XYN1, BGL1, PEP1, SLP1, GAP1, TRE81070, TRE120998 and TRE 123234.
The invention also provides a construction method of the recombinant trichoderma reesei, which comprises the following steps:
s1, constructing the recombinant expression plasmid;
s2, transforming the recombinant expression plasmid into trichoderma reesei;
s3, constructing the knockout plasmid;
and S4, transforming the knockout plasmid into the trichoderma reesei, and screening to obtain the recombinant trichoderma reesei.
Further, in step S4, trichoderma reesei containing both the recombinant expression plasmid and the knock-out plasmid is screened out, then transformants successfully undergoing homologous recombination in the genome are screened out, and transformants without the resistance marker are screened out by a marker recovery technology.
Preferred protocols for transformation of Trichoderma reesei include protoplast transformation, electrotransformation, and Agrobacterium tumefaciens conjugative transfer. Preferably, the recombinant Trichoderma reesei genetic engineering bacteria capable of expressing and secreting asparaginase can be obtained by conjugating Agrobacterium tumefaciens with Trichoderma reesei spores, and then screening out transformants with integrated heterologous protein genes in genomes.
The invention also provides application of the recombinant trichoderma reesei or the plasmid in asparaginase production.
The invention also provides a production method of asparaginase, which comprises the following steps:
s1, producing asparaginase by using the recombinant trichoderma reesei or the plasmid;
s2, separating the asparaginase from the system of S1.
Further, in step S1, the method for producing asparaginase comprises: inoculating the recombinant trichoderma reesei into a fermentation culture medium, wherein the inoculation amount is 1-20%, and the culture is carried out for 2-12 days at the temperature of 20-37 ℃.
The fermentation medium comprises a carbon source, a nitrogen source, inorganic salt, a growth factor and water; wherein the carbon source comprises: at least one of glucose, sucrose, lactose, cellulose, sophorose, xylose, xylan, molasses, straw, lignocellulose, or corn steep liquor; the nitrogen source includes: at least one of inorganic ammonium salt, nitrate and nitrogen-containing organic matter.
The invention also provides application of the asparaginase produced by the production method in flour food processing.
Further, the asparaginase produced by the production method is applied to baking, roasting, cooking, frying or frying of flour food.
During the food processing process by baking, roasting, cooking, frying and the like, asparagine in food raw materials can react with glucose to generate a carcinogen, namely acrylamide. The addition of asparaginase during food processing, which hydrolyzes asparagine to aspartic acid, significantly reduces acrylamide formation.
In the flour food industry, asparaginase powder is added to flour at 0-300ppm to make snacks such as bread and cake according to conventional protocols. And detecting the content of acrylamide in the bread by using a liquid phase method. The preferred concentration is 50-300ppm, with a 23% -43% reduction in acrylamide content.
In a typical fried tuber industrial process, liquid asparaginase is added to a blanched phosphate solution, and the blanched tuber is immersed in the asparaginase-containing phosphate solution at a temperature of about 45 ℃ to 75 ℃ and incubated for 1-10min; then carrying out the subsequent frying operation. The preferred asparaginase concentration is 50U/mL; preferably for 1-3min; the acrylamide content is reduced by 30-46%.
The invention discloses the following technical effects:
the recombinant trichoderma reesei disclosed by the invention utilizes a homologous recombination technology to replace coding sequences of proteins (cellulase CBH1, CBH2, EGL3, xylanase XYN2 and XYN 3) secreted and expressed in large quantities by trichoderma reesei strains into coding sequences of asparaginase proteins, so that the expression quantity and the product purity of asparaginase are improved, and the (semi) cellulase EGL1, XYN1 and BGL1 of the trichoderma reesei are knocked out to improve the product purity of the asparaginase; the proteases PEP1, SLP1, GAP1, TRE81070, TRE120998 or TRE123234 of Trichoderma reesei are knocked out respectively, and the PEP1 knock-out is found to improve the product stability of asparaginase obviously. In the expression process of the recombinant trichoderma reesei, asparaginase protein is secreted to the outside of cells under the guiding action of a proper signal peptide, the produced asparaginase has high yield, high purity and high stability, the activity of the extracellular protease is low, asparaginase genes from trichoderma reesei and other microorganisms can be effectively produced, and the produced asparaginase can be applied to food related industries.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows the construction of recombinant Trichoderma reesei, wherein A. Recombinant expression plasmid, B. Knock-out plasmid;
FIG. 2 is an electrophoretogram of a crude enzyme solution of example 2.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, 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. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
The experimental methods in the following examples, in which specific conditions are not specified, are generally performed according to conventional conditions, such as "molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989).
In the examples of the present invention, the formulation of the medium and reagents was as follows:
(1) Luria Bertani (LB) medium formula: yeast powder: 5g, peptone: 10g, sodium chloride: 10g, tap water to 1L, natural pH.
(2) The PDA culture medium formula comprises: and (3) potato: 200g, glucose: 20g, agar: 20g, the volume of tap water is fixed to 1L, and the pH value is natural.
(3) The formula of the MD culture medium is as follows: carbon source: 20g, yeast powder: 0.3g, peptone: 1g, urea:0.3g, (NH 4) 2 SO 4 ：1.4g，KH 2 PO 4 ：2.0g，CaCl 2 ：0.34g，MgSO 4 ·7H 2 O:0.3g, mandels microelement liquid: 1mL.
(4) Formulation of Mandels microelement liquid (1000 ×): feSO 4 ·7H 2 O：5g，CoCl·6H 2 O：2g，ZnSO 4 ·7H 2 O：1.4g，MnSO 4 ·H 2 O:1.6g, purified water is fixedTo 1L.
(5) Medium for conjugation transfer:
1M MES:21.32g MES, 100mL pH5.3 (5M KOH adjustment) by adding water, and membrane-passing sterilization.
b.5M KOH:7.013g KOH powder was taken to a volume of 25mL.
c.2M Acetosyringone (AS): weighed 0.01962g, dissolved in DMSO and made a constant volume of 0.5mL, and sterilized by passing through a membrane.
e.MM salts(1L)：NaCl：0.375g，(NH 4 ) 2 SO 4 ：1.250g，K 2 HPO 4 ：5.125g，KH 2 PO 4 ：3.625g，CaCl 2 ·2H 2 O：0.165g，MgSO 4 ·7H 2 O：1.250g，FeSO 4 ·7H 2 O：0.0062g。
Im (Induction Medium) liquid: 80mL MM salts,0.36g glucose, 1.26g glycerol, water to a constant volume of 192mL, 8 bottles of 24mL each, 1mL MES 1M and 25. Mu.L acetosyringone 2M at the time of inoculation.
(g) Induction Medium Plates solids: 160mL MM salts,0.36g glucose, 2.52g glycerol, adding water to a constant volume of 384mL, subpackaging 4 bottles with 96mL each bottle, adding 1.5g agar each bottle, sterilizing at 115 deg.C for 20min. 4mL of 1M MES and 100. Mu.L of 2M acetosyringone were added to each vial before pouring.
Trichoderma reesei used in the examples of the present invention was Trichoderma reesei Rut-C30 (ATCC 56765). This protocol is also applicable to other strains of Trichoderma reesei.
The plasmid extraction kit is purchased from AXYGEN company, the gel recovery kit is purchased from MAGEN company, the seamless cloning kit is selected from gold company, and the DNA restriction endonuclease and the ligase need NEB company. And similar products of other companies can be used for replacement.
Example 1 construction of strains
1. Construction of expression vector for substituting corresponding cellulase and xylanase genes with heterologous protein gene
The skeleton of the vector constructed by the invention is derived from LML2.1 constructed by the experiment, contains Cre-loxP elements, and can realize the repeated use of the screening marker (Scientific reports.2016,6 20761. The public is available from the applicant and can only use it for repeated experiments in accordance with the present invention.
Firstly, DNA sequences (terminators) with the length of about 600-1000 bp upstream of the initiation codon ATG (promoter) and downstream of the termination codon TAA (or TAG, TGA) of the Trichoderma reesei cellulase CBH1, CBH2, EGL2, EGL3 and xylanase XYN2, XYN3 coding genes are respectively amplified by PCR. The nucleotide sequence of the primer is shown in a sequence table SEQ ID No. 5-40:
CBH1 expression plasmid construction primers:
SEQ ID No.5：ATTACGAATTCTTAATTAACCTGTAAAGCCGCAATGCAGC；
SEQ ID No.6：CATGATGCGCAGTCCGCGGT；
SEQ ID No.7：GGACTGCGCATCATGCCTCTCAAGCCGATTCTCC；
SEQ ID No.8：CATTATACGAAGTTATTCTAGACTACGCATCCGTGCCCAGAG；
SEQ ID No.9：ACTAGTGAGCTCATTTAGCTCCGTGGCGAAAGCCT；
SEQ ID No.10：AGTGCCAAGCTTATTTCATCGTAACCGAGAATCCAGAGCTG；
CBH2 expression plasmid construction primers:
SEQ ID No.11：ATTACGAATTCTTAATTACGAGAATGGTGAGGACTGAGATAA；
SEQ ID No.12：CATGGTGCAATACACAGAGGGT；
SEQ ID No.13：GTGTATTGCACCATGCCTCTCAAGCCGATTCTCC；
SEQ ID No.14：CATTATACGAAGTTATTCTAGACTACGCATCCGTGCCCAGAG；
SEQ ID No.15：ACTAGTGAGCTCATTTGCTTTCGTGACCGGGCTTCAAA；
SEQ ID No.16：AGTGCCAAGCTTATTTATGCGATGCGGCTCAAGACTTC；
EGL2 expression plasmid construction primers:
SEQ ID No.17：ATTACGAATTCTTAATTAACGCAAGTGCCTGAGTGAAGAT；
SEQ ID No.18：CATTGTCGATGACGGGGAGATATTAT；
SEQ ID No.19：CCGTCATCGACAATGCCTCTCAAGCCGATTCTCC；
SEQ ID No.20：CATTATACGAAGTTATTCTAGACTACGCATCCGTGCCCAGAG；
SEQ ID No.21：ACTAGTGAGCTCATTTCACTCTGAGCTGAATGCAGAAGC；
SEQ ID No.22：AGTGCCAAGCTTATTTCACCAAGGCAACTCGTCCAG；
EGL3 expression plasmid construction primers:
SEQ ID No.23：ATTACGAATTCTTAATTAAGTGAGGAGATTGTGGCTACGA；
SEQ ID No.24：CATTGCGACGCTATGCTTGTG；
SEQ ID No.25：CATAGCGTCGCAATGCCTCTCAAGCCGATTCTCC；
SEQ ID No.26：CATTATACGAAGTTATTCTAGACTACGCATCCGTGCCCAGAG；
SEQ ID No.27：ACTAGTGAGCTCATTTAACCTGGAAACGTGAGATGTG；
SEQ ID No.28：AGTGCCAAGCTTATTTCCGAGTCCTGAGAGTTGTAGT；
primer construction of XYN2 expression plasmid:
SEQ ID No.29：ATTACGAATTCTTAATTAATTGTGATGCTGCTGCTGATG；
SEQ ID No.30：CATGTTGATGTCTTCTTGCTTCAG；
SEQ ID No.31：GAAGACATCAACATGCCTCTCAAGCCGATTCTCC；
SEQ ID No.32：CATTATACGAAGTTATTCTAGACTACGCATCCGTGCCCAGAG；
SEQ ID No.33：ACTAGTGAGCTCATTTAGGGGGCTCTTCTTTTGTGAT；
SEQ ID No.34：AGTGCCAAGCTTATTTCACGACGACAACACCTGGAT；
primer construction of XYN3 expression plasmid:
SEQ ID No.35：ATTACGAATTCTTAATTAATTCCGTGCGTGTCTGATGG；
SEQ ID No.36：CATATTGTCCGCCTCAATTGAAAC；
SEQ ID No.37：GAGGCGGACAATATGCCTCTCAAGCCGATTCTCC；
SEQ ID No.38：CATTATACGAAGTTATTCTAGACTACGCATCCGTGCCCAGAG；
SEQ ID No.39：ACTAGTGAGCTCATTTCTGCTCTGCTACATGGGTATG；
SEQ ID No.40：AGTGCCAAGCTTATTTGGATTCGGGCGTATGTTGAG。
next, the asparaginase sequence (from the start codon to the stop codon) was PCR amplified. DNA can be used as a template for amplification to retain the intron of the coding gene, or cDNA can be used as a template for amplification or the gene can be artificially synthesized to remove the intron of the gene. The nucleotide sequence of the primer is shown in SEQ ID No. 5-40 of the sequence table, and the nucleotide sequence of asparaginase is shown in SEQ ID No. 1-4 of the sequence table:
SEQ ID No.1：ATGCCTCTCAAGCCGATTCTCCTGTCTGCCCTGGCCAGTCTCGCCTCGGCCATGAAACTGTTAGTGCTGGGCACCGGCGGCACCATTGCAAGCGCCAAAACCGAAATGGGCTATAAAGCAGCACTGTCAGCAGATGATATTTTACAGTTAGCAGGTATTCGTCGCGAAGATGGCGCCAAAATTGAAACCCGTGATATTCTGAATTTAGATAGTACCTTAATTCAGCCGGAAGATTGGGTGACCATTGGTCGCGCCGTTTTTGAAGCCTTTGATGAATATGATGGCATTGTTATTACACATGGCACAGATACCTTAGCCTATACCTCTAGTGCACTGAGCTTTATGATTCGTAATCCGCCGATTCCGGTTGTTCTGACCGGCTCAATGCTGCCGATTACCGAACCGAATAGCGATGCCCCGCGCAATCTGCGTACCGCCCTGACCTTTGCACGTAAAGGCTTTCCGGGCATTTATGTGGCCTTTATGGATAAAATTATGCTGGGAACTCGTGTGTCTAAAGTTCATAGCTTAGGTCTGAATGCATTTCAGAGTATTAATTATCCGGATATTGCCTATGTTAAAGGTGATGAAGTGTTAGTGCGTCATAAACCGCGCATTGGTAATGGTGAACCGCTGTTTGATCCGGAATTAGATCCGAATGTGGTTCATATTCGCTTAACTCCAGGCCTGTCTCCGGAAGTTCTGCGTGCCGTTGCACGTGCCACCGATGGTATTGTTTTAGAAGGCTATGGCGCAGGTGGCATTCCGTATCGCGGTCGTAATCTGCTGGAAGTTGTGTCAGAAACCGCACGCGAAAAGCCAGTTGTGATGACCACCCAGGCCCTGTATGGCGGTGTGGATCTGACCCGCTATGAAGTTGGTCGTCGTGCCTTAGAAGCAGGCGTGATTCCGGCAGGCGATATGACCAAAGAAGCCACCTTAACCAAACTGATGTGGGCCCTGGGTCATACCCGCGATCTGGAAGAAATTCGCAAAATTATGGAACGCAATATTGCAGGCGAAATTACCGGCTCT；
SEQ ID No.2：ATGCCTCTCAAGCCGATTCTCCTGTCTGCCCTGGCCAGTCTCGCCTCGGCCTCTCCGCTGCTCTACTCGCGGACCACCAATGAAACCTTCGTCTTCACCAATGCCAATGGCCTCAACTTCACCCAGATGAACACCACCCTGCCGAACGTGACCATTTTCGCAACGGGTAGGTGGACCGAGTATACCTCAGGTAGTGCGACCGATAGTTAACCGCAACTCACAGGTGGTACCATCGCGGGCTCCGATTCCAGCTCAACCGCCACGACCGGCTACACCTCCGGAGCAGTCGGGGTCCTGTCCCTCATCGATGCGGTGCCATCCATGCTGGATGTGGCCAATGTTGCCGGCGTCCAGGTGGCCAACGTGGGAAGCGAGGATATCACCTCTGACATCCTGATTTCCATGTCCAAGAAGCTGAACCGCGTTGTATGTGAGGACCCGACCATGGCCGGTGCTGTCATCACCCACGGCACCGACACCCTCGAGGAGACTGCCTTCTTCCTGGACGCCACTGTCAACTGTGGCAAGCCAATTGTCATCGTGGGTGCCATGCGCCCATCCACGGCCATCTCAGCTGACGGGCCCTTCAATCTGCTCGAAGCCGTGACGGTGGCTGCCTCCACGTCGGCGCGCGATCGCGGTGCCATGGTGGTCATGAACGATCGCATTGCCTCGGCCTACTATGTGACCAAGACCAATGCCAACACTATGGACACCTTCAAGGCCATGGAGATGGGCTACCTTGGCGAGATGATCTCCAACACCCCTTTCTTCTTCTACCCGCCCGTCAAGCCAACCGGTAAGGTGGCCTTTGACATCACCAACGTGACTGAGATCCCCCGTGTGGACATTCTGTTTTCTTATGAGGACATGCACAACGACACCCTCTACAACGCCATCTCCAGTGGTGCCCAGGGAATTGTGGTGAGTGTGATTTCCTTGATCTCTCTCTATAAAACTTGGAATGGACGCTGATGAGAATAGATTGCCGGGGCTGGTGCTGGAGGCGTCACAACCTCCTTCAATGAGGCTATCGAGGATGTCATCAACCGTTTGGAGATCCCTGTCGTGCAGAGTATGCGCACAGTCAATGGGGAAGTGCCACTGTCAGACGTGAGCAGCGACACCGCCACCCACATCGCCAGTGGATACCTAAACCCGCAGAAGTCCCGCATTCTGTTGGGATTGCTGCTATCCCAGGGAAAGAATATCACCGAAATCGCTGACGTGTTTGCTCTGGGCACGGATGCGTAG；
SEQ ID No.3：ATGGGTGTCAATTTCAAAGTTCTTGCCCTGTCGGCCTTAGCTACTATTAGCCATGCTTCGCCTCTCCTATATCCTCGAGCCACAGACTCGAACGTCACCTATGTGTTCACCAACCCCAATGGCCTGAACTTTACTCAGATGAACACCACCCTGCCAAACGTCACTATCTTCGCGACAGGTACACACTACCCTTAGCCTACTCGCACAAGCCCCCCTTCATCACAACAAAAACTAACAAGAAAAAACAGGCGGCACAATCGCGGGCTCCAGCGCCGACAACACCGCAACAACAGGTTACAAAGCCGGTGCAGTCGGCATCCAGACACTGATCGACGCGGTCCCGGAAATGCTAAACGTTGCCAACGTCGCTGGCGTGCAAGTAACCAATGTCGGCAGCCCAGACATCACCTCCGACATTCTCCTGCGTCTCTCCAAACAGATCAACGAGGTGGTCTGCAACGACCCCACCATGGCCGGTGCAGTGGTCACCCACGGCACCGACACGCTCGAAGAATCCGCCTTCTTCCTCGACGCCACGGTCAACTGTCGCAAGCCCGTGGTCATCGTCGGCGCCATGCGCCCTTCAACCGCCATCTCGGCTGACGGCCCCCTCAACCTCCTGCAATCCGTCACCGTCGCCACGAGCCCCAAGGCCCGAGACCGCGGCGCCCTGATTGTCATGAACGACCGCATCGTATCCGCCTTCTACGCCTCCAAGACGAACGCCAACACCGTCGATACATTCAAGGCCATCGAAATGGGTAACCTGGGCGAGGTCGTCTCCAACAAACCCTACTTCTTCTACCCCCCAGTCAAGCCAACAGGCAAGACGGAAGTAGATATCCGGAACATCACCTCCATCCCCAGAGTCGACATCCTCTACTCATACGAAGACATGCACAATGACACCCTTTACTCCGCCATCGACAACGGCGCAAAGGGCATCGTTGTAAGTCTCGTCCACTCTCAATATGAACCCCATCTATCAAAAACACCCCCAGAACCCCTCCACCAAAATACTAATCCAAATAAAAAAACAGATCGCCGGCTCCGGCTCCGGCTCCGTCTCCACCCCCTTCAGCGCCGCCATGGAAGACATCACAACCAAACACAACATCCCCATCGTAGCCAGCACGCGCACCGGAAACGGGGAGGTGCCGTCCTCCGCCGAGTCGAGCCAGATCGCAAGCGGGTATTTGAACCCCGCAAAGTCACGCGTTTTGCTTGGCTTGTTGCTTGCCCAGGGGAAGAGTATTGAGGAAATGAGGGCGGTTTTTGAGCGGATTGGGGTTGCTTGA；
SEQ ID No.4：ATGACTGTACCCGCCCAGAATAAGAGCCATGTTATCATTGACAGAAACGGCATCATCGAAAATAGACACCTGGTCCACGCCGCGATAGTCGACTCAGCCGGAAAGGTTCTCTTCTCTCTCGGCGATCCATCGCGAGTGACACTCCTCCGATCTGCAGCCAAACCAGTTCAAGCACTAGCGGTTGCGGAGACTGGCGCCCTCGAGCATTTTGCTTTTGACGATGGTGATCTGGCTCTTATGTGTGCCTCGCATAGTAGCGAGGACCGGCATATCGAGCGGGCTCGACGAATGCTCGCTAAGTCGCAGCATACGGAGGACCAATTGCGGTGTGGTGGCCATCCTTCAATAAATCCGGCGATCAACAACGAATGGATCAAGCGGGACTTCGAACCGACGGCAGTCTATAACAATTGTTCCGGCAAACATGCGGGTATGCTGGCTGGGGCCAAAGCTATTGGTGCCTCCGCAGACAATTACCATCTCCTGACGCATCCAATCCAGGCCAGCGTCAAGAGAGTCGTGGAAGAAGTAGCTGGGTTAAGCGAGGAAGAGGTTCAGTGGGGGCTTGACGGCTGCAATATGCCGGCGCCTGCATTTCCCCTTTCTCACTTGGCCAAGGTATATGCTTCATTTGCCCAGGCATCAGACGCAGTAGCCAGTGGCAGTCCAGTCACGCCCAGGGTGCAGCTGATGGGAAAGATCTTCAATGCCATGGCTCAAATGCCCGAGATGGTTGGCGGCGAGGGTCGCTTCTGTACTATCTTAATGAGTGCTCTTGGTGGCTCTACCATCGGCAAGGTCGGTGCAGATGGCTGCTACGCCGTTGGAATGCGCGAGTCTGAACAGACGAAGGGAATTGGAGCAGAGGGCGCAGTTGGGATCGCCGTCAAGATTGAAGACGGCAATCTAGGTATACTGTATGCCGCGGTTGCTGAAATATTAGCGCAGCTGAAGCTCTGTAATCGGGAGGACATGCAGACTCTGGAGACATATCATAATCCGAAGATGAAGAACACCATGGGAATTGTCACTGGTACTGCGAGGTTTGACTTTGAGTTTCTTTGCACCAGGGGGGCAATTTTACTCTAA。
the Trichoderma reesei expression plasmid LML2.1 was digested with the DNA restriction enzymes PacI/XbaI. The promoter, asparaginase sequence was ligated into the PacI/XbaI site of the LML2.1 plasmid using either seamless cloning or DNA ligase to construct an intermediate plasmid. After the intermediate plasmid was successfully constructed, the intermediate plasmid was digested with SwaI, and the corresponding terminator sequence was ligated to the SwaI site to obtain the corresponding recombinant expression plasmid (see FIG. 1A). The expression vector used here mainly provides the function of Agrobacterium junction transfer site, so, in addition to LML2.1, any one of the following Agrobacterium binary vectors can be used: PZP100, pPZP201B, pPZP201BK, pBI121, pCAMBIA, pPK2, but not limited to these vectors.
2. Knock-out of cellulase, xylanase, and protease genes
Firstly, DNA sequences (downstream sequences) with the lengths of about 600-1000 bp upstream of an initiation codon ATG and about 600-1000 bp downstream of a termination codon TAA (or TAG and TGA) of Trichoderma reesei EGL1, XYN1, BGL1, PEP1, SLP1, GAP1, TRE81070, TRE120998 and TRE123234 encoding genes are respectively amplified by PCR. The nucleotide sequence of the primer is shown in a sequence table SEQ ID No. 41-76:
primer for construction of XYN1 knockout plasmid:
SEQ ID No.41：ATTACGAATTCTTAATTAAGGAGTCTAAGGCACACCAGTA；
SEQ ID No.42：CATTATACGAAGTTATTCTAGACGGCTGAACCTAGTCATCCT；
SEQ ID No.43：ATTACGAATTCTTAATTAATGACTAGGTTCAGCCGTTAAAG；
SEQ ID No.44：CATTATACGAAGTTATTCTAGATGATTATTTGTGCGTGTTTTCC；
primers for constructing BGL1 knockout plasmids:
SEQ ID No.45：ATTACGAATTCTTAATTAATGCTGTTACATTCAAGGTTGGA；
SEQ ID No.46：CATTATACGAAGTTATTCTAGATGGTCAAGACTGGTAGGAAGAA；
SEQ ID No.47：ACTAGTGAGCTCATTTCAGGATAGGCATCAGAGCAGTA；
SEQ ID No.48：AGTGCCAAGCTTATTTGAAGCATTCGGCGGTTGTT；
EGL1 knockout plasmid construction primers:
SEQ ID No.49：ATTACGAATTCTTAATTAAAAATCTGTCCTCGTCCTTTGTC；
SEQ ID No.50：CATTATACGAAGTTATTCTAGAGCATCCAGCGGTAGTTCCA；
SEQ ID No.51：ACTAGTGAGCTCATTTCGTCGTCTTCTCCAACATCC；
SEQ ID No.52：AGTGCCAAGCTTATTTGGTGCTTGACATGGCTTGA；
PEP1 knock-out plasmid construction primers:
SEQ ID No.53：ATTACGAATTCTTAATTAAAGAAGCAGAAAGGCTGGTGA；
SEQ ID No.54：CATTATACGAAGTTATTCTAGAATGGGATCAATGATGGAACG；
SEQ ID No.55：ACTAGTGAGCTCATTTCTTGAATATCGGAGAAGGTTGC；
SEQ ID No.56：AGTGCCAAGCTTATTTAAGCGTTGAGCAGGAAGACC；
SLP1 knock-out plasmid construction primers:
SEQ ID No.57：ATTACGAATTCTTAATTAATTCAAGCCAGGGAGAAAGAA；
SEQ ID No.58：CATTATACGAAGTTATTCTAGACACCATCAAGTATGCGTAGGG；
SEQ ID No.59：ACTAGTGAGCTCATTTACACTCTATCCCGCTTGTCTGA；
SEQ ID No.60：AGTGCCAAGCTTATTTGCTGGTACGGAATGGTATTCTTC；
primer for constructing GAP1 knockout plasmid:
SEQ ID No.61：ATTACGAATTCTTAATTAAAAATGGCATACCAGCTTTCA；
SEQ ID No.62：CATTATACGAAGTTATTCTAGAGCTTCGGACTCAACCAGGAC；
SEQ ID No.63：ACTAGTGAGCTCATTTTCAAGAAGAGGCAGAGGGTA；
SEQ ID No.64：AGTGCCAAGCTTATTTACGTGGAGAAATCGGAATAT；
TRE81070 knockout plasmid construction primers:
SEQ ID No.65：ATTACGAATTCTTAATTAAATCATGTTCTTTGTTGCGGTAG；
SEQ ID No.66：CATTATACGAAGTTATTCTAGACGGGAGGCTGGAGTATTG；
SEQ ID No.67：ACTAGTGAGCTCATTTTAAAGTGGTGGCGAGGTG；
SEQ ID No.68：AGTGCCAAGCTTATTTGGCGCAAGAAGCTGAAAT；
TRE120998 knockout plasmid construction primer:
SEQ ID No.69：ATTACGAATTCTTAATTAAGTCGTGCCTGAGGTCCAT；
SEQ ID No.70：CATTATACGAAGTTATTCTAGATCTGACGGTGAGGTTGCA；
SEQ ID No.71：ACTAGTGAGCTCATTTGACTGGCGGTCATTACGT；
SEQ ID No.72：AGTGCCAAGCTTATTTGCTCATCAATACCCGACTTT；
TRE123234 knockout plasmid construction primer:
SEQ ID No.73：ATTACGAATTCTTAATTAAAAGTCCTCCCGCACATCA；
SEQ ID No.74：CATTATACGAAGTTATTCTAGATGGCACGAAGAAGGTACTGA；
SEQ ID No.75：ACTAGTGAGCTCATTTCAGACTATGGTGGTTTGGG；
SEQ ID No.76：AGTGCCAAGCTTATTTAGAGCGTAGCGATGTGGT。
the Trichoderma reesei-expressing LML2.1 plasmid was digested with DNA restriction enzymes PacI/XbaI. The upstream sequence was ligated to the PacI/XbaI site of the LML2.1 plasmid using either seamless cloning or DNA ligase to construct an intermediate plasmid. After the intermediate plasmid is successfully constructed, the intermediate plasmid is digested by using a DNA restriction enzyme SwaI, and the corresponding downstream sequence is connected to the SwaI site of the intermediate plasmid to obtain the corresponding knockout plasmid (as shown in FIG. 1B). The expression vector used here mainly provides the function of Agrobacterium junction transfer site, so, in addition to LML2.1, any one of the following Agrobacterium binary vectors can be used: PZP100, pPZP201B, pPZP201BK, pBI121, pCAMBIA, pPK2, but not limited to these vectors.
3. Trichoderma reesei gene engineering bacterium construction for expressing asparaginase
The trichoderma reesei genetic engineering bacteria for expressing asparaginase are obtained by transforming trichoderma reesei through agrobacterium tumefaciens carrying an asparaginase expression vector in a combined transfer manner, and then recovering resistance markers; the preparation method comprises the following specific steps:
firstly, carrying a recombinant protein expression vector to electrically transform agrobacterium tumefaciens, and screening out positive agrobacterium tumefaciens by using kanamycin (50 mug/mL); transferring positive agrobacterium tumefaciens cultured by LB liquid to liquid IM, diluting the spores of trichoderma reesei to 10 5 ～10 8 Spore suspensions at gradient concentrations ranging in counts/mL. The suspension of Agrobacterium tumefaciens and Trichoderma reesei spores in the IM was mixed in equal volume and spotted onto a nitrocellulose membrane of a solid Industion Medium Plates (sterilized at 115 ℃ and then attached to the upper surface of the solid plate by aseptic technique on a clean bench). After incubating at 25 ℃ for 48h, the membrane was transferred by ultraclean bench sterile technique to PDA medium supplemented with 20g/L glucose, hygromycin (150. Mu.g/mL) and cefuroxime (400. Mu.g/mL) and the plate was incubated at 28 ℃ for 5-7 days, spores were collected, diluted to grow out in a single colony form on the plate, PDA plates (containing 20g/L glucose, 150. Mu.g/mL hygromycin and 150. Mu.g/mL) were coated, incubated at 28 ℃ for 48h,single clones were picked on PDA well plates (containing 20g/L glucose, 150. Mu.g/mL hygromycin and 150. Mu.g/mL cefuroxime), cultured at 28 ℃ for 5-7 days, spores were collected, and mycelia were scraped to extract the genome, verified by PCR and sent for sequencing.
Inoculating the obtained positive recombinant Trichoderma reesei strain to a Martin liquid (containing 20g/L of xylose) culture medium to induce resistance gene deletion, performing shake culture at 28 ℃ and 200rpm for 48h, then picking a small amount of hyphae to a PDA (containing 20g/L of glucose) plate, performing culture at 28 ℃ for 5-7 days, collecting spores, diluting the spores to a concentration on the plate to grow out in a single clone mode, coating the PDA plate (containing 20g/L of glucose or xylose), and performing culture at 28 ℃ for 48h. Single clones were picked up on PDA plates (containing 20g/L glucose or xylose) and cultured at 28 ℃ for 24h. Picking small pieces of agar with hyphae in the well plate to PDA well plate (containing 20g/L glucose, 150. Mu.g/ml hygromycin and 150. Mu.g/ml cefamycin), and verifying whether the resistance deletion is successful. After culturing at 28 ℃ for 5-7 days, successfully collected spores with resistance deletion are selected.
Example 2 fermentative production
1. Engineering bacteria express asparaginase in shake flask
Inoculating the obtained positive recombinant Trichoderma reesei strain into a Martin liquid (containing 20g/L of lactose) culture medium, performing shake culture at 28 deg.C and 200rpm for 48h, collecting the fermentation liquid 400 μ L, centrifuging at 12500 rpm for 5min, and collecting the supernatant of the fermentation liquid. The supernatant of the fermentation liquid of the Trichoderma reesei engineering strain is used as a crude enzyme liquid, and protein denaturation electrophoresis is carried out to analyze the purity of asparaginase (figure 2). As shown in fig. 2: band one-marker for protein molecular weight; strip II-original Trichoderma reesei fermentation liquor; after the third strip, namely the first round of transformation, the cbh1 promoter is used for expressing asparaginase fermentation liquor; after the fourth strip-the second round of transformation, the promoter cbh1 and cbh2 are used for expressing asparaginase fermentation liquor; after the fifth strip, the third and the fourth strips are transformed, the promoters cbh1, cbh2, egl2 and egl3 are used for expressing asparaginase fermentation liquor; after the six-band is transformed in the fifth and sixth rounds, the cbh1, cbh2, egl3, xyn2 and xyn3 promoters are used for expressing asparaginase fermentation liquor; seventhly, knocking out xyn1 after the seventh round of transformation; the eighth stripe, after the eighth round of transformation, egl1 and bgl1 are knocked out, and the purity of asparaginase is improved to 80 percent; after the ninth belt is transformed, the protease pep1 is knocked out, the stability of asparaginase is obviously improved, the crude enzyme solution can be stably stored in a refrigerator at 4 ℃ for 90 days, the enzyme activity is reduced by within 10 percent, and the effect of knocking out strains by other five proteases such as SLP1, GAP1, TRE81070, TRE120998 or TRE123234 is obviously better; the detection of the activity of the extracellular protease shows that after the pep1 of the protease is knocked out, the activity of the extracellular protease is reduced by 60 percent, and the effect of knocking out strains by other five proteases such as SLP1, GAP1, TRE81070, TRE120998 or TRE123234 is obviously better than that of knocking out strains by other five proteases.
2. Production of asparaginase by engineering bacteria in fermentation tank
The obtained positive recombinant Trichoderma reesei strain is inoculated in a Martin liquid (containing 20g/L of glucose), after shaking culture at 28 ℃ and 200rpm for 48h, 1L of fermentation liquid is taken and inoculated in a fermentation tank with 9L of fresh culture medium. The glucose concentration is maintained at 0.1-0.25% and the dissolved oxygen is maintained at 20% by feeding sophorose-glucose mixture (total sugar concentration is about 500g/L, wherein the content of sophorose is 5-20%). 30% sodium glutamate solution is used as nitrogen source feeding, and the feeding speed is 30mL/h.
3. Determination of asparaginase Activity
(1) Solution preparation
A:0.1M citric acid: 21.014g of citric acid was weighed, dissolved in purified water and made up to 1L.
B:0.2M Na 2 HPO 4 : weighing Na 2 HPO 4 28.39g, dissolved in purified water and made to 1L.
C: disodium phosphate-citric acid buffer: 11.5mL of (B) +8.5mL of (A).
D:189mM L-asparagine solution: 0.25g of L-asparagine was weighed out, dissolved in purified water and made to volume of 10mL.
E:1.5M trichloroacetic acid (TCA): 1.225g of trichloroacetic acid was weighed out, dissolved in purified water and brought to volume of 5mL.
F：6mM(NH 4 ) 2 SO 4 : weigh 0.7928 g (NH) 4 ) 2 SO 4 Dissolving with purified water and making the volume to 1L.
G: potassium sodium tartrate solution: heating 50 g of sodium potassium tartrate to dissolve in water, adding 1.25mL of Nessler reagent, stirring uniformly, diluting to 100mL, standing for 1 hour, centrifuging, and taking supernatant.
(2) Preparation of standard curve
After standing for 3 minutes, 50. Mu.L of the above mixture was mixed with 100. Mu.L of water and 50. Mu.L of Nessler reagent, and the absorbance OD was measured at 436nm after 1 minute. According to OD-NH 3 The amount of substance was plotted, and bad spots were excluded, and it was found that: each OD value represents 0.3056. Mu. Mol NH 3 。
(3) Asparaginase activity assay
Asparaginase units (ASNU) are defined as the amount of enzyme required to produce 1.0. Mu. Mol ammonia at 37 ℃ and pH 5.4,1 min.
Incubating the sample to be tested for 30min at 37 ℃; the control was incubated at 37 ℃ for 0min.
The reaction was stopped by placing on ice and adding 50. Mu.L of E (1.5M trichloroacetic acid).
The samples were mixed and centrifuged at 20000g for 2 minutes.
mu.L of the supernatant was taken, mixed with 100. Mu.L of water and 50. Mu.L of Nessler reagent, and the absorbance was measured at 436nm after 1 minute. The OD of the control (Δ OD) was subtracted from the OD of the sample.
4. Preparation of asparaginase
Filtering the culture of the engineering bacteria by a plate frame to obtain crude enzyme liquid containing asparaginase, and adding a universal protective agent to obtain liquid asparaginase; and (3) drying the crude enzyme liquid in a spray drying tower, adding a universal protective agent, and preparing into solid asparaginase powder. The activity of the liquid crude enzyme liquid reaches 500-2000U/mL, and the activity of the solid enzyme powder can be stabilized at 1000U/g.
EXAMPLE 3 use of the food processing procedure
1. Preparation of acrylamide standard solution and standard curve preparation
Accurately weighing 0.100g of acrylamide standard substance, dissolving the acrylamide standard substance by using methanol and diluting the acrylamide standard substance to 250mL to obtain standard solution of acrylamide with the concentration of 0.4mg/mL, sealing the standard solution and storing the standard solution in a refrigerator (0-4 ℃). During measurement, a proper amount of acrylamide standard solution is respectively diluted by methanol to prepare a series of standard solutions with the concentrations of 0.25, 0.50, 1.0, 2.0, 4.0, 5.0 and 6.0 mu g/mL.
Acrylamide has a maximum absorption peak at 210nm, the data is measured and recorded at the wavelength of 210nm, and a standard curve is drawn by taking the absorbance as a Y axis and the content of the acrylamide as an X axis. In the concentration range of 0.25-6 mug/L, the absorbance of the acrylamide and the concentration have good linear relation. Since the acrylamide aqueous solution is not very stable, the preparation is measured in general experiments. Zeroing with methanol.
Standard curve: y =0.0806X-0.0304 2 ＝0.9991。
2. Baking application experiments
1. Flour variety: ningxia Saibei snow high-gluten flour
2. The experimental method comprises the following steps: one-time fermentation method
Preparing materials: 400g of flour; 5.5g of yeast; 60g of sugar; 10g of salt; 32g of butter; 228g of water and 0-300ppm of asparaginase enzyme powder.
3. Fermentation temperature: 38 ℃; humidity: 80 percent; oven temperature: 180 ℃; baking time: and 20min.
3. Potato chip application experiment
The experimental method comprises the following steps: asparaginase was added to a phosphate solution (0.1M, pH 6.0) at 50 ℃ to a final concentration of 50U/mL. The blanched tuberous food is dipped into phosphate solution containing asparaginase, incubated for 0,1 or 3min, and then subjected to a subsequent frying treatment.
4. Extraction of acrylamide from samples
1. Placing a proper amount of sample to be detected in a culture dish, drying water in an oven at 85 ℃ for 2.5h, and fully grinding.
2. 2.00g of dried sample powder is weighed into a 7mL centrifuge tube, 5mL of methanol is added, the mixture is shaken for 5min at room temperature, and centrifuged for 15min at 8000r/min. Collect 2mL of supernatant from round 1.
3. Then 2mL of methanol was added to the precipitate, and the mixture was shaken at room temperature for 5min, and centrifuged at 8000r/min for 15min, and 2mL of the supernatant of the 2 nd time was collected. This operation was repeated once, and a total of 6mL of the supernatant was collected.
4. The C18 solid phase extraction cartridge was activated by passing 1mL of methanol through it and then 1mL of deionized water through it.
5. The supernatant was then 5mL of the intermediate clear solution was aspirated by a syringe with a needle while avoiding the oil layer, and the filtrate was collected through a 0.45 μm PVDF filter.
6. Then 5mL of the filtrate was passed through a pre-activated C18 solid phase extraction cartridge, the first 0.5mL of the filtrate was discarded, the remaining effluent was collected and the effluent sample was diluted 20-fold for assay.
7. 3mL was taken for analytical determination, i.e.absorbance was measured at a wavelength of 210nm, and methanol was used as a blank.
(5) Calculation of acrylamide content
Acrylamide = N (K μ g/L \65121; 9 mL)/2.0 g
＝90K(μg/kg)
Wherein N is the dilution factor (20 times); k is the acrylamide concentration (μ g/L) found on the standard curve;
9mL is the sample dissolution volume; 2.0g is the test amount of the sample.
The measurement results were as follows:
| sequence of | Sample (enzyme addition/treatment time) | Acrylamide content (μ g/kg) |
| 1 | Bread (0) | 365.7 |
| 2 | Bread (50 ppm) | 279.7 |
| 3 | Bread (100 ppm) | 271.7 |
| 4 | Bread (300 ppm) | 210.2 |
| 5 | Potato chips (0 min) | 785.1 |
| 6 | Potato chips (1 min) | 547.2 |
| 7 | Potato chips (3 min) | 421.8 |
The above-described embodiments are only intended to illustrate the preferred embodiments of the present invention, and not to limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Sequence listing
<110> university of east China's college of science
NINGXIA SUNSON INDUSTRY GROUP Co.,Ltd.
Cangzhou Xiasheng Enzyme Biotechnology Co.,Ltd.
<120> recombinant trichoderma reesei for producing asparaginase and construction method and application thereof
<160> 76
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1035
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgcctctca agccgattct cctgtctgcc ctggccagtc tcgcctcggc catgaaactg 60
ttagtgctgg gcaccggcgg caccattgca agcgccaaaa ccgaaatggg ctataaagca 120
gcactgtcag cagatgatat tttacagtta gcaggtattc gtcgcgaaga tggcgccaaa 180
attgaaaccc gtgatattct gaatttagat agtaccttaa ttcagccgga agattgggtg 240
accattggtc gcgccgtttt tgaagccttt gatgaatatg atggcattgt tattacacat 300
ggcacagata ccttagccta tacctctagt gcactgagct ttatgattcg taatccgccg 360
attccggttg ttctgaccgg ctcaatgctg ccgattaccg aaccgaatag cgatgccccg 420
cgcaatctgc gtaccgccct gacctttgca cgtaaaggct ttccgggcat ttatgtggcc 480
tttatggata aaattatgct gggaactcgt gtgtctaaag ttcatagctt aggtctgaat 540
gcatttcaga gtattaatta tccggatatt gcctatgtta aaggtgatga agtgttagtg 600
cgtcataaac cgcgcattgg taatggtgaa ccgctgtttg atccggaatt agatccgaat 660
gtggttcata ttcgcttaac tccaggcctg tctccggaag ttctgcgtgc cgttgcacgt 720
gccaccgatg gtattgtttt agaaggctat ggcgcaggtg gcattccgta tcgcggtcgt 780
aatctgctgg aagttgtgtc agaaaccgca cgcgaaaagc cagttgtgat gaccacccag 840
gccctgtatg gcggtgtgga tctgacccgc tatgaagttg gtcgtcgtgc cttagaagca 900
ggcgtgattc cggcaggcga tatgaccaaa gaagccacct taaccaaact gatgtgggcc 960
ctgggtcata cccgcgatct ggaagaaatt cgcaaaatta tggaacgcaa tattgcaggc 1020
gaaattaccg gctct 1035
<210> 2
<211> 1254
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgcctctca agccgattct cctgtctgcc ctggccagtc tcgcctcggc ctctccgctg 60
ctctactcgc ggaccaccaa tgaaaccttc gtcttcacca atgccaatgg cctcaacttc 120
acccagatga acaccaccct gccgaacgtg accattttcg caacgggtag gtggaccgag 180
tatacctcag gtagtgcgac cgatagttaa ccgcaactca caggtggtac catcgcgggc 240
tccgattcca gctcaaccgc cacgaccggc tacacctccg gagcagtcgg ggtcctgtcc 300
ctcatcgatg cggtgccatc catgctggat gtggccaatg ttgccggcgt ccaggtggcc 360
aacgtgggaa gcgaggatat cacctctgac atcctgattt ccatgtccaa gaagctgaac 420
cgcgttgtat gtgaggaccc gaccatggcc ggtgctgtca tcacccacgg caccgacacc 480
ctcgaggaga ctgccttctt cctggacgcc actgtcaact gtggcaagcc aattgtcatc 540
gtgggtgcca tgcgcccatc cacggccatc tcagctgacg ggcccttcaa tctgctcgaa 600
gccgtgacgg tggctgcctc cacgtcggcg cgcgatcgcg gtgccatggt ggtcatgaac 660
gatcgcattg cctcggccta ctatgtgacc aagaccaatg ccaacactat ggacaccttc 720
aaggccatgg agatgggcta ccttggcgag atgatctcca acaccccttt cttcttctac 780
ccgcccgtca agccaaccgg taaggtggcc tttgacatca ccaacgtgac tgagatcccc 840
cgtgtggaca ttctgttttc ttatgaggac atgcacaacg acaccctcta caacgccatc 900
tccagtggtg cccagggaat tgtggtgagt gtgatttcct tgatctctct ctataaaact 960
tggaatggac gctgatgaga atagattgcc ggggctggtg ctggaggcgt cacaacctcc 1020
ttcaatgagg ctatcgagga tgtcatcaac cgtttggaga tccctgtcgt gcagagtatg 1080
cgcacagtca atggggaagt gccactgtca gacgtgagca gcgacaccgc cacccacatc 1140
gccagtggat acctaaaccc gcagaagtcc cgcattctgt tgggattgct gctatcccag 1200
ggaaagaata tcaccgaaat cgctgacgtg tttgctctgg gcacggatgc gtag 1254
<210> 3
<211> 1298
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgggtgtca atttcaaagt tcttgccctg tcggccttag ctactattag ccatgcttcg 60
cctctcctat atcctcgagc cacagactcg aacgtcacct atgtgttcac caaccccaat 120
ggcctgaact ttactcagat gaacaccacc ctgccaaacg tcactatctt cgcgacaggt 180
acacactacc cttagcctac tcgcacaagc cccccttcat cacaacaaaa actaacaaga 240
aaaaacaggc ggcacaatcg cgggctccag cgccgacaac accgcaacaa caggttacaa 300
agccggtgca gtcggcatcc agacactgat cgacgcggtc ccggaaatgc taaacgttgc 360
caacgtcgct ggcgtgcaag taaccaatgt cggcagccca gacatcacct ccgacattct 420
cctgcgtctc tccaaacaga tcaacgaggt ggtctgcaac gaccccacca tggccggtgc 480
agtggtcacc cacggcaccg acacgctcga agaatccgcc ttcttcctcg acgccacggt 540
caactgtcgc aagcccgtgg tcatcgtcgg cgccatgcgc ccttcaaccg ccatctcggc 600
tgacggcccc ctcaacctcc tgcaatccgt caccgtcgcc acgagcccca aggcccgaga 660
ccgcggcgcc ctgattgtca tgaacgaccg catcgtatcc gccttctacg cctccaagac 720
gaacgccaac accgtcgata cattcaaggc catcgaaatg ggtaacctgg gcgaggtcgt 780
ctccaacaaa ccctacttct tctacccccc agtcaagcca acaggcaaga cggaagtaga 840
tatccggaac atcacctcca tccccagagt cgacatcctc tactcatacg aagacatgca 900
caatgacacc ctttactccg ccatcgacaa cggcgcaaag ggcatcgttg taagtctcgt 960
ccactctcaa tatgaacccc atctatcaaa aacaccccca gaacccctcc accaaaatac 1020
taatccaaat aaaaaaacag atcgccggct ccggctccgg ctccgtctcc acccccttca 1080
gcgccgccat ggaagacatc acaaccaaac acaacatccc catcgtagcc agcacgcgca 1140
ccggaaacgg ggaggtgccg tcctccgccg agtcgagcca gatcgcaagc gggtatttga 1200
accccgcaaa gtcacgcgtt ttgcttggct tgttgcttgc ccaggggaag agtattgagg 1260
aaatgagggc ggtttttgag cggattgggg ttgcttga 1298
<210> 4
<211> 1089
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atgactgtac ccgcccagaa taagagccat gttatcattg acagaaacgg catcatcgaa 60
aatagacacc tggtccacgc cgcgatagtc gactcagccg gaaaggttct cttctctctc 120
ggcgatccat cgcgagtgac actcctccga tctgcagcca aaccagttca agcactagcg 180
gttgcggaga ctggcgccct cgagcatttt gcttttgacg atggtgatct ggctcttatg 240
tgtgcctcgc atagtagcga ggaccggcat atcgagcggg ctcgacgaat gctcgctaag 300
tcgcagcata cggaggacca attgcggtgt ggtggccatc cttcaataaa tccggcgatc 360
aacaacgaat ggatcaagcg ggacttcgaa ccgacggcag tctataacaa ttgttccggc 420
aaacatgcgg gtatgctggc tggggccaaa gctattggtg cctccgcaga caattaccat 480
ctcctgacgc atccaatcca ggccagcgtc aagagagtcg tggaagaagt agctgggtta 540
agcgaggaag aggttcagtg ggggcttgac ggctgcaata tgccggcgcc tgcatttccc 600
ctttctcact tggccaaggt atatgcttca tttgcccagg catcagacgc agtagccagt 660
ggcagtccag tcacgcccag ggtgcagctg atgggaaaga tcttcaatgc catggctcaa 720
atgcccgaga tggttggcgg cgagggtcgc ttctgtacta tcttaatgag tgctcttggt 780
ggctctacca tcggcaaggt cggtgcagat ggctgctacg ccgttggaat gcgcgagtct 840
gaacagacga agggaattgg agcagagggc gcagttggga tcgccgtcaa gattgaagac 900
ggcaatctag gtatactgta tgccgcggtt gctgaaatat tagcgcagct gaagctctgt 960
aatcgggagg acatgcagac tctggagaca tatcataatc cgaagatgaa gaacaccatg 1020
ggaattgtca ctggtactgc gaggtttgac tttgagtttc tttgcaccag gggggcaatt 1080
ttactctaa 1089
<210> 5
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
attacgaatt cttaattaac ctgtaaagcc gcaatgcagc 40
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
catgatgcgc agtccgcggt 20
<210> 7
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ggactgcgca tcatgcctct caagccgatt ctcc 34
<210> 8
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cattatacga agttattcta gactacgcat ccgtgcccag ag 42
<210> 9
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
actagtgagc tcatttagct ccgtggcgaa agcct 35
<210> 10
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
agtgccaagc ttatttcatc gtaaccgaga atccagagct g 41
<210> 11
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
attacgaatt cttaattacg agaatggtga ggactgagat aa 42
<210> 12
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
catggtgcaa tacacagagg gt 22
<210> 13
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gtgtattgca ccatgcctct caagccgatt ctcc 34
<210> 14
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cattatacga agttattcta gactacgcat ccgtgcccag ag 42
<210> 15
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
actagtgagc tcatttgctt tcgtgaccgg gcttcaaa 38
<210> 16
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
agtgccaagc ttatttatgc gatgcggctc aagacttc 38
<210> 17
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
attacgaatt cttaattaac gcaagtgcct gagtgaagat 40
<210> 18
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
cattgtcgat gacggggaga tattat 26
<210> 19
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ccgtcatcga caatgcctct caagccgatt ctcc 34
<210> 20
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
cattatacga agttattcta gactacgcat ccgtgcccag ag 42
<210> 21
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
actagtgagc tcatttcact ctgagctgaa tgcagaagc 39
<210> 22
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
agtgccaagc ttatttcacc aaggcaactc gtccag 36
<210> 23
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
attacgaatt cttaattaag tgaggagatt gtggctacga 40
<210> 24
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
cattgcgacg ctatgcttgt g 21
<210> 25
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
catagcgtcg caatgcctct caagccgatt ctcc 34
<210> 26
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
cattatacga agttattcta gactacgcat ccgtgcccag ag 42
<210> 27
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
actagtgagc tcatttaacc tggaaacgtg agatgtg 37
<210> 28
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
agtgccaagc ttatttccga gtcctgagag ttgtagt 37
<210> 29
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
attacgaatt cttaattaat tgtgatgctg ctgctgatg 39
<210> 30
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
catgttgatg tcttcttgct tcag 24
<210> 31
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
gaagacatca acatgcctct caagccgatt ctcc 34
<210> 32
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
cattatacga agttattcta gactacgcat ccgtgcccag ag 42
<210> 33
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
actagtgagc tcatttaggg ggctcttctt ttgtgat 37
<210> 34
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
agtgccaagc ttatttcacg acgacaacac ctggat 36
<210> 35
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
attacgaatt cttaattaat tccgtgcgtg tctgatgg 38
<210> 36
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
catattgtcc gcctcaattg aaac 24
<210> 37
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
gaggcggaca atatgcctct caagccgatt ctcc 34
<210> 38
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
cattatacga agttattcta gactacgcat ccgtgcccag ag 42
<210> 39
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
actagtgagc tcatttctgc tctgctacat gggtatg 37
<210> 40
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
agtgccaagc ttatttggat tcgggcgtat gttgag 36
<210> 41
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
attacgaatt cttaattaag gagtctaagg cacaccagta 40
<210> 42
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
cattatacga agttattcta gacggctgaa cctagtcatc ct 42
<210> 43
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
attacgaatt cttaattaat gactaggttc agccgttaaa g 41
<210> 44
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
cattatacga agttattcta gatgattatt tgtgcgtgtt ttcc 44
<210> 45
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
attacgaatt cttaattaat gctgttacat tcaaggttgg a 41
<210> 46
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
cattatacga agttattcta gatggtcaag actggtagga agaa 44
<210> 47
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
actagtgagc tcatttcagg ataggcatca gagcagta 38
<210> 48
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
agtgccaagc ttatttgaag cattcggcgg ttgtt 35
<210> 49
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
attacgaatt cttaattaaa aatctgtcct cgtcctttgt c 41
<210> 50
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
cattatacga agttattcta gagcatccag cggtagttcc a 41
<210> 51
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
actagtgagc tcatttcgtc gtcttctcca acatcc 36
<210> 52
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
agtgccaagc ttatttggtg cttgacatgg cttga 35
<210> 53
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
attacgaatt cttaattaaa gaagcagaaa ggctggtga 39
<210> 54
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
cattatacga agttattcta gaatgggatc aatgatggaa cg 42
<210> 55
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
actagtgagc tcatttcttg aatatcggag aaggttgc 38
<210> 56
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
agtgccaagc ttatttaagc gttgagcagg aagacc 36
<210> 57
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
attacgaatt cttaattaat tcaagccagg gagaaagaa 39
<210> 58
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
cattatacga agttattcta gacaccatca agtatgcgta ggg 43
<210> 59
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
actagtgagc tcatttacac tctatcccgc ttgtctga 38
<210> 60
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
agtgccaagc ttatttgctg gtacggaatg gtattcttc 39
<210> 61
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
attacgaatt cttaattaaa aatggcatac cagctttca 39
<210> 62
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
cattatacga agttattcta gagcttcgga ctcaaccagg ac 42
<210> 63
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
actagtgagc tcattttcaa gaagaggcag agggta 36
<210> 64
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
agtgccaagc ttatttacgt ggagaaatcg gaatat 36
<210> 65
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
attacgaatt cttaattaaa tcatgttctt tgttgcggta g 41
<210> 66
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
cattatacga agttattcta gacgggaggc tggagtattg 40
<210> 67
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
actagtgagc tcattttaaa gtggtggcga ggtg 34
<210> 68
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
agtgccaagc ttatttggcg caagaagctg aaat 34
<210> 69
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
attacgaatt cttaattaag tcgtgcctga ggtccat 37
<210> 70
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
cattatacga agttattcta gatctgacgg tgaggttgca 40
<210> 71
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
actagtgagc tcatttgact ggcggtcatt acgt 34
<210> 72
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
agtgccaagc ttatttgctc atcaataccc gacttt 36
<210> 73
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
attacgaatt cttaattaaa agtcctcccg cacatca 37
<210> 74
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
cattatacga agttattcta gatggcacga agaaggtact ga 42
<210> 75
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
actagtgagc tcatttcaga ctatggtggt ttggg 35
<210> 76
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
agtgccaagc ttatttagag cgtagcgatg tggt 34
Claims (7)
1. A recombinant Trichoderma reesei for producing asparaginase, wherein the recombinant Trichoderma reesei heterologously expresses asparaginase with a nucleotide sequence shown as SEQ ID No. 2; in the recombinant trichoderma reesei, the original genes of cellulase CBH1, CBH2, EGL3 and xylanase XYN2 and XYN3 in the trichoderma reesei are replaced by the gene of asparaginase, and the original genes of cellulase EGL1, BGL1, xylanase XYN1 and protease PEP1 in the trichoderma reesei are knocked out.
2. A group of plasmids for constructing the recombinant trichoderma reesei of claim 1, which comprises a recombinant expression plasmid and a knockout plasmid, wherein the recombinant expression plasmid sequentially comprises a promoter sequence of a cellulase or xylanase gene required to be replaced, a gene sequence encoding the asparaginase, a recoverable resistance screening marker based on Cre-loxP mediation and a terminator sequence of the cellulase or xylanase gene required to be replaced; the knockout plasmid sequentially comprises an upstream homology arm sequence of a cellulase, xylanase or protease gene to be knocked out, a recoverable resistance screening marker based on Cre-loxP mediation and a downstream homology arm sequence of the cellulase, xylanase or protease gene to be knocked out.
3. The method for constructing recombinant trichoderma reesei according to claim 1, comprising the steps of:
s1, constructing a recombinant expression plasmid as described in claim 2;
s2, transforming the recombinant expression plasmid into trichoderma reesei;
s3, constructing the knockout plasmid as described in claim 2;
and S4, transforming the knockout plasmid into the trichoderma reesei, and screening to obtain the recombinant trichoderma reesei.
4. The construction method according to claim 3, wherein in step S4, trichoderma reesei containing both the recombinant expression plasmid and the knock-out plasmid is screened, transformants successfully undergoing homologous recombination in the genome are screened, and transformants without the resistance marker are screened by a marker recovery technique.
5. Use of the recombinant trichoderma reesei of claim 1 or the plasmid of claim 2 in the production of asparaginase.
6. A method for producing asparaginase, comprising the steps of:
s1, producing asparaginase by using the recombinant Trichoderma reesei of claim 1 or the plasmid of claim 2;
and S2, separating the asparaginase from the system of the S1.
7. The method of claim 6, wherein in step S1, the method of producing asparaginase comprises: inoculating the recombinant trichoderma reesei into a fermentation culture medium, wherein the inoculation amount is 1-20%, and the culture is carried out for 2-12 days at the temperature of 20-37 ℃.
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| CN202110667266.1A CN113388532B (en) | 2021-06-16 | 2021-06-16 | Recombinant trichoderma reesei for producing asparaginase and construction method and application thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105492604A (en) * | 2013-08-23 | 2016-04-13 | 诺维信公司 | Rregulated PEPC expression |
| CN106132995A (en) * | 2014-03-28 | 2016-11-16 | 诺维信公司 | Protein crystal resolubilization at a low ph |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE102007027825A1 (en) * | 2007-06-13 | 2008-12-18 | C-Lecta Gmbh | Amidohydrolases for the treatment of food or beverages |
| CN101560513A (en) * | 2009-05-15 | 2009-10-21 | 邢苗 | Trichoderma reesei expression cassette, recombinant strain and application thereof |
| RU2645252C2 (en) * | 2012-01-05 | 2018-02-19 | Глюкос Финланд Ой | Felamentous fungi cell with protease deficit and methods of its application |
| CN108064266A (en) * | 2014-07-21 | 2018-05-22 | 格利科斯芬兰公司 | The preparation of the glycoprotein with mammal sample N- glycan in filamentous fungi |
| CN104630186A (en) * | 2015-02-07 | 2015-05-20 | 大连理工大学 | Preparation method and application of saccharide mixed solution for induced production of cellulase |
| CN108588060B (en) * | 2017-03-07 | 2020-12-01 | 武汉康复得生物科技股份有限公司 | Recombinant oxalate decarboxylase expressed by filamentous fungus host cell |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105492604A (en) * | 2013-08-23 | 2016-04-13 | 诺维信公司 | Rregulated PEPC expression |
| CN106132995A (en) * | 2014-03-28 | 2016-11-16 | 诺维信公司 | Protein crystal resolubilization at a low ph |
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