阅读说明：本技术 一种提高双功能纤维素酶的甘露聚糖酶活的方法及纤维素酶突变体rmx-m和应用 (Method for improving mannanase activity of bifunctional cellulase, cellulase mutant RMX-M and application ) 是由 罗会颖 郑洁 姚斌 黄火清 王亚茹 柏映国 苏小运 王苑 涂涛 张�杰 于 2020-08-05 设计创作，主要内容包括：本发明涉及农业生物技术领域,具体涉及一种提高双功能纤维素酶的甘露聚糖酶活的方法及纤维素酶突变体RMX-M和应用。本发明通过氨基酸序列如SEQ ID NO：1所示的野生型纤维素酶的E218位点实施定点突变获得E218H突变体。结果表明,以羧甲基纤维素钠和角豆胶为底物两种底物测定时,酶促反应的最适pH值不变,最适温度降低5℃；以角豆胶为底物时,突变体的甘露聚糖比活力比野生型提高了约80%；在以羧甲基纤维素钠为底物时,与野生型RMX的纤维素比活相比,突变体RMX-M的比活略有降低,实现了在纤维素酶活损失较小的基础上提高了其降解半纤维素底物甘露聚糖的能力。(The invention relates to the technical field of agricultural biology, in particular to a method for improving mannanase activity of bifunctional cellulase, a cellulase mutant RMX-M and application. The invention adopts the amino acid sequence shown as SEQ ID NO: site-directed mutagenesis is carried out on E218 site of the wild cellulase shown in 1 to obtain an E218H mutant. The result shows that when the test is carried out by taking the sodium carboxymethylcellulose and the carob bean gum as two substrates, the optimum pH value of the enzymatic reaction is not changed, and the optimum temperature is reduced by 5 ℃; when carob bean is used as a substrate, the specific activity of mannan of the mutant is improved by about 80 percent compared with that of the wild type; when sodium carboxymethylcellulose is used as a substrate, compared with the cellulose specific activity of wild type RMX, the specific activity of the mutant RMX-M is slightly reduced, so that the capability of degrading the hemicellulose substrate mannan is improved on the basis of less loss of the cellulose activity.)

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, the amino acid His at the 218 th site of the wild-type cellulase is mutated into the amino acid Elu.

2. The 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 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 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 bifunctional cellulase with improved mannanase activity.

7. The use of the bifunctional cellulase mutant RMX-M with increased mannanase activity as claimed in claim 2.

Technical Field

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

Background

Non-starch polysaccharides (NSP) are a generic term for carbohydrates other than starch in plants, and include cellulose, hemicellulose, pectin, and the like. The non-starch polysaccharide is the main component of the feed fiber, and the fiber surrounds the nutrient substances in the feed inside the cells, so that the degradation and absorption of the nutrient substances by the animals are inhibited to a certain extent. Enzymes which can degrade non-starch polysaccharides widely exist in nature, and comprise cellulase, mannase, xylanase, glucanase and the like, and the enzymes can effectively degrade NSP in the feed, improve the nutritional value of the feed and improve the growth performance of animals.

The degradation of non-starch polysaccharide in the feed usually needs the compound action of a plurality of enzymes, but the addition of the compound enzyme increases the use cost of the feed and becomes an important factor for limiting the wide application of the feed. A multifunctional enzyme with two or more functions is an effective way to simplify the feed processing technology and reduce the feed cost. The multifunctional enzyme which catalyzes two or even more substrates by single catalytic domain enzyme is obtained, or the range of the enzyme for efficiently acting on the substrates is widened by utilizing molecular improvement, so that the significance of obtaining the high-efficiency NSP enzyme with functional diversity is great.

The improvement of enzyme molecules by means of protein engineering is also a research hotspot in the field of enzyme engineering at present, but because the research on the amino acid sequence and the function of the enzyme is limited, the mutation scheme designed according to the enzyme mutation strategy is difficult to obtain the expected technical effect.

Disclosure of Invention

The invention aims to provide a method for improving the mannanase activity of bifunctional cellulase.

The invention further aims to provide the bifunctional cellulase mutant with improved mannanase activity.

Still another object of the present invention is to provide the above gene encoding bifunctional cellulase mutants with improved mannanase activity.

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

It is still another object of the present invention to provide a recombinant strain comprising the above gene.

It is a further object of the present invention to provide a method for preparing a bifunctional cellulase having improved mannanase activity.

In order to change the substrate specificity of the cellulase, the invention has the following amino acid sequence shown as SEQ ID NO: 1, mutating the wild-type cellulase.

The invention carries out site-directed mutagenesis on 218 site of wild cellulase, and carries out site-directed mutagenesis on His of the 218 site to Elu, thereby obtaining a cellulase mutant RMX-M with changed substrate specificity, wherein the amino acid sequence of the cellulase mutant RMX-M is shown as SEQ ID NO: 2, respectively.

The method for preparing the bifunctional cellulase with improved activity of the mannosidase 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 RMX-M with altered substrate specificity.

The invention adopts the amino acid sequence shown as SEQ ID NO: site-directed mutagenesis is carried out on E218 site of the wild cellulase shown in 1 to obtain an E218H mutant. The result shows that when the test is carried out by taking the sodium carboxymethylcellulose and the carob bean gum as two substrates, the optimum pH value of the enzymatic reaction is not changed, and the optimum temperature is reduced by 5 ℃; when carob bean is used as a substrate, the specific activity of mannan of the mutant is improved by about 80 percent compared with that of the wild type; when sodium carboxymethylcellulose is used as a substrate, compared with the cellulose specific activity of wild type RMX, the specific activity of the mutant RMX-M is slightly reduced, so that the capability of degrading the hemicellulose substrate mannan is improved on the basis of less loss of the cellulose activity.

Drawings

FIG. 1 shows the optimum pH of the cellulase mutant of the present invention with the wild type.

FIG. 2 shows the optimal temperature for the cellulase mutant of the present invention compared to the wild type.

FIG. 3 is a graph comparing the specific activity of the cellulase mutant of the present invention with that of the wild type.

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.