CN111676209B - Method for improving mannanase activity of bifunctional cellulase, cellulase mutant RMX-M and application - Google Patents

Abstract

The invention relates to the technical field of agricultural biology, in particular to a method for improving the mannanase activity of bifunctional cellulase, a cellulase mutant RMX-M and applications. In the present invention, the E218H mutant is obtained by site-directed mutation of the E218 site of the wild-type cellulase whose amino acid sequence is shown in SEQ ID NO: 1. The results showed that the optimum pH value of the enzymatic reaction remained unchanged and the optimum temperature decreased by 5℃ when the two substrates were determined using sodium carboxymethyl cellulose and carob bean gum as substrates; when carob bean gum was used as the substrate , the specific activity of mannan of the mutant was increased by about 80% compared with that of the wild type; when sodium carboxymethylcellulose was used as the substrate, compared with the specific activity of cellulose of the wild type RMX, the specific activity of the mutant RMX‑M was higher than that of the wild type. The cellulase activity was slightly reduced, and the ability to degrade the hemicellulose substrate mannan was improved on the basis of less loss of cellulase activity.

Description

A kind of method for improving the mannanase activity of bifunctional cellulase and cellulase Mutant RMX-M and applications

technical field

The invention relates to the technical field of agricultural biotechnology, in particular to a method for improving the mannanase activity of a bifunctional cellulase, a cellulase mutant RMX-M and applications.

Background technique

Non-starch polysaccharide (NSP) is a general term for carbohydrates other than starch in plants, including cellulose, hemicellulose and pectin. Non-starch polysaccharides are the main components of feed fibers, because these fibers surround the nutrients in the feed inside the cells, which inhibit the degradation and absorption of nutrients by animals to a certain extent. Enzymes that can degrade non-starch polysaccharides widely exist in nature, including cellulase, mannanase, xylanase, glucanase, etc. Improve animal growth performance.

The degradation of non-starch polysaccharides in feed often requires the compound action of multiple enzymes. However, the addition of compound enzymes increases the cost of feed use and becomes an important factor limiting its wide application. A multifunctional enzyme with two or more functions at the same time is an effective way to simplify the feed processing technology and reduce the feed cost. It is of great significance to obtain a multifunctional enzyme with a single catalytic domain enzyme that catalyzes two or even multiple substrates, or to use molecular modification to broaden the scope of the enzyme's efficient substrates, and to obtain a high-efficiency NSP enzyme with functional diversity.

The use of protein engineering methods to improve enzyme molecules is also a research hotspot in the field of enzyme engineering. However, due to the limited research on the relationship between the amino acid sequence and function of enzymes, it is difficult to obtain the expected technical effect based on the mutation scheme designed according to the enzyme mutation strategy.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a method for improving the mannanase activity of bifunctional cellulase.

Another object of the present invention is to provide a bifunctional cellulase mutant with improved mannanase activity.

Another object of the present invention is to provide the above-mentioned gene encoding a bifunctional cellulase mutant with increased mannanase activity.

Still another object of the present invention is to provide a recombinant vector comprising the above mutant gene.

Another object of the present invention is to provide a recombinant strain comprising the above-mentioned gene.

Another object of the present invention is to provide a method for preparing a bifunctional cellulase with improved mannanase activity.

In the present invention, in order to change the substrate specificity of cellulase, the wild-type cellulase whose amino acid sequence is shown in SEQ ID NO: 1 is mutated.

In the present invention, site-directed mutation of the 218 site of the wild-type cellulase is performed, and the His site-directed mutation of the 218 site is changed to Elu to obtain a cellulase mutant RMX-M with altered substrate specificity, the amino acid sequence of which is as shown in SEQ ID NO: 2 shown.

The method for preparing the bifunctional cellulase with improved basal mannanase activity according to the present invention comprises the following steps:

1) Transform the host cell with the above-mentioned recombinant vector to obtain a recombinant strain;

2) Cultivate the recombinant strain to induce the expression of recombinant cellulase;

3) Recovery and purification of the expressed cellulase RMX-M with altered substrate specificity.

In the present invention, the E218H mutant is obtained by site-directed mutation of the E218 site of the wild-type cellulase whose amino acid sequence is shown in SEQ ID NO: 1. The results showed that the optimum pH value of the enzymatic reaction remained unchanged and the optimum temperature decreased by 5℃ when the two substrates were determined with sodium carboxymethyl cellulose and carob bean gum as substrates; when carob bean gum was used as the substrate , the specific activity of mannan of the mutant was increased by about 80% compared with that of the wild type; when sodium carboxymethylcellulose was used as the substrate, compared with the specific activity of cellulose of the wild type RMX, the specific activity of the mutant RMX-M was higher than that of the wild type. The cellulase activity was slightly reduced, and the ability to degrade the hemicellulose substrate mannan was improved on the basis of less loss of cellulase activity.

Description of drawings

Figure 1 shows the pH optima of the cellulase mutants of the present invention and the wild type.

Figure 2 shows the optimum temperature of cellulase mutants of the present invention and wild type.

Fig. 3 is a graph comparing the specific activity of the cellulase mutant of the present invention and the wild type.

Detailed ways

Test Materials and Reagents

1. Strain and vector: expression host Pichiapastoris GS115, expression plasmid vector pPIC9r.

2. Biochemical reagents: restriction endonuclease was purchased from NEB company, ligase was purchased from Promaga company, point mutation kit was purchased from Quanjijin company, and sodium carboxymethyl cellulose was purchased from Sigma company. Others are domestic analytical reagents (all can be purchased from common biochemical reagent companies).

3. Culture medium:

LB 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 biology experimental methods that are not described in detail in this example are all carried out by referring to the specific methods listed in the book "Molecular Cloning Experiment Guide" (Third Edition) by J. Sambrook, or according to the kit. and product manual.

Example 1 Preparation of cellulase mutant recombinant vector pPIC9r-RMX-M with altered substrate specificity

The wild-type cellulase encoding gene (removing the signal peptide encoding sequence) before mutation was cloned into the expression vector pPIC-9r, and the recombinant vector was named pPIC9r-RMX ; the recombinant vector pPIC9r-RMX was used as the template, and the primers carrying the mutation site were used as the template. It was amplified to obtain a recombinant vector carrying the mutant sequence, named pPIC9r-RMX-M .

Table 1 Cellulase mutant RMX-M specific primers with altered substrate specificity

primer Sequence (5'<i>—</i>3') ^<i>a</i> Length (bp) RMX-E218H-F CATCCAACAAGATATTGTACcacATGCACCAATACT 36 RMX-E218H-R gtgGTACAATATCTTGTTGGATGGATCCTTC 31

Example 2 Preparation of cellulase mutants with altered substrate specificity.

(1) Mass expression of cellulase mutant RMX-M at shake flask level in Pichia pastoris

The obtained recombinant plasmid pPIC9r-RMX-M containing the mutant gene RMX-M with altered substrate specificity was transformed into Pichia pastoris GS115 to obtain a recombinant yeast strain GS115 /RMX-M . Take the GS115 strain containing the recombinant plasmid, inoculate it into a 1 L Erlenmeyer flask of 300 mL BMGY medium, place it at 30 °C, and cultivate it on a shaker at 220 rpm for 48 h; centrifuge the culture medium at 4000 g for 5 min, discard the supernatant, and use 200 mL of precipitation. The cells were resuspended in BMMY medium containing 0.5% methanol, and then placed again at 30°C and induced to culture at 220 rpm. 1 mL of methanol was added every 12 h, and the supernatant was taken for enzyme activity detection.

(2) Purification of recombinant cellulase

The supernatant of recombinant cellulase expressed in shake flasks was collected and concentrated by passing through a 10 kDa membrane pack while replacing the medium with low-salt buffer, leaving approximately 20 ml of protein concentrate. The recombinant cellulase RMX-M concentrated to a certain multiple is purified by ion exchange chromatography. Specifically, 10.0 mL of cellulase RMX and mutant RMX-M concentrates were pre-equilibrated with 10 mmol/L Tris-HCl (pH 8.0) on a HiTrap Q HP anion column, and then 10.0 mL of the concentrated solution containing 1 mol/L NaCl was used. 10 mmol/L Tris-HCl (pH 8.0) was used for linear gradient elution, the enzyme activity of the gradient eluted protein solution was detected by DNS method, and the purity of the gradient eluted protein solution was determined by SDS-PAGE gel electrophoresis. detection.

Example 3 Activity Analysis of Cellulase Mutants and Wild Types with Improved Recombinant Mannanase Activity

The basic enzymatic properties of recombinant cellulase RMX and mutant RMX-M were determined by dinitrosalicylic acid (DNS) method. The specific method is as follows: under the conditions of pH 5.0 and 75 ℃, 1 mL of reaction system includes 100 µL of appropriate diluted enzyme solution, 900 µL of substrate, react for 10 min, and add 1.5 mL of DNS to terminate the reaction; boil in boiling water for 5 min and then cool down To room temperature, the OD value was measured at a wavelength of 540 nm. The assay method of mannanase is the same as that of cellulase. Definition of endocellulase activity unit: Under certain conditions, the amount of enzyme required to decompose the substrate to generate 1 μmoL of glucose per minute is 1 activity unit (U). Mannanase activity unit definition: Under certain conditions, the amount of enzyme required to decompose the substrate to generate 1 μmoL of mannose per minute is 1 activity unit (U). The enzyme solution used in the study of enzymatic properties should be electrophoretic pure.

(1) Analysis and comparison of optimum pH

Example 2 The purified expressed wild-type cellulase RMX and mutant RMX-M were subjected to enzymatic reactions at different pH to determine their optimum pH. The buffer used is a citric acid-disodium hydrogen phosphate series buffer system of pH 2.0-7.0. The purified cellulase RMX and mutant RMX-M were tested in buffer systems of different pH and 75 ℃ with sodium carboxymethyl cellulose and carob bean gum as substrates. and RMX-M are both pH 5.0.

(2) Analysis and comparison of optimum temperature

The purified cellulase was tested under the respective optimum pH conditions, using sodium carboxymethyl cellulose and carob bean gum as substrates, and the enzyme activity at different temperatures from 20 to 80 °C was determined. The analysis results are shown in Figure 2. RMX and RMX-M at 75°C and 70°C, respectively.

(3) Comparison of specific activity analysis

Example 2 Purified cellulase wild-type RMX and mutant RMX-M were subjected to enzymatic reaction with sodium carboxymethyl cellulose and carob bean gum at pH 5.0, 75 °C and pH 5.0, 70 °C to Its enzymatic activity was determined.

The specific activity measurement results are shown in Figure 3. When using sodium carboxymethylcellulose as the substrate, the specific activity of the wild-type RMX was 1214 U/mg, and the specific activity of the mutant RMX-M was 1007 U/mg. , which is about 20% lower than that of the wild type; when carob bean gum is used as the substrate, the specific activity of the mannan of the wild type RMX is 251 U/mg, and the specific activity of the mutant RMX-M is 454 U/mg, which is higher than that of the wild type. The cellulase type was increased by about 80%, and the ability to degrade the hemicellulose substrate mannan was improved on the basis of less loss of cellulase activity.

sequence listing

<110> Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences

<120> A method for improving mannanase activity of bifunctional cellulase and cellulase mutant RMX-M and application

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<170> SIPOSequenceListing 1.0

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<211> 322

<212> PRT

<213> Artificial Sequence

<400> 1

Ala Pro Glu Ser Ser Leu Asp Lys Arg Ala Gly Asn Phe Lys Phe Phe

1 5 10 15

Gly Val Asn Glu Ala Gly Pro Glu Phe Gly Asn Gln Asn Leu Pro Gly

20 25 30

Val Tyr Asn Lys Asp Tyr Val Phe Pro Thr Leu Ser Thr Tyr Asp Thr

35 40 45

Phe Ile Ser Lys Gly Phe Asn Thr Phe Arg Leu Asn Ile Gln Met Glu

50 55 60

Arg Leu Ala Pro Asn Ala Ile Asn Gly Asn Leu Asp Thr Thr Tyr Leu

65 70 75 80

Asn Met Ile Lys Glu Gln Val Asn Tyr Val Thr Gly Lys Gly Ala Tyr

85 90 95

Met Met Ile Asn Pro His Asn Tyr Gly Arg Tyr Tyr Gly Gln Ile Tyr

100 105 110

Arg Asp Thr Gln Ser Phe Gly Gln Phe Trp Ala His Leu Ala Gln Glu

115 120 125

Phe Lys Ser Asn Ser Arg Val Ile Phe Asp Thr Asp Asn Glu Phe His

130 135 140

Asp Glu Pro Gly Gln Leu Val Ala Asp Leu Asn Gln Ala Ala Ile Asn

145 150 155 160

Ala Ile Arg Ala Thr Gly Ala Thr Asn Gln Tyr Ile Ala Val Glu Gly

165 170 175

Asn Ala Trp Thr Gly Ala Trp Thr Trp Thr Thr Ala Lys Gly Thr Asp

180 185 190

Gly Leu Thr Asn Ala Gln Thr Met Gly Asn Leu Lys Asp Pro Ser Asn

195 200 205

Lys Ile Leu Tyr Glu Met His Gln Tyr Leu Asp Ser Asp Gly Ser Gly

210 215 220

Thr Ser Thr Thr Cys Val Ser Ser Thr Ile Gly Ser Glu Arg Leu Lys

225 230 235 240

Ala Ala Thr Gln Trp Leu Arg Ala Asn Gly Lys Lys Lys Gly Leu Leu Gly

245 250 255

Glu Tyr Ala Gly Ala Val Asn Pro Thr Cys Gln Ala Ala Val Lys Asp

260 265 270

Met Leu Ser Tyr Met Val Lys Asn Lys Asp Val Trp Glu Gly Ala Val

275 280 285

Trp Trp Ala Ala Gly Pro Trp Trp Gly Asp Tyr Met Phe Ser Ile Glu

290 295 300

Pro Thr Asn Gly Pro Ala Tyr Asn Thr Tyr Val Pro Leu Ile Thr Gln

305 310 315 320

Tyr Ala

<210> 2

<211> 322

<212> PRT

<213> Artificial Sequence

<400> 2

Ala Pro Glu Ser Ser Leu Asp Lys Arg Ala Gly Asn Phe Lys Phe Phe

1 5 10 15

Gly Val Asn Glu Ala Gly Pro Glu Phe Gly Asn Gln Asn Leu Pro Gly

20 25 30

Val Tyr Asn Lys Asp Tyr Val Phe Pro Thr Leu Ser Thr Tyr Asp Thr

35 40 45

Phe Ile Ser Lys Gly Phe Asn Thr Phe Arg Leu Asn Ile Gln Met Glu

50 55 60

Arg Leu Ala Pro Asn Ala Ile Asn Gly Asn Leu Asp Thr Thr Tyr Leu

65 70 75 80

Asn Met Ile Lys Glu Gln Val Asn Tyr Val Thr Gly Lys Gly Ala Tyr

85 90 95

Met Met Ile Asn Pro His Asn Tyr Gly Arg Tyr Tyr Gly Gln Ile Tyr

100 105 110

Arg Asp Thr Gln Ser Phe Gly Gln Phe Trp Ala His Leu Ala Gln Glu

115 120 125

Phe Lys Ser Asn Ser Arg Val Ile Phe Asp Thr Asp Asn Glu Phe His

130 135 140

Asp Glu Pro Gly Gln Leu Val Ala Asp Leu Asn Gln Ala Ala Ile Asn

145 150 155 160

Ala Ile Arg Ala Thr Gly Ala Thr Asn Gln Tyr Ile Ala Val Glu Gly

165 170 175

Asn Ala Trp Thr Gly Ala Trp Thr Trp Thr Thr Ala Lys Gly Thr Asp

180 185 190

Gly Leu Thr Asn Ala Gln Thr Met Gly Asn Leu Lys Asp Pro Ser Asn

195 200 205

Lys Ile Leu Tyr His Met His Gln Tyr Leu Asp Ser Asp Gly Ser Gly

210 215 220

Thr Ser Thr Thr Cys Val Ser Ser Thr Ile Gly Ser Glu Arg Leu Lys

225 230 235 240

Ala Ala Thr Gln Trp Leu Arg Ala Asn Gly Lys Lys Lys Gly Leu Leu Gly

245 250 255

Glu Tyr Ala Gly Ala Val Asn Pro Thr Cys Gln Ala Ala Val Lys Asp

260 265 270

Met Leu Ser Tyr Met Val Lys Asn Lys Asp Val Trp Glu Gly Ala Val

275 280 285

Trp Trp Ala Ala Gly Pro Trp Trp Gly Asp Tyr Met Phe Ser Ile Glu

290 295 300

Pro Thr Asn Gly Pro Ala Tyr Asn Thr Tyr Val Pro Leu Ile Thr Gln

305 310 315 320

Tyr Ala

Claims (7)

1. A method for improving mannanase activity of a bifunctional cellulase, the method comprising contacting the bifunctional cellulase with an amino acid sequence as set forth in SEQ ID NO: 1, mutating the amino acid Glu at the 213 th site of the wild type cellulase to the amino acid His.

2. A high bifunctional cellulase mutant RMX-M with improved mannanase activity is characterized in that the amino acid sequence of the cellulase mutant RMX-M is shown as SEQ ID NO: 2, respectively.

3. A gene encoding the high bifunctional cellulase mutant RMX-M with increased mannanase activity according to claim 2.

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

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

6. A method for preparing a high bifunctional cellulase with improved mannanase activity, 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 high-bifunctional cellulase with improved mannanase activity.

7. The use of the highly bifunctional cellulase mutant RMX-M with increased mannanase activity according to claim 2 for the degradation of sodium carboxymethylcellulose and carob bean gum.

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