阅读说明：本技术 一种提高纤维素酶催化效率的方法及突变体5i77-m2 (Method for improving cellulase catalytic efficiency and mutant 5I77-M2 ) 是由 罗会颖 郑洁 秦星 黄火清 王亚茹 王晓璐 王苑 苏小运 柏映国 涂涛 张�杰 于 2021-10-27 设计创作，主要内容包括：本发明涉及基因工程领域,具体涉及一种提高纤维素酶催化效率的方法及突变体5I77-M2。所述方法包括将氨基酸序列如SEQ ID NO：1所示的野生型纤维素酶的第300位的氨基酸Thr突变为氨基酸Pro和将第307位点的氨基酸Asp突变为氨基酸Pro、以及第193位的氨基酸Asn突变为Ala的步骤。酶促反应的最适pH值、最适温度并未发生变化；以羧甲基纤维素钠为底物时,本发明的突变体5i77-M2的比活力比突变体5I77-M提高了50%。(The invention relates to the field of genetic engineering, and particularly relates to a method for improving the catalytic efficiency of cellulase and a mutant 5I 77-M2. The method comprises the following steps of mixing the amino acid sequence shown as SEQ ID NO: 1, the amino acid Thr at the 300 th position of the wild-type cellulase is mutated into the amino acid Pro, the amino acid Asp at the 307 th position is mutated into the amino acid Pro, and the amino acid Asn at the 193 th position is mutated into Ala. The optimum pH value and the optimum temperature of the enzymatic reaction are not changed; when sodium carboxymethylcellulose is used as a substrate, the specific activity of the mutant 5I77-M2 is improved by 50% compared with that of the mutant 5I 77-M.)

1. A method for increasing the catalytic efficiency of a cellulase enzyme, said method comprising contacting the cellulase enzyme with an amino acid sequence as set forth in SEQ ID NO: 1, the amino acid Thr at the 300 th position of the wild-type cellulase is mutated into the amino acid Pro, the amino acid Asp at the 307 th position is mutated into the amino acid Pro, and the amino acid Asn at the 193 th position is mutated into Ala.

2. A cellulase mutant with improved catalytic efficiency 5I77-M2, wherein the amino acid sequence of the cellulase mutant is shown as SEQ ID NO: 2, respectively.

3. A gene encoding the cellulase mutant 5I77-M2 with improved catalytic efficiency according to claim 2.

4. The gene of claim 3, wherein the nucleotide sequence of the gene is as shown in SEQ ID NO: 3, respectively.

5. A recombinant expression vector comprising the gene of claim 3.

6. A recombinant strain comprising the gene of claim 3.

7. A method of making a cellulase mutant with improved catalytic efficiency, comprising the steps of:

transforming a host strain with a recombinant expression vector comprising the gene of claim 3;

inducing the recombinant strain to express cellulase;

separating and purifying to obtain the cellulase with improved catalytic activity.

8. Use of a cellulase mutant with improved catalytic efficiency according to claim 2.

Technical Field

The invention relates to the field of genetic engineering, and particularly relates to a method for improving the catalytic efficiency of cellulase and a mutant 5I 77-M2.

Background

Cellulose is the main component constituting plant cell walls, is the most abundant organic matter on earth, and is the largest renewable biomass resource nowadays. Cellulose has received considerable attention in recent years as being converted to glucose or other fermentable sugars and hence biofuels or other products. Among the existing cellulose conversion technologies, cellulase is considered as a key factor and is also gradually becoming one of the hot spots of research.

Cellulases are a class of hydrolytic enzymes that degrade cellulose into cellooligosaccharides or glucose, and can hydrolyze beta-1, 4-glucosidic bonds, i.e., the chemical bonds in the cellulose molecule that link glucose units. According to the existing research, most of cellulose degradation and transformation in nature are carried out by fungi and bacteria, and the microorganisms develop various mechanisms for extracellular high-efficiency degradation of natural crystalline cellulose by producing a series of cellulase systems.

The catalytic activity has been widely paid attention as an important index for measuring the industrial application value of the enzyme. Although the following enzyme mutation strategies have been disclosed so far, since research between amino acid sequences and functions of enzymes is limited, it is difficult to obtain the intended technical effects by designing a mutation scheme according to the enzyme mutation strategy.

Disclosure of Invention

The invention aims to provide a method for improving the catalytic efficiency of cellulase.

It is a further object of the present invention to provide cellulase mutant 5i77-M2 with improved catalytic efficiency.

It is still another object of the present application to provide a gene encoding the cellulase mutant 5i77-M2 with improved catalytic efficiency as described above.

It is a further object of the present application to provide the use of the cellulase mutant 5i77-M2 with improved catalytic efficiency as described above.

The method for improving the catalytic efficiency of the cellulase comprises the following steps of: 1, wherein the amino acid Thr at the 300 th position of the wild-type cellulase is mutated into the amino acid Pro, the amino acid Asp at the 307 th position is mutated into the amino acid Pro, and the amino acid Asn at the 193 th position is mutated into Ala.

According to previous studies in this application, the amino acid sequence has been shown as SEQ ID NO: 1, carrying out spotting on the wild-type cellulase, and mutating amino acid Thr at the 300 th site of the wild-type cellulase to amino acid Pro and mutating amino acid Asp at the 307 th site to amino acid Pro to obtain the mutant 5I 77-M. The results show that the optimum pH value and the optimum temperature of the mutant 5I77-M are not changed compared with the wild cellulase, and the specific activity of the mutant 5I77-M is improved by about 50 percent compared with the wild cellulase when sodium carboxymethylcellulose is used as a substrate.

On this basis, the amino acid Asn at position 193 of the present application was further mutated to Ala.

According to the cellulase mutant 5i77-M2 with improved catalytic efficiency, the amino acid sequence of the mutant is SEQ ID NO: 2, respectively.

The present invention provides a gene encoding the cellulase mutant 5i77-M2 with improved catalytic efficiency. Specifically, the nucleotide sequence of the gene is shown as SEQ ID NO: 3, respectively.

The invention also provides a recombinant vector containing the gene for coding the cellulase mutant 5i77-M2 with improved catalytic efficiency.

The present invention also provides a recombinant strain comprising the above gene encoding cellulase mutant 5i77-M2 with improved catalytic efficiency.

The invention also provides a method for preparing the cellulase mutant with improved catalytic efficiency, which comprises the following steps:

1) transforming host cells by using the recombinant vector to obtain a recombinant strain;

2) culturing the recombinant strain, and inducing the recombinant cellulase to express;

3) recovering and purifying the expressed cellulase mutant with improved catalytic efficiency.

According to the specific implementation mode of the invention, the pichia pastoris GS115 is transformed by the recombinant vector containing the mutant gene, and the positive transformant is initially screened by carrying out enzyme activity measurement on the fermentation liquor at the level of the tubules. And (3) performing large-bottle induction on a transformant with the highest enzyme activity, and performing protein concentration and purification on the crude enzyme solution. The purity of the purified mutants and wild type was checked by SDS-PAGE electrophoresis. The purified protein was used as a target, and the basic enzymatic properties of the wild type and the mutant were measured by the DNS method. The results show that the optimum pH value and the optimum temperature of the enzymatic reaction are not changed; when sodium carboxymethylcellulose is used as a substrate, the specific activity of the mutant 5I77-M2 is improved by 50% compared with that of the mutant 5I 77-M.

Drawings

FIG. 1 shows the optimum pH of cellulase mutants 5I77-M and 5I 77-M2;

FIG. 2 shows the optimal temperatures for cellulase mutants 5I77-M and 5I 77-M2.

Detailed Description

Test materials and reagents

1. Bacterial strain and carrier: expression hostPichiapastorisGS115, expression plasmid vector pPIC9 r.

2. Biochemical reagents: restriction enzymes were purchased from NEB, ligase from Promaga, point mutation kit from total gold, and sodium carboxymethylcellulose from Sigma. The others are domestic analytical pure reagents (all can be purchased from common biochemical reagents).

3. Culture medium:

LB culture medium: 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0

YPD medium: 1% yeast extract, 2% peptone, 2% glucose

MD solid medium: 2% glucose, 1.5% agarose, 1.34% YNB, 0.00004% Biotin

BMGY medium: 1% yeast extract, 2% peptone, 1% glycerol (V/V), 1.34% YNB, 0.00004% Biotin.

BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 0.5% methanol (V/V).

4. The molecular biological experiments, which are not described in detail in this example, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) j. sambrook, or according to the kit and product instructions.

EXAMPLE 1 cellulase mutant recombinant vectors with improved catalytic ActivitypPIC9r-5I77-MPreparation of 5i77-M2

Cloning cellulase wild type (before mutation) sequence fragment (removing signal peptide) to expression vector pPIC-9r, and naming recombinant vectorpPIC9r-5I77(ii) a With recombinant vectorspPIC9r-5I77As a template, the mutant site is amplified by a primer to obtain a recombinant vector carrying the mutant sequence, which is namedpPIC9r-5I77-M2。

TABLE 1 cellulase mutants with improved catalytic Activity5I77-M2Specific primer

Example 2 preparation of cellulase mutants.

(1) Cellulase mutants5I77-M2Large scale expression at shake flask level in pichia pastoris.

The obtained gene containing the mutant5I77-M2The recombinant plasmid of (1)pPIC9r-5I77-M2Transforming Pichia pastoris GS115 to obtain recombinant yeast strain GS115/5I77-M2. Taking a GS115 strain containing the recombinant plasmid, inoculating the strain into a 1L triangular flask of 300 mL BMGY medium, and carrying out shake culture at 30 ℃ and 220 rpm for 48 h; the culture broth was centrifuged at 4000 g for 5 min, the supernatant was discarded, and the pellet was resuspended in 200 mL BMMY medium containing 0.5% methanol and again placed at 30 ℃ for induction culture at 220 rpm. 1 mL of methanol was added every 12 h, and the supernatant was used for enzyme activity detection.

(2) Purification of recombinant proteases

The shake flask-expressed recombinant cellulase supernatant was collected and concentrated through a 10 kDa membrane pack while the medium was replaced with low salt buffer, and finally about 20 ml of protein concentrate remained. Concentrating to obtain recombinant cellulase 5I77-M, and purifying by ion exchange chromatography. Specifically, 10.0 mL of cellulase 5I77 and mutant 5I77-M concentrated solution was subjected to HiTrap Q HP anion column equilibrated with 10 mmol/L Tris-HCl (pH 8.0) in advance, then subjected to linear gradient elution with 10 mmol/L Tris-HCl (pH 8.0) containing 1 mol/L NaCl, and the protein solution subjected to gradient elution was subjected to enzyme activity detection by the DNS method and purity detection by SDS-PAGE gel electrophoresis.

Example 3 Activity analysis of cellulase mutants having improved recombinant catalytic Activity

The basic enzymological properties of the recombinant endo-cellulase were determined by the dinitrosalicylic acid (DNS) method. The specific method comprises the following steps: under the conditions of pH4.0 and 75 ℃, reacting for 10 min in a 1 mL reaction system comprising 100 muL of appropriate diluted enzyme solution and 900 muL of substrate, and adding 1.5 mL of DNS to terminate the reaction; boiling in water bath for 5 min, cooling to room temperature, and measuring OD value at 540 nm wavelength. Endocellulase activity unit definition: under certain conditions, the amount of enzyme required to decompose the substrate to 1. mu. moL of glucose per minute was 1 activity unit (U). The enzyme solution used for the enzymology property research needs to reach the electrophoresis purity.

(1) Comparison of optimum pH analysis

The purified (example 2) expressed cellulase 5I77 and mutant 5I77-M2 were subjected to enzymatic reactions at different pH to determine their optimum pH. The buffer solution is a citric acid disodium hydrogen phosphate series buffer system with the pH value of 2.0-7.0. The results of the optimum pH of the purified cellulase 5I77 and the mutant 5I77-M2 in a buffer system with different pH values and at 75 ℃ are shown in figure 1: the optimum pH values of 5I77 and 5I77-M2 were both 4.0.

(2) Comparison of optimum temperature analysis

The purified endo-cellulase is subjected to enzyme activity measurement at different temperatures (30-80 ℃) under the condition of pH4.0 (taking sodium carboxymethyl cellulose as a substrate) to determine the optimal temperature of the recombinase. The experimental result shows that the optimal reaction temperature of the wild cellulase 5I77 and the mutant 5I77-M2 is 75 ℃, and the enzyme activity is still more than 50% at 80 ℃ (figure 2).

(3) Analytical comparison of catalytic efficiency

The purified cellulase mutant 5I77-M2 (example 2) was subjected to an enzymatic reaction with a wild-type cellulase mutant 5I77-M at pH4.0 and 75 ℃ to determine the enzymatic activity and kinetic parameters thereof, wherein the 300 th amino acid Thr of the wild-type cellulase of the present invention was mutated to the amino acid Pro, and the 307 th amino acid Asp was mutated to the amino acid Pro to obtain the mutant 5I 77-M.

The specific activity assay results are shown in Table 2, the specific activity of mutant 5I77-M is 2313 +/-39U/mg, and the specific activity of mutant 5I77-M2 is 3472 +/-42U/mg. Compared with 5I77-M, 5I77-M2KThe M value does not vary much, 5I77-MK _mValues of 6.51. + -. 0.4 mg/ml, of mutant 5I77-M2KThe value of m is 6.56. + -. 0.4 mg/ml. But instead of the other end of the tubeV _maxThe change in the value is significant and,V _max5I77-M is 3029 + -103. mu. mol/min/mg, mutant 5I77-M2 is increased to 5084 + -156. mu. mol/min/mg. The conversion number is 1766 + -76S^-1Increased to 2965 +/-81S^-1Finally, finallyk _cat/K _mThe concentration is increased from 271 +/-36 to 451 +/-43 ml/s/mg. As can be seen from the above-mentioned data,V _maxthe improvement results in further improvement of the catalytic efficiency of the mutant 5I 77-M2.

TABLE 2

Sequence listing

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