CN114703165A - Beta-glucosidase mutant and application thereof - Google Patents
Beta-glucosidase mutant and application thereof Download PDFInfo
- Publication number
- CN114703165A CN114703165A CN202210376425.7A CN202210376425A CN114703165A CN 114703165 A CN114703165 A CN 114703165A CN 202210376425 A CN202210376425 A CN 202210376425A CN 114703165 A CN114703165 A CN 114703165A
- Authority
- CN
- China
- Prior art keywords
- glucosidase
- mutant
- gly
- ala
- asp
- 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
- 108010047754 beta-Glucosidase Proteins 0.000 title claims abstract description 89
- 102000006995 beta-Glucosidase Human genes 0.000 title claims abstract description 82
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims description 32
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- 239000013598 vector Substances 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 17
- -1 oligosaccharide compound Chemical class 0.000 claims description 11
- 239000013612 plasmid Substances 0.000 claims description 11
- 229930182470 glycoside Natural products 0.000 claims description 10
- 241000588724 Escherichia coli Species 0.000 claims description 7
- 230000003301 hydrolyzing effect Effects 0.000 claims description 6
- 239000003446 ligand Substances 0.000 claims description 6
- 241001506991 Komagataella phaffii GS115 Species 0.000 claims description 5
- 230000002255 enzymatic effect Effects 0.000 claims description 5
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims description 4
- 238000006555 catalytic reaction Methods 0.000 claims description 4
- 230000007062 hydrolysis Effects 0.000 claims description 4
- 238000006460 hydrolysis reaction Methods 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 229920001542 oligosaccharide Polymers 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 3
- 150000002338 glycosides Chemical class 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 2
- 230000003197 catalytic effect Effects 0.000 claims 1
- 238000012258 culturing Methods 0.000 claims 1
- 230000001939 inductive effect Effects 0.000 claims 1
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- 239000008103 glucose Substances 0.000 abstract description 31
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- MGDFPGCFVJFITQ-CIUDSAMLSA-N Pro-Glu-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O MGDFPGCFVJFITQ-CIUDSAMLSA-N 0.000 description 4
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- 230000006798 recombination Effects 0.000 description 4
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- IFBHRQDFSNCLOZ-UHFFFAOYSA-N 2-(hydroxymethyl)-6-(4-nitrophenoxy)oxane-3,4,5-triol Chemical compound OC1C(O)C(O)C(CO)OC1OC1=CC=C([N+]([O-])=O)C=C1 IFBHRQDFSNCLOZ-UHFFFAOYSA-N 0.000 description 3
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- FFEUXEAKYRCACT-PEDHHIEDSA-N Arg-Ile-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CCCNC(N)=N)[C@@H](C)CC)C(O)=O FFEUXEAKYRCACT-PEDHHIEDSA-N 0.000 description 2
- CVXXSWQORBZAAA-SRVKXCTJSA-N Arg-Lys-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCN=C(N)N CVXXSWQORBZAAA-SRVKXCTJSA-N 0.000 description 2
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Abstract
Description
技术领域technical field
本发明属于微生物与基因工程技术领域，具体涉及一种葡萄糖耐受性提高的β-葡萄糖苷酶突变体及其应用。The invention belongs to the technical field of microorganisms and genetic engineering, and in particular relates to a β-glucosidase mutant with improved glucose tolerance and application thereof.
背景技术Background technique
β-葡萄糖苷酶(β-Glucosidases EC 3.2.1.21)，全称β-D-葡萄糖苷水解酶，属水解酶类，又称纤维二糖酶、龙胆二糖酶或苦杏仁苷酶。该酶可以水解结合于末端的非还原性的β-D-糖苷键，同时释放出β-D-葡萄糖以及相应的配基。β-葡萄糖苷酶因对多种糖苷类化合物具有水解活性，可作用于多种生物活性物质，其水解产生的配基多为功能性苷元或芳香成分；有的β-葡萄糖苷酶还具有转糖苷活性，可转化生成功能性低聚糖，因此在食品保健，生物能源等领域具有广阔的应用前景。β-Glucosidase (β-Glucosidases EC 3.2.1.21), the full name of β-D-glucoside hydrolase, is a hydrolase, also known as cellobiase, gentiobiase or amygdalinase. The enzyme can hydrolyze the non-reducing β-D-glycosidic bond bound to the terminal, releasing β-D-glucose and the corresponding ligand at the same time. Because β-glucosidase has hydrolysis activity on various glycoside compounds, it can act on a variety of biologically active substances, and the ligands produced by its hydrolysis are mostly functional aglycones or aromatic components; some β-glucosidases also have It has transglycosidic activity and can be converted into functional oligosaccharides, so it has broad application prospects in the fields of food health care and bioenergy.
由于在实际生产过程中，酶常处于不利环境，β-葡萄糖苷酶受到产物-葡萄糖的影响，导致生产效率低下，底物反映不充分等问题的出现。通常采用增加酶量、同步糖化发酵法(SSF)等措施来缓解产物的抑制作用，但这些方法工艺复杂，污染大、成本较高。为使催化反应高效地进行，目前亟需获得具有高耐受葡萄糖的β-葡萄糖苷酶。同时葡萄糖耐受性作为β-葡萄糖苷酶的关键性质在生物燃料、食品保健和生物合成等生产过程中也越来越受到重视。具有高耐受性的β-葡萄糖苷酶因可降低葡萄糖对酶活力的抑制作用，而显示出独特的应用潜能。CN102220302A以来自海洋未培养微生物来源的β-葡萄糖苷酶为基础，通过PCR定点突变，获得突变基因，相对野生型β-葡萄糖苷酶蛋白，获得的突变蛋白的葡萄糖耐受浓度提高了13倍。CN103266096A通过将β-葡萄糖苷酶基因Pbgl的386位的色氨酸突变成半胱氨酸，得到β-葡萄糖苷酶突变体Pbgl-W386C，葡萄糖的耐受性是突变前的11.88倍，提高了10.88倍。CN105754973A通过将链霉菌的β-葡萄糖苷酶S-bgl6分子上的233位色氨酸突变成天冬氨酸而得到β-葡萄糖苷酶突变体，和突变前相比，葡萄糖的耐受性提高了208.9倍。CN107142254A将来源于嗜酸耐热脂环酸芽孢杆菌Alicyclobacillus sp.A4的β-葡萄糖苷酶的315位氨基酸由组氨酸突变为精氨酸，得到较高葡萄糖耐受性β-葡萄糖苷酶突变体，在相同条件下，葡萄糖对突变体和野生型的酶活促进作用分别为212％和163％。In the actual production process, the enzyme is often in an unfavorable environment, and the β-glucosidase is affected by the product-glucose, resulting in low production efficiency and insufficient substrate reflection. Generally, measures such as increasing the amount of enzyme and simultaneous saccharification and fermentation (SSF) are used to alleviate the inhibitory effect of the product, but these methods are complicated in process, large in pollution and high in cost. In order to carry out the catalytic reaction efficiently, there is an urgent need to obtain β-glucosidase with high glucose tolerance. At the same time, glucose tolerance, as a key property of β-glucosidase, has been paid more and more attention in the production process of biofuel, food health care and biosynthesis. β-glucosidase with high tolerance has shown unique potential for application because it can reduce the inhibitory effect of glucose on enzyme activity. CN102220302A is based on β-glucosidase from marine uncultured microorganisms, and the mutant gene is obtained by PCR site-directed mutation. Compared with the wild-type β-glucosidase protein, the glucose tolerance concentration of the obtained mutant protein is increased by 13 times. CN103266096A obtained β-glucosidase mutant Pbgl-W386C by mutating tryptophan at position 386 of β-glucosidase gene Pbgl into cysteine, the glucose tolerance was 11.88 times higher than that before mutation, and improved 10.88 times. CN105754973A The β-glucosidase mutant is obtained by mutating tryptophan at position 233 on the S-bgl6 molecule of Streptomyces β-glucosidase to aspartic acid. Compared with before the mutation, the glucose tolerance is improved 208.9 times. CN107142254A The 315th amino acid of β-glucosidase derived from Alicyclobacillus sp. A4 is mutated from histidine to arginine to obtain a higher glucose tolerance β-glucosidase mutation Under the same conditions, the effect of glucose on the enzyme activity of mutant and wild type was 212% and 163%, respectively.
发明内容SUMMARY OF THE INVENTION
针对当前产业需求和现有技术的不足，本发明主要目的是通过定点突变技术构建一种具有良好的葡萄糖耐受性的β-葡萄糖苷酶突变体。In view of the current industrial demand and the deficiencies of the prior art, the main purpose of the present invention is to construct a β-glucosidase mutant with good glucose tolerance through site-directed mutagenesis technology.
为了实现上述目的，本发明采取如下的技术方案：In order to achieve the above object, the present invention adopts the following technical scheme:
第一方面，本发明提供一种β-葡萄糖苷酶突变体，所述β-葡萄糖苷酶突变体的氨基酸序列如SEQ ID NO：4所示。In a first aspect, the present invention provides a β-glucosidase mutant, and the amino acid sequence of the β-glucosidase mutant is shown in SEQ ID NO: 4.
第二方面，本发明提供编码所述β-葡萄糖苷酶突变体的基因。In a second aspect, the present invention provides a gene encoding the β-glucosidase mutant.
第三方面，本发明提供包含所述β-葡萄糖苷酶突变体的基因的载体。In a third aspect, the present invention provides a vector comprising the gene of the β-glucosidase mutant.
第四方面，本发明提供包含如上所述基因或载体的重组菌株。In a fourth aspect, the present invention provides a recombinant strain comprising the above-mentioned gene or vector.
第五方面，本发明提供如上所述β-葡萄糖苷酶突变体的用途，其用于水解糖苷或寡糖化合物非还原性末端的β-D-葡萄糖苷键，产生β-D-葡萄糖与相应配基；并且，所述突变体相比野生型具有提高的葡萄糖耐受性。In a fifth aspect, the present invention provides the use of the β-glucosidase mutant as described above, which is used to hydrolyze the β-D-glucosidic bond at the non-reducing end of a glycoside or oligosaccharide compound to produce β-D-glucose with the corresponding ligand; and, the mutant has improved glucose tolerance compared to the wild type.
第六方面，本发明提供制备如上所述β-葡萄糖苷酶突变体的方法，该方法通过利用本发明所述β-葡萄糖苷酶突变体的编码基因或本发明所述载体进行基因重组和表达。In a sixth aspect, the present invention provides a method for preparing the β-glucosidase mutant as described above, by using the gene encoding the β-glucosidase mutant of the present invention or the vector of the present invention for gene recombination and expression .
第七方面，本发明提供一种水解糖苷类化合物尤其是纤维二糖或纤维寡糖的方法，该方法包括使糖苷类化合物与本发明所述β-葡萄糖苷酶突变体或与本发明所述重组菌株接触的步骤，在能够通过所述β-葡萄糖苷酶突变体或所述重组菌株的酶促催化作用水解化合物的β-D-葡萄糖苷键的条件下进行。In a seventh aspect, the present invention provides a method for hydrolyzing a glycoside compound, especially cellobiose or cello-oligosaccharide, the method comprising combining the glycoside compound with the β-glucosidase mutant of the present invention or with the present invention The step of contacting the recombinant strain is carried out under conditions capable of hydrolyzing the β-D-glucosidic bond of the compound by the enzymatic catalysis of the β-glucosidase mutant or the recombinant strain.
有益效果：Beneficial effects:
本发明通过定点突变技术获得的β-葡萄糖苷酶突变体，通过基因工程技术在毕赤酵母系统中进行了重组和表达，相同条件下，本发明突变体可在400mmol/L葡萄糖的激活下最高达到239.0％的相对活力，在葡萄糖浓度为1.5mol/L时，其相对活力仍能达到112.61％，相比野生型具有显著提高的葡萄糖耐受能力。本发明的突变体在生物燃料、食品保健和生物合成等领域具有较高的应用价值。The β-glucosidase mutant obtained by the site-directed mutagenesis technology of the present invention has been recombined and expressed in the Pichia pastoris system by the genetic engineering technology. Under the same conditions, the mutant of the present invention can be activated by 400 mmol/L glucose. The relative activity reached 239.0%. When the glucose concentration was 1.5mol/L, the relative activity could still reach 112.61%. Compared with the wild type, it had significantly improved glucose tolerance. The mutant of the invention has high application value in the fields of biofuel, food health care, biosynthesis and the like.
附图说明Description of drawings
图1：实施例中野生型β-葡萄糖苷酶基因的PCR扩增电泳图；其中：1为阴性对照，2为DNA Marker，3为野生型bglHi基因。Figure 1: The PCR amplification electropherogram of the wild-type β-glucosidase gene in the example; wherein: 1 is the negative control, 2 is the DNA Marker, and 3 is the wild-type bglHi gene.
图2：实施例中野生型与突变体在不同浓度葡萄糖激活下的相对酶活力。Figure 2: Relative enzyme activities of wild-type and mutants in the examples under different concentrations of glucose activation.
图3：实施例中突变体的纯化蛋白电泳验证；其中：1为蛋白Marker，2、3为重组菌株GS115/pPIC9K-bglHiM，4为对照菌株GS115/pPIC9K。Figure 3: The purified protein electrophoresis verification of the mutant in the example; wherein: 1 is the protein marker, 2 and 3 are the recombinant strain GS115/pPIC9K-bglHiM, and 4 is the control strain GS115/pPIC9K.
图4：本发明β-葡萄糖苷酶突变体的氨基酸序列。Figure 4: Amino acid sequence of the β-glucosidase mutant of the present invention.
具体实施方式Detailed ways
下面通过具体的实施方案进一步叙述本发明。除非特别说明，以下实施方案所涉及的技术手段、材料等均可以是本领域技术人员所公知的，可以在已知的能解决相应技术问题的手段和材料中选择合适的。另外，实施方案应理解为说明性的，而非限制本发明的范围，本发明的实质和范围仅由权利要求书所限定。对于本领域技术人员而言，在不背离本发明实质和范围的前提下，对这些实施方案中的物料成分和用量进行的各种改变或改动也属于本发明的保护范围。The present invention is further described below through specific embodiments. Unless otherwise specified, the technical means, materials, etc. involved in the following embodiments may be known to those skilled in the art, and appropriate ones may be selected from known means and materials that can solve corresponding technical problems. In addition, the embodiments are to be understood as illustrative, rather than limiting, of the scope of the invention, the spirit and scope of the invention being limited only by the claims. For those skilled in the art, on the premise of not departing from the spirit and scope of the present invention, various changes or modifications to the material components and dosages in these embodiments also belong to the protection scope of the present invention.
第一方面，本发明提供一种β-葡萄糖苷酶突变体，所述β-葡萄糖苷酶突变体的氨基酸序列如SEQ ID NO：4所示。In a first aspect, the present invention provides a β-glucosidase mutant, and the amino acid sequence of the β-glucosidase mutant is shown in SEQ ID NO: 4.
第二方面，本发明提供编码所述β-葡萄糖苷酶突变体的基因。In a second aspect, the present invention provides a gene encoding the β-glucosidase mutant.
根据本发明一种优选的实施方式，所述基因的核苷酸序列如SEQ ID NO：3所示。According to a preferred embodiment of the present invention, the nucleotide sequence of the gene is shown in SEQ ID NO:3.
第三方面，本发明提供包含所述β-葡萄糖苷酶突变体的基因的载体。所述载体可以是本领域技术人员已知的通过基因重组制备蛋白质的载体之一，例如表达载体。In a third aspect, the present invention provides a vector comprising the gene of the β-glucosidase mutant. The vector may be one of those known to those skilled in the art for producing proteins by genetic recombination, such as an expression vector.
根据本发明一种优选的实施方式，所述载体是pPIC9K质粒。According to a preferred embodiment of the present invention, the vector is pPIC9K plasmid.
第四方面，本发明提供包含如上所述基因或载体的重组菌株。所述重组菌株可以是适合于从本发明的基因或载体生产本发明的β-葡萄糖苷酶突变体的任何宿主，例如毕赤酵母。In a fourth aspect, the present invention provides a recombinant strain comprising the above-mentioned gene or vector. The recombinant strain may be any host suitable for producing the beta-glucosidase mutant of the present invention from the gene or vector of the present invention, such as Pichia pastoris.
根据本发明一种优选的实施方式，所述宿主是毕赤酵母GS115。According to a preferred embodiment of the present invention, the host is Pichia GS115.
根据本发明另一种具体实施方式，所述宿主是大肠杆菌JM109。According to another specific embodiment of the present invention, the host is Escherichia coli JM109.
第五方面，本发明提供如上所述β-葡萄糖苷酶突变体的用途，其用于水解糖苷或寡糖化合物非还原性末端的β-D-葡萄糖苷键，产生β-D-葡萄糖与相应配基；并且，所述突变体相比野生型具有提高的葡萄糖耐受性。In a fifth aspect, the present invention provides the use of the β-glucosidase mutant as described above, which is used to hydrolyze the β-D-glucosidic bond at the non-reducing end of a glycoside or oligosaccharide compound to produce β-D-glucose with the corresponding ligand; and, the mutant has improved glucose tolerance compared to the wild type.
第六方面，本发明提供制备如上所述β-葡萄糖苷酶突变体的方法，该方法通过利用本发明所述β-葡萄糖苷酶突变体的编码基因或本发明所述载体进行基因重组和表达。可以使用本领域技术人员已知的基因重组方法和表达宿主，并选择适合于宿主表达的培养基和培养条件。所述方法还可以包括回收所述β-葡萄糖苷酶突变体的步骤，该回收步骤可能涉及了将β-葡萄糖苷酶突变体从宿主的培养物或表达产物中进行分离或纯化的步骤，可以使用本领域技术人员已知的任何方法进行。In a sixth aspect, the present invention provides a method for preparing the β-glucosidase mutant as described above, by using the gene encoding the β-glucosidase mutant of the present invention or the vector of the present invention for gene recombination and expression . Genetic recombination methods and expression hosts known to those skilled in the art can be used, and media and culture conditions suitable for host expression can be selected. The method may also include the step of recovering the β-glucosidase mutant, which may involve a step of isolating or purifying the β-glucosidase mutant from the host culture or expression product, which may be This is done using any method known to those skilled in the art.
根据本发明一种优选的实施方式，所述突变体的制备方法如下：将突变体基因与质粒pPIC9K进行经EcoR I和Avr II相同双酶切并连接获得重组载体，转化入大肠杆菌宿主JM109中，质粒经Sac I线性化后最终电转到毕赤酵母GS115中进行表达，经阴离子交换柱等纯化蛋白并验证其酶活力，获得了一种相比野生型具有提高的葡萄糖耐受性的β-葡萄糖苷酶突变体。According to a preferred embodiment of the present invention, the preparation method of the mutant is as follows: the mutant gene and the plasmid pPIC9K are digested with the same double enzymes of EcoR I and Avr II and connected to obtain a recombinant vector, which is transformed into E. coli host JM109 , the plasmid was linearized by Sac I and finally electrotransferred to Pichia GS115 for expression. The protein was purified by anion exchange column and its enzymatic activity was verified, and a β- Glucosidase mutants.
第七方面，本发明提供一种水解糖苷类化合物尤其是纤维二糖或纤维寡糖的方法，该方法包括使糖苷类化合物与本发明所述β-葡萄糖苷酶突变体或与本发明所述重组菌株接触的步骤，在能够通过所述β-葡萄糖苷酶突变体或所述重组菌株的酶促催化作用水解化合物的β-D-葡萄糖苷键的条件下进行。In a seventh aspect, the present invention provides a method for hydrolyzing a glycoside compound, especially cellobiose or cello-oligosaccharide, the method comprising combining the glycoside compound with the β-glucosidase mutant of the present invention or with the present invention The step of contacting the recombinant strain is carried out under conditions capable of hydrolyzing the β-D-glucosidic bond of the compound by the enzymatic catalysis of the β-glucosidase mutant or the recombinant strain.
本发明中采用如下定义：The following definitions are adopted in the present invention:
1、氨基酸和DNA核酸序列的命名法1. Nomenclature of Amino Acids and DNA Nucleic Acid Sequences
氨基酸残基使用公认IUPAC命名法，用三字母缩写或单字母符号形式。DNA核酸序列采用公认IUPAC命名法。Amino acid residues are in the form of three-letter abbreviations or one-letter symbols using accepted IUPAC nomenclature. DNA nucleic acid sequences use accepted IUPAC nomenclature.
2、β-葡萄糖苷酶突变体的标识2. Identification of β-glucosidase mutants
采用“原始氨基酸位置替换的氨基酸”来表示β-葡萄糖苷酶突变体中突变的氨基酸。本发明突变体是如SEQ ID NO：2所示的β-葡萄糖苷酶的氨基酸序列中第331位的苏氨酸(T)替换成谷氨酰胺(Q)，以T331Q或Thr331Gln表示。突变体的基因突变位点如下：The amino acid substituted in the original amino acid position is used to refer to the mutated amino acid in the beta-glucosidase mutant. The mutant of the present invention is the amino acid sequence of β-glucosidase shown in SEQ ID NO: 2 where the threonine (T) at position 331 is replaced by glutamine (Q), which is represented by T331Q or Thr331Gln. The gene mutation sites of the mutants are as follows:
以下将通过具体的实施例对本发明进行更详细描述。如无特别说明，以下实施例中：The present invention will be described in more detail below through specific embodiments. Unless otherwise specified, in the following examples:
本发明所用培养基及酶活测定方法：Culture medium and enzyme activity assay method used in the present invention:
LB培养基：蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L，固体培养基加入琼脂18g/L，其他成分一致。LB medium: peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, solid medium with agar 18g/L, other ingredients are the same.
MD固体培养基：葡萄糖20g/L，琼脂18g/L。MD solid medium: glucose 20g/L, agar 18g/L.
YPD培养基：蛋白胨20g/L，酵母粉10g/L，葡萄糖20g/L，固体培养基加入琼脂18g/L，其他成分一致。YPD medium: peptone 20g/L, yeast powder 10g/L, glucose 20g/L, solid medium with agar 18g/L, other ingredients are the same.
YPG培养基：蛋白胨20g/L，酵母粉10g/L，丙三醇20g/L。YPG medium: peptone 20g/L, yeast powder 10g/L, glycerol 20g/L.
BMGY培养基：酵母粉10g，蛋白胨20g溶于700mL水121℃灭菌20min，使用时60℃下加入100mL pH 6.0，1mol/L磷酸钾缓冲溶液，100mL 10×YNB，2mL 500×生物素，100mL 10×甘油。BMGY medium: 10g yeast powder, 20g peptone dissolved in 700mL water, sterilized at 121℃ for 20min, add 100mL pH 6.0, 1mol/L potassium phosphate buffer solution at 60℃ when using, 100mL 10×YNB, 2mL 500×biotin, 100mL 10x glycerol.
BMMY培养基：酵母粉10g，蛋白胨20g溶于700mL水121℃灭菌20min，使用时60℃下加入100mL 1mol/L磷酸钾缓冲液pH 6.0，100mL10×YNB，2mL 500×生物素，2.5mL甲醇。BMMY medium: 10g yeast powder, 20g peptone dissolved in 700mL water, sterilized at 121℃ for 20min, add 100mL 1mol/L potassium phosphate buffer pH 6.0 at 60℃ when in use, 100mL 10×YNB, 2mL 500×biotin, 2.5mL methanol .
MES缓冲液：称重3.91gMES溶于水，用Tris碱溶液调节pH至6.5，加水定容至1L。MES buffer: Weigh 3.91g MES and dissolve it in water, adjust the pH to 6.5 with Tris alkali solution, and add water to make up to 1L.
buffer A：终浓度为20mmol/L、pH 6.5MES缓冲液，过除菌膜备用。Buffer A: The final concentration is 20 mmol/L, pH 6.5 MES buffer, pass through the sterilized membrane for use.
buffer B：终浓度为20mmol/L、pH 6.5MES缓冲液和终浓度为1mol/L的NaCl溶液，过0.22μm除菌膜备用。Buffer B: MES buffer with a final concentration of 20 mmol/L, pH 6.5, and NaCl solution with a final concentration of 1 mol/L, pass through a 0.22 μm sterilization membrane for use.
G418筛选培养基：按照浓度(0.5mg/mL，1mg/mL，2mg/mL)融化YPD培养基于55℃左右下加入混匀使用；其他抗性筛选培养基加入相应浓度的抗性。G418 screening medium: melt the YPD culture according to the concentration (0.5mg/mL, 1mg/mL, 2mg/mL), add and mix at about 55°C; add the corresponding concentration of resistance to other resistance screening medium.
大肠杆菌感受态制备培养基Escherichia coli competent preparation medium
大肠杆菌复苏，LB培养基。E. coli recovery, LB medium.
洗液培养基：每升中加入CaCl2 11.1g；Wash medium: add CaCl 2 11.1g per liter;
重悬液培养基：1.5mL甘油，8.5mL 11.1g/L CaCl2溶液；Resuspension medium: 1.5mL glycerol, 8.5mL 11.1g/L CaCl 2 solution;
毕赤酵母感受态制备培养基：Pichia pastoris competent preparation medium:
YPD培养基：蛋白胨20g/L，酵母粉10g/L，葡萄糖20g/L；YPD medium: peptone 20g/L, yeast powder 10g/L, glucose 20g/L;
无菌水：过膜水经121℃，20min灭菌；Sterile water: The membrane water is sterilized at 121°C for 20min;
1mol/L山梨醇：每100mL去离子水溶解18.21g山梨醇，121℃灭菌20min，4℃保存备用。1 mol/L sorbitol: dissolve 18.21 g of sorbitol per 100 mL of deionized water, sterilize at 121 °C for 20 min, and store at 4 °C for later use.
本发明所用β-葡萄糖苷酶的酶活检测主要是基于酶解反应释放的配基与葡萄糖，以人工合成底物对硝基苯基-β-D-吡喃葡萄糖苷(pNPG)为底物，其水解产物为对硝基苯(pNP)和葡萄糖，对硝基苯在400nm下有特异的吸光值。The enzyme activity detection of β-glucosidase used in the present invention is mainly based on the ligand and glucose released by the enzymatic hydrolysis reaction, and the artificially synthesized substrate p-nitrophenyl-β-D-glucopyranoside (pNPG) is used as the substrate , its hydrolysis products are p-nitrobenzene (pNP) and glucose, p-nitrobenzene has a specific absorbance value at 400nm.
具体检测方法如下：取100μL适当稀释的酶液与2.9mL底物溶液(pNPG溶解在磷酸氢二钠—柠檬酸缓冲液，终浓度为3mmol/L，pH 5.0)混合，50℃下反应4min，加入1mL 1mol/L Na2CO3溶液终止反应。以100℃煮沸10min的灭活发酵液上清液作为对照。测定反应液OD400nm的值，参照标准曲线计算pNP的含量。将反应体系中每分钟产生1μmol对硝基苯(pNP)所需的酶量定义为1个酶活力单位(U)。The specific detection method is as follows: mix 100 μL of appropriately diluted enzyme solution with 2.9 mL of substrate solution (pNPG is dissolved in disodium hydrogen phosphate-citric acid buffer, the final concentration is 3 mmol/L, pH 5.0), and react at 50 ° C for 4 min. The reaction was terminated by adding 1 mL of 1 mol/L Na 2 CO 3 solution. The inactivated fermentation broth supernatant was boiled at 100 °C for 10 min as a control. The OD 400 nm value of the reaction solution was measured, and the content of pNP was calculated with reference to the standard curve. The amount of enzyme required to produce 1 μmol of p-nitrobenzene (pNP) per minute in the reaction system was defined as 1 enzyme activity unit (U).
对硝基苯(pNP)标准曲线如下；配制0.1mg/mL对硝基苯酚母液，分别稀释到浓度为0.002mg/mL、0.004mg/mL、0.006mg/mL、0.008mg/mL、0.010mg/mL的稀释液，以无菌水为空白，在400nm下测定吸光值。以OD值为纵坐标，pNP浓度(mg/mL)为横坐标，做出pNP标准曲线，其线性回归方程Y＝40.8X-0.026；R2＝0.9988。The standard curve of p-nitrobenzene (pNP) is as follows; prepare 0.1mg/mL p-nitrophenol stock solution and dilute to the concentration of 0.002mg/mL, 0.004mg/mL, 0.006mg/mL, 0.008mg/mL, 0.010mg/mL respectively. mL of diluent, take sterile water as blank, and measure the absorbance at 400 nm. Taking the OD value as the ordinate and the pNP concentration (mg/mL) as the abscissa, a pNP standard curve was drawn, and its linear regression equation was Y=40.8X-0.026; R 2 =0.9988.
其中，酶液和底物溶液混合之前，底物溶液需在50℃水浴中预热2min以上。Among them, before the enzyme solution and the substrate solution are mixed, the substrate solution needs to be preheated in a 50°C water bath for more than 2 minutes.
β-葡萄糖苷酶酶活力计算公式：酶活力S＝X×V1×1000×n/139×t×V2 β-Glucosidase enzyme activity calculation formula: enzyme activity S=X×V 1 ×1000×n/139×t×V 2
式中，X为p-NP的量(mg/mL)；V1为反应液体积(mL)；V2为酶液的体积(mL)；n为酶液稀释倍数；t为反应时间(min)。In the formula, X is the amount of p-NP (mg/mL); V 1 is the volume of the reaction solution (mL); V 2 is the volume of the enzyme solution (mL); n is the dilution ratio of the enzyme solution; t is the reaction time (min ).
酶活力定义：在50℃条件下将反应体系中每分钟产生1μmol对硝基苯(pNP)所需的酶量定义为1个酶活力单位(U)。Definition of enzyme activity: The amount of enzyme required to produce 1 μmol of p-nitrobenzene (pNP) per minute in the reaction system at 50°C was defined as 1 unit of enzyme activity (U).
实施例1：野生型β-葡萄糖苷酶bglHi重组菌株的构建Example 1: Construction of wild-type β-glucosidase bglHi recombinant strain
1.1 合成并扩增野生型β-葡萄糖苷酶基因bglHi1.1 Synthesis and amplification of wild-type β-glucosidase gene bglHi
根据GenBank:KF588650.1获得特异腐质霉(Humicola insolens)来源的野生型β-葡萄糖苷酶bglHi基因序列，为了适用于毕赤酵母表达，进行了密码子的优化，优化后的核苷酸序列如SEQ ID NO：1所示，委托生物公司合成其序列，并通过PCR进行扩增，引物序列如下：According to GenBank: KF588650.1, the wild-type β-glucosidase bglHi gene sequence derived from Humicola insolens was obtained. In order to be suitable for Pichia pastoris expression, the codons were optimized. The optimized nucleotide sequence As shown in SEQ ID NO: 1, a biological company was commissioned to synthesize its sequence and amplify it by PCR. The primer sequences are as follows:
引物P1：F 5’-ATGTCTTTGCCTCCAGACTTC-3’；Primer P1: F 5'-ATGTCTTTGCCTCCAGACTTC-3';
引物P2：R 5’-CGACTCTTTGATTAGAAAGGAGTAA-3’；Primer P2: R 5'-CGACTCTTTGATTAGAAAGGAGTAA-3';
以P1和P2作为上、下游引物，以bglHi的野生型基因为模板进行扩增；Use P1 and P2 as upstream and downstream primers, and use the wild-type gene of bglHi as a template for amplification;
其扩增的反应体系为：The amplification reaction system is:
扩增程序的设置为：预变性：95℃5min；变性：98℃10s；退火：50℃20s；延伸：72℃10s；反应32个循环；延伸：72℃10min。The settings of the amplification program were: pre-denaturation: 95°C for 5 min; denaturation: 98°C for 10s; annealing: 50°C for 20s; extension: 72°C for 10s; reaction for 32 cycles; extension: 72°C for 10 min.
将PCR产物进行琼脂糖凝胶电泳，可以看到野生型β-葡萄糖苷酶基因的条带，共1431bp(如图1所示)，再由小量DNA回收试剂盒回收PCR产物，得到了野生型β-葡萄糖苷酶基因，即bglHi。The PCR product was subjected to agarose gel electrophoresis, and the band of the wild-type β-glucosidase gene was seen, with a total of 1431 bp (as shown in Figure 1). The PCR product was recovered by a small amount of DNA recovery kit to obtain the wild type β-glucosidase gene, namely bglHi.
1.2 表达载体的线性化1.2 Linearization of expression vectors
提取pPIC9K质粒，其提取过程参照试剂盒的操作手册进行。经EcoR I和Avr II双酶切后进行琼脂糖凝胶电泳，再由DNA凝胶回收试剂盒回收产物，得到了线性化载体序列。The pPIC9K plasmid was extracted, and the extraction process was carried out according to the operation manual of the kit. After double digestion with EcoR I and Avr II, agarose gel electrophoresis was carried out, and the product was recovered by DNA gel recovery kit to obtain the linearized vector sequence.
1.3 重组菌株的构建1.3 Construction of recombinant strains
将经EcoR I和Avr II双酶切的目标片段(bglHi)和载体片段连接形成重组质粒pPIC9K-bglHi，将重组质粒转化进大肠杆菌JM109中，经测序验证序列如SEQ ID NO：1。The target fragment (bglHi) double digested by EcoR I and Avr II and the vector fragment were connected to form a recombinant plasmid pPIC9K-bglHi, and the recombinant plasmid was transformed into Escherichia coli JM109. The sequence was verified by sequencing as SEQ ID NO: 1.
实施例2：利用定点突变方法获得β-葡萄糖苷酶突变体Example 2: Obtaining β-glucosidase mutants by site-directed mutagenesis
2.1基于定点突变技术进行定向突变，构建新型β-葡萄糖苷酶突变体，设计引物如下：2.1 Targeted mutation based on site-directed mutagenesis technology to construct a new β-glucosidase mutant. The designed primers are as follows:
引物P3：5’-CAGTTGTTCTACAACAAGTACGGTGATTGTATCGGTCCA G-3’；Primer P3: 5'-CAGTTGTTCTACAACAAGTACGGTGATTGTATCGGTCCA G-3';
引物P4：R 5’-CTCCAAGTTTCCCAAAAAGTCGTCCTC-3’；Primer P4: R 5'-CTCCAAGTTTCCCAAAAAGTCGTCCTC-3';
在定点突变PCR反应体系中，以P3和P4作为上下游引物，以重组质粒pPIC9K-bglHi为模板，进行定向突变PCR。In the site-directed mutagenesis PCR reaction system, P3 and P4 were used as the upstream and downstream primers, and the recombinant plasmid pPIC9K-bglHi was used as the template to carry out the directed mutagenesis PCR.
其扩增的反应体系为：The amplification reaction system is:
扩增条件为：预变性：95℃5min；变性：98℃10s；退火：62℃20s；延伸：72℃1min；反应32个循环；延伸：72℃10min。Amplification conditions were: pre-denaturation: 95°C for 5 min; denaturation: 98°C for 10s; annealing: 62°C for 20s; extension: 72°C for 1 min; reaction for 32 cycles; extension: 72°C for 10 min.
2.2将获得的带有β-葡萄糖苷酶突变体基因的线性质粒自连成环，得到重组质粒pPIC9K-bglHiM，并化转入大肠杆菌JM109中，将重组质粒pPIC9K-bglHi和pPIC9K-bglHiM转化毕赤酵母中，获得能表达β-葡萄糖苷酶突变体的重组菌株GS115/pPIC9K-bglHiM。2.2 The obtained linear plasmid with the β-glucosidase mutant gene was self-linked into a circle to obtain a recombinant plasmid pPIC9K-bglHiM, which was transformed into E. coli JM109, and the recombinant plasmids pPIC9K-bglHi and pPIC9K-bglHiM were transformed In S. cerevisiae, a recombinant strain GS115/pPIC9K-bglHiM capable of expressing a β-glucosidase mutant was obtained.
实施例3：β-葡萄糖苷酶突变体的验证Example 3: Validation of β-glucosidase mutants
3.1 摇瓶验证3.1 Shake flask validation
将实施例2获得的重组菌株参考《毕赤酵母表达手册》，从筛选出高拷贝的重组酵母中划线分离单菌落，并将其与对照菌P.pastoris GS115/pPIC9K的单菌落分别接到30mLYPG培养基(含有终浓度为50μg/mL卡那霉素)中，30℃、180r/min培养16-18h；按2％接种量分别将种子液接入50mL BMGY液体培养基中，30℃，180r/min培养至OD600达到5-6(约16-20h)；收集菌液于灭菌的50mL离心管中，8000r/min，离心5min，倒掉上清，用20mL BMMY培养基重悬菌体进行细胞洗涤，重复洗涤3次，最后用BMMY培养基重悬菌体(菌体终浓度OD600＝1)；饥饿1h后开始添加诱导剂甲醇，每隔24h加入0.5％甲醇诱导表达，并定时取样分析，即可制备获得β-葡萄糖苷酶酶液。The recombinant strain obtained in Example 2 was referred to "Pichia pastoris expression manual", and a single colony was streaked and isolated from the recombinant yeast with high copy, and it was received with the single colony of the control bacteria P. pastoris GS115/pPIC9K respectively. In 30 mL YPG medium (containing a final concentration of 50 μg/mL kanamycin), culture at 30 °C and 180 r/min for 16-18 h; insert the seed liquid into 50 mL BMGY liquid medium at 30 °C according to the inoculum volume of 2%. Cultivate at 180r/min until OD 600 reaches 5-6 (about 16-20h); collect the bacterial liquid in a sterilized 50mL centrifuge tube, centrifuge at 8000r/min for 5min, pour off the supernatant, and resuspend the bacteria in 20mL BMMY medium The cells were washed for 3 times, and finally the cells were resuspended in BMMY medium (the final concentration of cells was OD 600 = 1); after 1 h of starvation, the inducer methanol was added, and 0.5% methanol was added every 24 h to induce expression, and The β-glucosidase enzyme solution can be prepared and obtained by regularly sampling and analyzing.
分别测定上述酶液的β-葡萄糖苷酶对葡萄糖耐受能力，将突变体与野生型β-葡萄糖苷酶在不同葡萄糖浓度下的耐受能力比较，得到1株β-葡萄糖苷酶对葡萄糖耐受能力显著强于野生型的菌株。The glucose tolerance of β-glucosidase in the above enzyme solution was measured respectively, and the tolerance of mutant and wild-type β-glucosidase at different glucose concentrations was compared, and a β-glucosidase strain was obtained. The susceptibility was significantly stronger than that of the wild-type strain.
3.2摇瓶复验、纯化及酶活性研究3.2 Shake flask re-examination, purification and enzymatic activity study
将上述重组菌株其与对照菌P.pastoris GS115/pPIC9K的单菌落分别接到30mLYPG培养基(含有终浓度为50μg/mL卡那霉素)中，30℃、180r/min培养16-18h；按2％接种量分别将种子液接入50mL BMGY液体培养基中，30℃，180r/min培养至OD600达到5-6(约16-20h)；收集菌液于灭菌的50mL离心管中，8000r/min，离心5min，倒掉上清，用20mL BMMY培养基重悬菌体进行细胞洗涤，重复洗涤3次，最后用BMMY培养基重悬菌体(菌体终浓度OD600＝1)；饥饿1h后开始添加诱导剂甲醇，每隔24h加入0.5％甲醇诱导表达，培养96h。将发酵液置于50mL无菌离心管中，于4℃，12000r/min离心20min，除去菌体；将发酵液上清过0.22μm除菌膜后置于30kD超滤管中进行超滤浓缩，4℃，5000r/min离心60min，大约制取1mL浓缩液。将浓缩后的发酵液转移到透析袋中进行透析，将透析袋放入装有去离子水的烧杯中，在4℃磁力搅拌下透析除去发酵液中的盐离子。将透析好的酶液置于10mL离心管中，8000r/min，4℃离心10min，去除不溶的底部杂质并过0.22μm的水系膜除去不溶杂质；平衡阴离子交换柱(SourseS)：20mmol/L的MES缓冲液作为buffer A，用buffer A冲洗柱子，冲洗3-5个柱体积，以平衡阴离子交换柱；上样：将样品全部上样至已平衡好的柱子中，上样流速设为0.5mL/min，用buffer A开始冲洗3-5个柱体积，自上样完成起即开始收集蛋白峰。样品平衡好后，用buffer B进行洗脱，并设置不同的梯度，直到达到100％NaCl浓度。洗脱流速设为1mL/min，每出现一个洗脱峰，换一个收集管，收集所有洗脱峰并分别编号。将纯化后的酶液上样进行5％浓缩胶、12％分离胶的聚丙烯酰胺凝胶电泳(SDS-PAGE)分析，结果如图3所示。图3的泳道4为对照菌株GS115/pPIC9K的发酵上清液，对照组基本无蛋白分泌，因为毕赤酵母GS115经过特异性改造，已经很少或不分泌任何胞外蛋白，泳道2或3为重组菌株GS115/pPIC9K-bglHiM的纯化后的酶液，可看见明显的蛋白条带，其表观分子量约为60kDa。测定纯化后的酶液的β-葡萄糖苷酶酶活力和葡萄糖耐受能力，得到一种β-葡萄糖苷酶突变体T331Q，其β-葡萄糖苷酶酶活力可在400mmol/L葡萄糖的激活下最高达到239.0％的相对活力，在葡萄糖浓度为1.5mol/L时，其相对活力仍能达到112.61％(酶活力对比如图2所示)。The single colony of the above recombinant strain and the control strain P. pastoris GS115/pPIC9K were respectively received in 30 mL YPG medium (containing a final concentration of 50 μg/mL kanamycin), and cultured at 30 ° C and 180 r/min for 16-18 h; press 2% of the inoculum amount, respectively, insert the seed liquid into 50 mL of BMGY liquid medium, and cultivate it at 30 ° C and 180 r/min until the OD 600 reaches 5-6 (about 16-20 h); collect the bacterial liquid in a sterilized 50 mL centrifuge tube, 8000 r/min, centrifuge for 5 min, discard the supernatant, resuspend the cells in 20 mL of BMMY medium for cell washing, repeat the
实施例4：β-葡萄糖苷酶突变体测序Example 4: Sequencing of beta-glucosidase mutants
将上述重组菌株提取β-葡萄糖苷酶基因序列，并进行测序(金唯智生物科技有限公司)，结果表明，扩增得到了β-葡萄糖苷酶突变体基因核苷酸序列如SEQ ID NO：3所示，将该编码基因命名为bglHiM。The β-glucosidase gene sequence was extracted from the above recombinant strain, and sequenced (Jinweizhi Biotechnology Co., Ltd.), the results showed that the nucleotide sequence of the β-glucosidase mutant gene was obtained by amplification, such as SEQ ID NO: 3 As shown, the encoding gene was named bglHiM.
将上述获得的β-葡萄糖苷酶bglHiM的氨基酸序列分别与野生型β-葡萄糖苷酶bglHi的氨基酸序列SEQ ID NO：1进行对比分析，结果显示：与野生型β-葡萄糖苷酶bglHi相比，β-葡萄糖苷酶bglHiM的第331位氨基酸由Thr突变为Gln(如图4所示)。The amino acid sequence of the above-obtained β-glucosidase bglHiM was compared with the amino acid sequence SEQ ID NO: 1 of the wild-type β-glucosidase bglHi, and the results showed that compared with the wild-type β-glucosidase bglHi, The amino acid 331 of β-glucosidase bglHiM was mutated from Thr to Gln (as shown in Figure 4).
虽然本发明已经以较佳实施例公开如上，但其并非用以限定本发明，任何本领域技术人员，在不脱离本发明的精神和原理的情况下，可以对这些实施例进行各种形式和细节的变化、修改、替换和变型，本发明的范围由权利要求及其等同物所限定。Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various forms and forms of these embodiments without departing from the spirit and principle of the present invention. Changes, modifications, substitutions and alterations of the details, the scope of the invention is defined by the claims and their equivalents.
序列表sequence listing
<120> 一种β-葡萄糖苷酶突变体及其应用<120> A β-glucosidase mutant and its application
<160> 8<160> 8
<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
<210> 1<210> 1
<211> 1431<211> 1431
<212> DNA<212> DNA
<213> 特异腐质霉(Humicola insolens)<213> Humicola insolens
<400> 1<400> 1
atgtctttgc ctccagactt caagtgggga tttgctactg ccgcctacca gattgagggt 60atgtctttgc ctccagactt caagtgggga tttgctactg ccgcctacca gattgagggt 60
tccgttaacg aggatggtcg tggtccatct atctgggaca ccttctgcgc catcccagga 120tccgttaacg aggatggtcg tggtccatct atctgggaca ccttctgcgc catcccagga 120
aagattgctg acggttcttc tggtgctgtc gcttgcgact cttacaagcg tactaaggaa 180aagattgctg acggttcttc tggtgctgtc gcttgcgact cttacaagcg tactaaggaa 180
gatattgcct tgttgaagga attgggtgct aactcctacc gtttctccat ctcctggtcc 240gatattgcct tgttgaagga attgggtgct aactcctacc gtttctccat ctcctggtcc 240
agaattatcc ctttgggtgg acgtaacgac ccaatcaatc aaaagggaat cgatcattac 300agaattatcc ctttgggtgg acgtaacgac ccaatcaatc aaaagggaat cgatcattac 300
gttaagttcg ttgatgactt gatcgaagcc ggtattaccc ctttcattac cttgttccac 360gttaagttcg ttgatgactt gatcgaagcc ggtattaccc ctttcattac cttgttccac 360
tgggacttgc cagacgcttt ggacaaaaga tacggaggtt ttttgaacaa ggaagagttt 420tgggacttgc cagacgcttt ggacaaaaga tacggaggtt ttttgaacaa ggaagagttt 420
gccgctgact tcgagaacta cgctagaatt atgttcaagg ctatccctaa atgtaagcat 480gccgctgact tcgagaacta cgctagaatt atgttcaagg ctatccctaa atgtaagcat 480
tggattacct ttaacgagcc atggtgctcc gccatcttgg gatacaacac cggttacttt 540tggattacct ttaacgagcc atggtgctcc gccatcttgg gatacaacac cggttacttt 540
gccccaggtc acacttccga cagatccaag tctccagttg gtgattctgc cagagagcct 600gccccaggtc acacttccga cagatccaag tctccagttg gtgattctgc cagagagcct 600
tggatcgttg gtcataacat cttgatcgcc cacgccagag ctgttaaggc ctaccgtgag 660tggatcgttg gtcataacat cttgatcgcc cacgccagag ctgttaaggc ctaccgtgag 660
gatttcaagc caacccaggg tggtgagatc ggtatcaccc ttaacggaga cgctactttg 720gatttcaagc caacccaggg tggtgagatc ggtatcaccc ttaacggaga cgctactttg 720
ccttgggatc cagaagaccc agctgacatt gaggcttgcg atagaaagat cgagttcgct 780ccttgggatc cagaagaccc agctgacatt gaggcttgcg atagaaagat cgagttcgct 780
atctcctggt tcgccgaccc aatctacttc ggaaagtacc cagactccat gagaaagcag 840atctcctggt tcgccgaccc aatctacttc ggaaagtacc cagactccat gagaaagcag 840
ttgggagaca gattgccaga gttcactcca gaggaagtcg ccttggttaa gggttctaat 900ttgggagaca gattgccaga gttcactcca gaggaagtcg ccttggttaa gggttctaat 900
gatttttacg gtatgaacca ctacaccgcc aactacatca agcacaagac tggtgtccct 960gatttttacg gtatgaacca ctacaccgcc aactacatca agcacaagac tggtgtccct 960
ccagaggacg actttttggg aaacttggag actttgttct acaacaagta cggtgattgt 1020ccagaggacg actttttggg aaacttggag actttgttct acaacaagta cggtgattgt 1020
atcggtccag agacccaatc cttttggttg cgtccacacg ctcaaggatt cagagacttg 1080atcggtccag agacccaatc cttttggttg cgtccacacg ctcaaggatt cagagacttg 1080
ttgaattggt tgtctaagag atacggttac ccaaaaattt acgttactga gaacggtacc 1140ttgaattggt tgtctaagag atacggttac ccaaaaattt acgttactga gaacggtacc 1140
tccttgaagg gtgagaacga catgcctttg gagcaggtct tggaggacga cttcagagtt 1200tccttgaagg gtgagaacga catgcctttg gagcaggtct tggaggacga cttcagagtt 1200
aagtacttta acgactacgt tagagctatg gctgccgctg ttgctgagga cggatgcaac 1260aagtacttta acgactacgt tagagctatg gctgccgctg ttgctgagga cggatgcaac 1260
gttcgtggtt atttggcctg gtctttgctt gacaactttg agtgggccga gggatacgag 1320gttcgtggtt atttggcctg gtctttgctt gacaactttg agtgggccga gggatacgag 1320
accagattcg gtgtcaccta cgttgattac gccaacgacc agaagcgtta cccaaagaag 1380accagattcg gtgtcaccta cgttgattac gccaacgacc agaagcgtta cccaaagaag 1380
tccgctaagt ccttgaaacc acttttcgac tctttgatta gaaaggagta a 1431tccgctaagt ccttgaaacc acttttcgac tctttgatta gaaaggagta a 1431
<210> 2<210> 2
<211> 476<211> 476
<212> PRT<212> PRT
<213> 特异腐质霉(Humicola insolens)<213> Humicola insolens
<400> 2<400> 2
Met Ser Leu Pro Pro Asp Phe Lys Trp Gly Phe Ala Thr Ala Ala TyrMet Ser Leu Pro Pro Asp Phe Lys Trp Gly Phe Ala Thr Ala Ala Tyr
1 5 10 151 5 10 15
Gln Ile Glu Gly Ser Val Asn Glu Asp Gly Arg Gly Pro Ser Ile TrpGln Ile Glu Gly Ser Val Asn Glu Asp Gly Arg Gly Pro Ser Ile Trp
20 25 30 20 25 30
Asp Thr Phe Cys Ala Ile Pro Gly Lys Ile Ala Asp Gly Ser Ser GlyAsp Thr Phe Cys Ala Ile Pro Gly Lys Ile Ala Asp Gly Ser Ser Gly
35 40 45 35 40 45
Ala Val Ala Cys Asp Ser Tyr Lys Arg Thr Lys Glu Asp Ile Ala LeuAla Val Ala Cys Asp Ser Tyr Lys Arg Thr Lys Glu Asp Ile Ala Leu
50 55 60 50 55 60
Leu Lys Glu Leu Gly Ala Asn Ser Tyr Arg Phe Ser Ile Ser Trp SerLeu Lys Glu Leu Gly Ala Asn Ser Tyr Arg Phe Ser Ile Ser Trp Ser
65 70 75 8065 70 75 80
Arg Ile Ile Pro Leu Gly Gly Arg Asn Asp Pro Ile Asn Gln Lys GlyArg Ile Ile Pro Leu Gly Gly Arg Asn Asp Pro Ile Asn Gln Lys Gly
85 90 95 85 90 95
Ile Asp His Tyr Val Lys Phe Val Asp Asp Leu Ile Glu Ala Gly IleIle Asp His Tyr Val Lys Phe Val Asp Asp Leu Ile Glu Ala Gly Ile
100 105 110 100 105 110
Thr Pro Phe Ile Thr Leu Phe His Trp Asp Leu Pro Asp Ala Leu AspThr Pro Phe Ile Thr Leu Phe His Trp Asp Leu Pro Asp Ala Leu Asp
115 120 125 115 120 125
Lys Arg Tyr Gly Gly Phe Leu Asn Lys Glu Glu Phe Ala Ala Asp PheLys Arg Tyr Gly Gly Phe Leu Asn Lys Glu Glu Phe Ala Ala Asp Phe
130 135 140 130 135 140
Glu Asn Tyr Ala Arg Ile Met Phe Lys Ala Ile Pro Lys Cys Lys HisGlu Asn Tyr Ala Arg Ile Met Phe Lys Ala Ile Pro Lys Cys Lys His
145 150 155 160145 150 155 160
Trp Ile Thr Phe Asn Glu Pro Trp Cys Ser Ala Ile Leu Gly Tyr AsnTrp Ile Thr Phe Asn Glu Pro Trp Cys Ser Ala Ile Leu Gly Tyr Asn
165 170 175 165 170 175
Thr Gly Tyr Phe Ala Pro Gly His Thr Ser Asp Arg Ser Lys Ser ProThr Gly Tyr Phe Ala Pro Gly His Thr Ser Asp Arg Ser Lys Ser Pro
180 185 190 180 185 190
Val Gly Asp Ser Ala Arg Glu Pro Trp Ile Val Gly His Asn Ile LeuVal Gly Asp Ser Ala Arg Glu Pro Trp Ile Val Gly His Asn Ile Leu
195 200 205 195 200 205
Ile Ala His Ala Arg Ala Val Lys Ala Tyr Arg Glu Asp Phe Lys ProIle Ala His Ala Arg Ala Val Lys Ala Tyr Arg Glu Asp Phe Lys Pro
210 215 220 210 215 220
Thr Gln Gly Gly Glu Ile Gly Ile Thr Leu Asn Gly Asp Ala Thr LeuThr Gln Gly Gly Glu Ile Gly Ile Thr Leu Asn Gly Asp Ala Thr Leu
225 230 235 240225 230 235 240
Pro Trp Asp Pro Glu Asp Pro Ala Asp Ile Glu Ala Cys Asp Arg LysPro Trp Asp Pro Glu Asp Pro Ala Asp Ile Glu Ala Cys Asp Arg Lys
245 250 255 245 250 255
Ile Glu Phe Ala Ile Ser Trp Phe Ala Asp Pro Ile Tyr Phe Gly LysIle Glu Phe Ala Ile Ser Trp Phe Ala Asp Pro Ile Tyr Phe Gly Lys
260 265 270 260 265 270
Tyr Pro Asp Ser Met Arg Lys Gln Leu Gly Asp Arg Leu Pro Glu PheTyr Pro Asp Ser Met Arg Lys Gln Leu Gly Asp Arg Leu Pro Glu Phe
275 280 285 275 280 285
Thr Pro Glu Glu Val Ala Leu Val Lys Gly Ser Asn Asp Phe Tyr GlyThr Pro Glu Glu Val Ala Leu Val Lys Gly Ser Asn Asp Phe Tyr Gly
290 295 300 290 295 300
Met Asn His Tyr Thr Ala Asn Tyr Ile Lys His Lys Thr Gly Val ProMet Asn His Tyr Thr Ala Asn Tyr Ile Lys His Lys Thr Gly Val Pro
305 310 315 320305 310 315 320
Pro Glu Asp Asp Phe Leu Gly Asn Leu Glu Thr Leu Phe Tyr Asn LysPro Glu Asp Asp Phe Leu Gly Asn Leu Glu Thr Leu Phe Tyr Asn Lys
325 330 335 325 330 335
Tyr Gly Asp Cys Ile Gly Pro Glu Thr Gln Ser Phe Trp Leu Arg ProTyr Gly Asp Cys Ile Gly Pro Glu Thr Gln Ser Phe Trp Leu Arg Pro
340 345 350 340 345 350
His Ala Gln Gly Phe Arg Asp Leu Leu Asn Trp Leu Ser Lys Arg TyrHis Ala Gln Gly Phe Arg Asp Leu Leu Asn Trp Leu Ser Lys Arg Tyr
355 360 365 355 360 365
Gly Tyr Pro Lys Ile Tyr Val Thr Glu Asn Gly Thr Ser Leu Lys GlyGly Tyr Pro Lys Ile Tyr Val Thr Glu Asn Gly Thr Ser Leu Lys Gly
370 375 380 370 375 380
Glu Asn Asp Met Pro Leu Glu Gln Val Leu Glu Asp Asp Phe Arg ValGlu Asn Asp Met Pro Leu Glu Gln Val Leu Glu Asp Asp Phe Arg Val
385 390 395 400385 390 395 400
Lys Tyr Phe Asn Asp Tyr Val Arg Ala Met Ala Ala Ala Val Ala GluLys Tyr Phe Asn Asp Tyr Val Arg Ala Met Ala Ala Ala Val Ala Glu
405 410 415 405 410 415
Asp Gly Cys Asn Val Arg Gly Tyr Leu Ala Trp Ser Leu Leu Asp AsnAsp Gly Cys Asn Val Arg Gly Tyr Leu Ala Trp Ser Leu Leu Asp Asn
420 425 430 420 425 430
Phe Glu Trp Ala Glu Gly Tyr Glu Thr Arg Phe Gly Val Thr Tyr ValPhe Glu Trp Ala Glu Gly Tyr Glu Thr Arg Phe Gly Val Thr Tyr Val
435 440 445 435 440 445
Asp Tyr Ala Asn Asp Gln Lys Arg Tyr Pro Lys Lys Ser Ala Lys SerAsp Tyr Ala Asn Asp Gln Lys Arg Tyr Pro Lys Lys Ser Ala Lys Ser
450 455 460 450 455 460
Leu Lys Pro Leu Phe Asp Ser Leu Ile Arg Lys GluLeu Lys Pro Leu Phe Asp Ser Leu Ile Arg Lys Glu
465 470 475465 470 475
<210> 3<210> 3
<211> 1431<211> 1431
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 3<400> 3
atgtctttgc ctccagactt caagtgggga tttgctactg ccgcctacca gattgagggt 60atgtctttgc ctccagactt caagtgggga tttgctactg ccgcctacca gattgagggt 60
tccgttaacg aggatggtcg tggtccatct atctgggaca ccttctgcgc catcccagga 120tccgttaacg aggatggtcg tggtccatct atctgggaca ccttctgcgc catcccagga 120
aagattgctg acggttcttc tggtgctgtc gcttgcgact cttacaagcg tactaaggaa 180aagattgctg acggttcttc tggtgctgtc gcttgcgact cttacaagcg tactaaggaa 180
gatattgcct tgttgaagga attgggtgct aactcctacc gtttctccat ctcctggtcc 240gatattgcct tgttgaagga attgggtgct aactcctacc gtttctccat ctcctggtcc 240
agaattatcc ctttgggtgg acgtaacgac ccaatcaatc aaaagggaat cgatcattac 300agaattatcc ctttgggtgg acgtaacgac ccaatcaatc aaaagggaat cgatcattac 300
gttaagttcg ttgatgactt gatcgaagcc ggtattaccc ctttcattac cttgttccac 360gttaagttcg ttgatgactt gatcgaagcc ggtattaccc ctttcattac cttgttccac 360
tgggacttgc cagacgcttt ggacaaaaga tacggaggtt ttttgaacaa ggaagagttt 420tgggacttgc cagacgcttt ggacaaaaga tacggaggtt ttttgaacaa ggaagagttt 420
gccgctgact tcgagaacta cgctagaatt atgttcaagg ctatccctaa atgtaagcat 480gccgctgact tcgagaacta cgctagaatt atgttcaagg ctatccctaa atgtaagcat 480
tggattacct ttaacgagcc atggtgctcc gccatcttgg gatacaacac cggttacttt 540tggattacct ttaacgagcc atggtgctcc gccatcttgg gatacaacac cggttacttt 540
gccccaggtc acacttccga cagatccaag tctccagttg gtgattctgc cagagagcct 600gccccaggtc acacttccga cagatccaag tctccagttg gtgattctgc cagagagcct 600
tggatcgttg gtcataacat cttgatcgcc cacgccagag ctgttaaggc ctaccgtgag 660tggatcgttg gtcataacat cttgatcgcc cacgccagag ctgttaaggc ctaccgtgag 660
gatttcaagc caacccaggg tggtgagatc ggtatcaccc ttaacggaga cgctactttg 720gatttcaagc caacccaggg tggtgagatc ggtatcaccc ttaacggaga cgctactttg 720
ccttgggatc cagaagaccc agctgacatt gaggcttgcg atagaaagat cgagttcgct 780ccttgggatc cagaagaccc agctgacatt gaggcttgcg atagaaagat cgagttcgct 780
atctcctggt tcgccgaccc aatctacttc ggaaagtacc cagactccat gagaaagcag 840atctcctggt tcgccgaccc aatctacttc ggaaagtacc cagactccat gagaaagcag 840
ttgggagaca gattgccaga gttcactcca gaggaagtcg ccttggttaa gggttctaat 900ttgggagaca gattgccaga gttcactcca gaggaagtcg ccttggttaa gggttctaat 900
gatttttacg gtatgaacca ctacaccgcc aactacatca agcacaagac tggtgtccct 960gatttttacg gtatgaacca ctacaccgcc aactacatca agcacaagac tggtgtccct 960
ccagaggacg actttttggg aaacttggag cagttgttct acaacaagta cggtgattgt 1020ccagaggacg actttttggg aaacttggag cagttgttct acaacaagta cggtgattgt 1020
atcggtccag agacccaatc cttttggttg cgtccacacg ctcaaggatt cagagacttg 1080atcggtccag agacccaatc cttttggttg cgtccacacg ctcaaggatt cagagacttg 1080
ttgaattggt tgtctaagag atacggttac ccaaaaattt acgttactga gaacggtacc 1140ttgaattggt tgtctaagag atacggttac ccaaaaattt acgttactga gaacggtacc 1140
tccttgaagg gtgagaacga catgcctttg gagcaggtct tggaggacga cttcagagtt 1200tccttgaagg gtgagaacga catgcctttg gagcaggtct tggaggacga cttcagagtt 1200
aagtacttta acgactacgt tagagctatg gctgccgctg ttgctgagga cggatgcaac 1260aagtacttta acgactacgt tagagctatg gctgccgctg ttgctgagga cggatgcaac 1260
gttcgtggtt atttggcctg gtctttgctt gacaactttg agtgggccga gggatacgag 1320gttcgtggtt atttggcctg gtctttgctt gacaactttg agtgggccga gggatacgag 1320
accagattcg gtgtcaccta cgttgattac gccaacgacc agaagcgtta cccaaagaag 1380accagattcg gtgtcaccta cgttgattac gccaacgacc agaagcgtta cccaaagaag 1380
tccgctaagt ccttgaaacc acttttcgac tctttgatta gaaaggagta a 1431tccgctaagt ccttgaaacc acttttcgac tctttgatta gaaaggagta a 1431
<210> 4<210> 4
<211> 476<211> 476
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 4<400> 4
Met Ser Leu Pro Pro Asp Phe Lys Trp Gly Phe Ala Thr Ala Ala TyrMet Ser Leu Pro Pro Asp Phe Lys Trp Gly Phe Ala Thr Ala Ala Tyr
1 5 10 151 5 10 15
Gln Ile Glu Gly Ser Val Asn Glu Asp Gly Arg Gly Pro Ser Ile TrpGln Ile Glu Gly Ser Val Asn Glu Asp Gly Arg Gly Pro Ser Ile Trp
20 25 30 20 25 30
Asp Thr Phe Cys Ala Ile Pro Gly Lys Ile Ala Asp Gly Ser Ser GlyAsp Thr Phe Cys Ala Ile Pro Gly Lys Ile Ala Asp Gly Ser Ser Gly
35 40 45 35 40 45
Ala Val Ala Cys Asp Ser Tyr Lys Arg Thr Lys Glu Asp Ile Ala LeuAla Val Ala Cys Asp Ser Tyr Lys Arg Thr Lys Glu Asp Ile Ala Leu
50 55 60 50 55 60
Leu Lys Glu Leu Gly Ala Asn Ser Tyr Arg Phe Ser Ile Ser Trp SerLeu Lys Glu Leu Gly Ala Asn Ser Tyr Arg Phe Ser Ile Ser Trp Ser
65 70 75 8065 70 75 80
Arg Ile Ile Pro Leu Gly Gly Arg Asn Asp Pro Ile Asn Gln Lys GlyArg Ile Ile Pro Leu Gly Gly Arg Asn Asp Pro Ile Asn Gln Lys Gly
85 90 95 85 90 95
Ile Asp His Tyr Val Lys Phe Val Asp Asp Leu Ile Glu Ala Gly IleIle Asp His Tyr Val Lys Phe Val Asp Asp Leu Ile Glu Ala Gly Ile
100 105 110 100 105 110
Thr Pro Phe Ile Thr Leu Phe His Trp Asp Leu Pro Asp Ala Leu AspThr Pro Phe Ile Thr Leu Phe His Trp Asp Leu Pro Asp Ala Leu Asp
115 120 125 115 120 125
Lys Arg Tyr Gly Gly Phe Leu Asn Lys Glu Glu Phe Ala Ala Asp PheLys Arg Tyr Gly Gly Phe Leu Asn Lys Glu Glu Phe Ala Ala Asp Phe
130 135 140 130 135 140
Glu Asn Tyr Ala Arg Ile Met Phe Lys Ala Ile Pro Lys Cys Lys HisGlu Asn Tyr Ala Arg Ile Met Phe Lys Ala Ile Pro Lys Cys Lys His
145 150 155 160145 150 155 160
Trp Ile Thr Phe Asn Glu Pro Trp Cys Ser Ala Ile Leu Gly Tyr AsnTrp Ile Thr Phe Asn Glu Pro Trp Cys Ser Ala Ile Leu Gly Tyr Asn
165 170 175 165 170 175
Thr Gly Tyr Phe Ala Pro Gly His Thr Ser Asp Arg Ser Lys Ser ProThr Gly Tyr Phe Ala Pro Gly His Thr Ser Asp Arg Ser Lys Ser Pro
180 185 190 180 185 190
Val Gly Asp Ser Ala Arg Glu Pro Trp Ile Val Gly His Asn Ile LeuVal Gly Asp Ser Ala Arg Glu Pro Trp Ile Val Gly His Asn Ile Leu
195 200 205 195 200 205
Ile Ala His Ala Arg Ala Val Lys Ala Tyr Arg Glu Asp Phe Lys ProIle Ala His Ala Arg Ala Val Lys Ala Tyr Arg Glu Asp Phe Lys Pro
210 215 220 210 215 220
Thr Gln Gly Gly Glu Ile Gly Ile Thr Leu Asn Gly Asp Ala Thr LeuThr Gln Gly Gly Glu Ile Gly Ile Thr Leu Asn Gly Asp Ala Thr Leu
225 230 235 240225 230 235 240
Pro Trp Asp Pro Glu Asp Pro Ala Asp Ile Glu Ala Cys Asp Arg LysPro Trp Asp Pro Glu Asp Pro Ala Asp Ile Glu Ala Cys Asp Arg Lys
245 250 255 245 250 255
Ile Glu Phe Ala Ile Ser Trp Phe Ala Asp Pro Ile Tyr Phe Gly LysIle Glu Phe Ala Ile Ser Trp Phe Ala Asp Pro Ile Tyr Phe Gly Lys
260 265 270 260 265 270
Tyr Pro Asp Ser Met Arg Lys Gln Leu Gly Asp Arg Leu Pro Glu PheTyr Pro Asp Ser Met Arg Lys Gln Leu Gly Asp Arg Leu Pro Glu Phe
275 280 285 275 280 285
Thr Pro Glu Glu Val Ala Leu Val Lys Gly Ser Asn Asp Phe Tyr GlyThr Pro Glu Glu Val Ala Leu Val Lys Gly Ser Asn Asp Phe Tyr Gly
290 295 300 290 295 300
Met Asn His Tyr Thr Ala Asn Tyr Ile Lys His Lys Thr Gly Val ProMet Asn His Tyr Thr Ala Asn Tyr Ile Lys His Lys Thr Gly Val Pro
305 310 315 320305 310 315 320
Pro Glu Asp Asp Phe Leu Gly Asn Leu Glu Gln Leu Phe Tyr Asn LysPro Glu Asp Asp Phe Leu Gly Asn Leu Glu Gln Leu Phe Tyr Asn Lys
325 330 335 325 330 335
Tyr Gly Asp Cys Ile Gly Pro Glu Thr Gln Ser Phe Trp Leu Arg ProTyr Gly Asp Cys Ile Gly Pro Glu Thr Gln Ser Phe Trp Leu Arg Pro
340 345 350 340 345 350
His Ala Gln Gly Phe Arg Asp Leu Leu Asn Trp Leu Ser Lys Arg TyrHis Ala Gln Gly Phe Arg Asp Leu Leu Asn Trp Leu Ser Lys Arg Tyr
355 360 365 355 360 365
Gly Tyr Pro Lys Ile Tyr Val Thr Glu Asn Gly Thr Ser Leu Lys GlyGly Tyr Pro Lys Ile Tyr Val Thr Glu Asn Gly Thr Ser Leu Lys Gly
370 375 380 370 375 380
Glu Asn Asp Met Pro Leu Glu Gln Val Leu Glu Asp Asp Phe Arg ValGlu Asn Asp Met Pro Leu Glu Gln Val Leu Glu Asp Asp Phe Arg Val
385 390 395 400385 390 395 400
Lys Tyr Phe Asn Asp Tyr Val Arg Ala Met Ala Ala Ala Val Ala GluLys Tyr Phe Asn Asp Tyr Val Arg Ala Met Ala Ala Ala Val Ala Glu
405 410 415 405 410 415
Asp Gly Cys Asn Val Arg Gly Tyr Leu Ala Trp Ser Leu Leu Asp AsnAsp Gly Cys Asn Val Arg Gly Tyr Leu Ala Trp Ser Leu Leu Asp Asn
420 425 430 420 425 430
Phe Glu Trp Ala Glu Gly Tyr Glu Thr Arg Phe Gly Val Thr Tyr ValPhe Glu Trp Ala Glu Gly Tyr Glu Thr Arg Phe Gly Val Thr Tyr Val
435 440 445 435 440 445
Asp Tyr Ala Asn Asp Gln Lys Arg Tyr Pro Lys Lys Ser Ala Lys SerAsp Tyr Ala Asn Asp Gln Lys Arg Tyr Pro Lys Lys Ser Ala Lys Ser
450 455 460 450 455 460
Leu Lys Pro Leu Phe Asp Ser Leu Ile Arg Lys GluLeu Lys Pro Leu Phe Asp Ser Leu Ile Arg Lys Glu
465 470 475465 470 475
<210> 5<210> 5
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 5<400> 5
atgtctttgc ctccagactt c 21atgtctttgc ctccagactt c 21
<210> 6<210> 6
<211> 25<211> 25
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 6<400> 6
cgactctttg attagaaagg agtaa 25cgactctttg attagaaagg agtaa 25
<210> 7<210> 7
<211> 40<211> 40
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 7<400> 7
cagttgttct acaacaagta cggtgattgt atcggtccag 40cagttgttct acaacaagta cggtgattgt atcggtccag 40
<210> 8<210> 8
<211> 27<211> 27
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 8<400> 8
ctccaagttt cccaaaaagt cgtcctc 27ctccaagttt cccaaaaagt cgtcctc 27
Claims (9)
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| CN117402858A (en) * | 2023-12-13 | 2024-01-16 | 北京科为博生物科技有限公司 | Beta-glucosidase mutant with improved heat resistance |
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| CN105229141A (en) * | 2013-03-15 | 2016-01-06 | 拉勒曼德匈牙利流动管理有限责任公司 | For the expression of the beta-glucosidase enzyme of the hydrolysis of lignocellulose and relevant oligopolymer |
| CN110438136A (en) * | 2019-08-30 | 2019-11-12 | 中国水产科学研究院黄海水产研究所 | The gene of beta-glucosidase and its mutant, amino acid sequence and application |
| CN111518792A (en) * | 2020-05-26 | 2020-08-11 | 江西师范大学 | A β-glucosidase mutant TLE of Penicillium oxalicum 16 and its application |
| CN111621490A (en) * | 2020-05-26 | 2020-09-04 | 江西师范大学 | Beta-glucosidase mutant SVS of penicillium oxalicum 16 and application thereof |
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| CN105229141A (en) * | 2013-03-15 | 2016-01-06 | 拉勒曼德匈牙利流动管理有限责任公司 | For the expression of the beta-glucosidase enzyme of the hydrolysis of lignocellulose and relevant oligopolymer |
| US10612061B2 (en) * | 2013-03-15 | 2020-04-07 | Lallemand Hungary Liquidity Management Llc | Expression of beta-glucosidases for hydrolysis of lignocellulose and associated oligomers |
| CN110438136A (en) * | 2019-08-30 | 2019-11-12 | 中国水产科学研究院黄海水产研究所 | The gene of beta-glucosidase and its mutant, amino acid sequence and application |
| CN111518792A (en) * | 2020-05-26 | 2020-08-11 | 江西师范大学 | A β-glucosidase mutant TLE of Penicillium oxalicum 16 and its application |
| CN111621490A (en) * | 2020-05-26 | 2020-09-04 | 江西师范大学 | Beta-glucosidase mutant SVS of penicillium oxalicum 16 and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117402858A (en) * | 2023-12-13 | 2024-01-16 | 北京科为博生物科技有限公司 | Beta-glucosidase mutant with improved heat resistance |
| CN117402858B (en) * | 2023-12-13 | 2024-03-15 | 北京科为博生物科技有限公司 | Beta-glucosidase mutant with improved heat resistance |
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