Beta-1, 3-1, 4-glucanase mutant with high catalytic activity at animal body temperature and application thereof

文档序号：1884789 发布日期：2021-11-26 浏览：23次中文

阅读说明：本技术 一组动物体温下高催化活力β-1,3-1,4-葡聚糖酶突变体及其应用 (Beta-1, 3-1, 4-glucanase mutant with high catalytic activity at animal body temperature and application thereof ) 是由游帅葛研谢晨张温馨王雪陈奕文张伊欣王俊于 2021-06-04 设计创作，主要内容包括：一组动物体温下高催化活力β-1,3-1,4-葡聚糖酶突变体及其应用,包括BisGlu16B-D213A、BisGlu16B-D253A和BisGlu16B-D213A/D253A突变体。三个突变体在65℃下的半衰期分别较野生型延长11min、28min和30min。动物肠道体温环境在37℃左右,酶在37℃下的催化活力提高有利于该酶在动物肠道中更好的发挥降解功能；且三种葡聚糖酶突变体的热稳定性均比野生型酶有不同程度的提高,这样的性质能够使得酶能够更好的抵抗高温制粒导致的酶失活。这样一种在动物体温下催化活力高、热稳定性优良的酸性葡聚糖酶在饲料加工、啤酒酿造及寡糖生产等领域中具有极大的应用潜力。(A group of animal body beta-1, 3-1, 4-glucanase mutants with high catalytic activity at room temperature and applications thereof comprise BisGlu16B-D213A, BisGlu16B-D253A and BisGlu16B-D213A/D253A mutants. Half-lives of the three mutants at 65 ℃ are respectively prolonged by 11min, 28min and 30min compared with the wild type. The body temperature environment of the animal intestinal tract is about 37 ℃, and the improvement of the catalytic activity of the enzyme at 37 ℃ is beneficial to better playing the degradation function of the enzyme in the animal intestinal tract; and the thermal stability of the three glucanase mutants is improved to a different extent compared with that of the wild enzyme, so that the enzyme can better resist enzyme inactivation caused by high-temperature granulation. The acidic dextranase with high catalytic activity and excellent thermal stability at animal body temperature has great application potential in the fields of feed processing, beer brewing, oligosaccharide production and the like.)

1. A group of animal body beta-1, 3-1, 4-glucanase mutants with high catalytic activity at room temperature is characterized by comprising BisGlu16B-D213A, BisGlu16B-D253A and BisGlu16B-D213A/D253A mutants, wherein the nucleotide sequence of BisGlu16B-D213A is shown in SEQ ID NO. 1; the nucleotide sequence of BisGlu16B-D253A is shown as SEQ ID NO. 2; the nucleotide sequence of BisGlu16B-D213A/D253A is shown as SEQ ID NO. 3; wherein, the sequence without signal peptide is used for the labeling of all mutation sites.

2. A group of animal body beta-1, 3-1, 4-glucanase mutants with high catalytic activity at room temperature is characterized in that the amino acid sequence of BisGlu16B-D213A is shown in SEQ ID NO. 4; the amino acid sequence of BisGlu16B-D253A is shown as SEQ ID NO. 5; the amino acid sequence of BisGlu16B-D213A/D253A is shown in SEQ ID NO.6, wherein the reference numerals of all mutation sites use the sequence without signal peptide removal.

3. A recombinant vector comprising any one of the nucleotide sequences set forth in claim 1.

4. A recombinant strain comprising the recombinant vector of claim 3.

5. Use of the recombinant strain of claim 4 for the preparation of a feed additive.

Technical Field

The invention relates to the fields of gene engineering and protein engineering, in particular to a group of beta-1, 3-1, 4-glucanase mutants with high catalytic activity at animal body temperature and application thereof.

Background

Cellulosic materials, including cellulose, hemicellulose and lignin, are the most abundant renewable resources common in nature. Cellulose is a macromolecular polymer formed by connecting D-glucopyranose with beta-1, 4 glycosidic bonds, and is also a main component of plant cell walls. Hemicellulose generally refers to other polysaccharide substances in the natural plant cell wall besides pectin and cellulose components, and mainly includes glucan, galactomannan and galactoglucomannan. Among these, β -glucans are a class of non-starch glucose polymers comprising β -glucose residues linked by β -glycosidic bonds. As a structural element, beta-glucan is widely present in plant cell walls, cereal seeds and some fungi and algae, wherein the beta-glucan content in barley and oat is highest and can reach 2-20g/100g dry weight (65% soluble) and 3-8g/100g dry weight (82% soluble), respectively (Khoury, 10.1155/2012/851362).

The beta-glucanase is a general term for endo-beta-1, 3-1, 4-glucanase and exo-beta-1, 3-1, 4-glucanase. The beta-1, 3 and beta-1, 4 glycosidic linkages of the glucan, which function as the domain, break down the beta-glucan into oligosaccharides. In the feed industry, beta-glucanase improves intestinal contents of monogastric animals, reduces chyme viscosity, promotes digestion and absorption of nutrients, and improves feed utilization rate by degrading beta-glucan (Beckmann 10.1002/jobm.200510107). In the beer processing industry, the beta-glucanase can efficiently hydrolyze beta-glucan in malt into glucose and oligosaccharide, destroy cell walls, release a large amount of cell contents, improve the wort yield, reduce the mash viscosity so as to shorten the mash filtration time, obtain clear wort, and make the prepared beer light in color and uniform and durable in foam (Gongchubo, 2002, Guangzhou food industry science and technology).

Aiming at the requirements of food industries such as feed industry, beer brewing industry and the like, the method still has great significance for obtaining the beta-glucanase with excellent characteristics.

Disclosure of Invention

The technical problem to be solved is as follows: the invention provides a group of beta-1, 3-1, 4-glucanase mutants with high catalytic activity at animal temperature and application thereof.

The technical scheme is as follows: a group of animal body beta-1, 3-1, 4-glucanase mutants with high catalytic activity at room temperature comprises BisGlu16B-D213A, BisGlu16B-D253A and BisGlu16B-D213A/D253A mutants, wherein the nucleotide sequence of BisGlu16B-D213A is shown in SEQ ID NO. 1; the nucleotide sequence of BisGlu16B-D253A is shown as SEQ ID NO. 2; the nucleotide sequence of BisGlu16B-D213A/D253A is shown as SEQ ID NO. 3; wherein, the sequence without signal peptide is used for the labeling of all mutation sites.

The amino acid sequence of BisGlu16B-D213A is shown in SEQ ID NO. 4; the amino acid sequence of BisGlu16B-D253A is shown as SEQ ID NO. 5; the amino acid sequence of BisGlu16B-D213A/D253A is shown in SEQ ID NO.6, wherein the reference numerals of all mutation sites use the sequence without signal peptide removal.

A recombinant vector comprising any one of the nucleotide sequences described above.

A recombinant strain contains the recombinant vector.

The recombinant strain is applied to the preparation of feed additives.

Has the advantages that: the invention provides a glucanase mutant which has excellent property and is suitable for catalyzing degradation of hemicellulose under the condition of animal body temperature. The optimum pH of the glucanase mutant is between 2 and 4.5, and the stability of the mutant D213A/D253A is better than that of the wild type at the pH of 1 to 12. In the aspect of optimum temperature adaptability, the optimum temperature of all three mutants is 60 ℃, but the relative enzyme activities of the mutants at 37 ℃ are respectively improved by 96%, 1.4 times and 89% compared with the wild type, and the relative enzyme activities of the mutants at 70 ℃ are respectively improved by 3.3 times, 70% and 1.1 times compared with the wild type. At 37 ℃, when oat glucan is taken as a substrate, the specific activities of three mutants of BisGlu16B-D213A, BisGlu16B-D253A and D213A/D253A are respectively improved by 78%, 52% and 90% compared with wild type BisGlu16B, and the catalytic efficiency is respectively improved by 5%, 16% and 58% compared with wild type; when lichenin is used as a substrate, the specific activities of the three mutants are respectively improved by 49 percent, 43 percent and 9 percent compared with the wild type, and the catalytic efficiencies are respectively improved by 55 percent, 45 percent and 1.1 times compared with the wild type; when laminarin is used as a substrate, the specific activities of the three mutants are respectively improved by 36 percent, 25 percent and 93 percent compared with the wild type, and the catalytic efficiency is not obviously different from that of the wild type. The combined mutant D213A/D253A has the most obvious catalytic activity improvement, and the catalytic efficiency is improved by 1.1 times (lichenin is used as a substrate) compared with the wild type by 93 percent (laminarin is used as a substrate) with the highest specific activity. In terms of thermostability, the half-lives of the three mutants at 65 ℃ were extended by 11min, 28min and 30min, respectively, compared to the wild type. The body temperature environment of the animal intestinal tract is about 37 ℃, and the improvement of the catalytic activity of the enzyme at 37 ℃ is beneficial to better playing the degradation function of the enzyme in the animal intestinal tract; and the thermal stability of the three glucanase mutants is improved to a different extent compared with that of the wild enzyme, so that the enzyme can better resist enzyme inactivation caused by high-temperature granulation. The acidic dextranase with high catalytic activity and excellent thermal stability at animal body temperature has great application potential in the fields of feed processing, beer brewing, oligosaccharide production and the like.

Drawings

FIG. 1 shows protein purification of high catalytic activity dextranase mutant and wild type;

FIG. 2 is the optimum pH of the mutant glucanase with high catalytic activity at 40 ℃ and the wild type;

FIG. 3 shows the pH stability of the mutant glucanase with high catalytic activity at 40 ℃ and the wild type;

FIG. 4 is the optimum temperature of the high catalytic activity dextranase mutant and the wild type;

FIG. 5 shows the thermostability of the mutant glucanase with high catalytic activity at 50 ℃ and the wild type.

Detailed Description

The invention is further described below with reference to the accompanying drawings and specific embodiments.

1. Bacterial strain and carrier: the expression host Pichia pastoris GS115 and the expression plasmid vector pPIC9r are self-prepared in a laboratory;

2. enzymes and other biochemical reagents: taq enzyme was purchased from Gentle gold, endonuclease was purchased from Fermentas, ligase was purchased from Promaga, and oat glucan was purchased from Sigma; other reagents are domestic analytical pure reagents (all purchased from the national pharmaceutical group);

3. culture medium:

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

(2) YPD medium: 1% yeast extract, 2% peptone, 2% glucose;

(3) MD solid medium: 2% glucose, 1.5% agarose, 1.34% YNB, 0.00004% Biotin;

(4) MM solid medium: 1.5% agarose, 1.34% YNB, 0.00004% Biotin, 0.5% methanol;

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

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

(7) the original nucleotide sequence of BisGlu16B is shown in SEQ ID NO. 7.

Example 1 construction of high catalytic Activity dextranase mutants

Random mutation is carried out on glucanase genes by adopting an error-prone PCR method by taking glucanase BisGlu16B from GH16 family as a starting material, and primers are shown in Table 1. The specific parameters are as follows: working concentration upregulation using TaqDNA polymeraseTo 2 times of normal PCR, Mg²⁺Concentration up-regulation to 4.7 times of normal PCR, increase dNTP concentration to 5 times of normal level (error-prone PCR research progress and application [ J)].2013,27(05):607-612.)。

Example 2 preparation of high catalytic Activity dextranase mutants

Carrying out double enzyme digestion (EcoR I + Not I) on an expression vector pPIC9r, carrying out double enzyme digestion (EcoR I + Not I) on genes encoding the glucanase mutant with high catalytic activity, connecting a gene fragment encoding a mature glucanase mutant with high catalytic activity after enzyme digestion with an expression vector pPIC9r to obtain a recombinant plasmid containing the glucanase mutant gene with high catalytic activity, converting the recombinant plasmid into pichia pastoris GS115 to obtain a recombinant yeast strain, screening the activity of the enzyme at 37 ℃ by using a high-throughput screening plate to obtain a mutant with higher activity than that of a wild type, carrying out gene sequencing on the mutant, setting primers as shown in table 1, and constructing a combined mutant by a site-directed mutagenesis method, wherein the primers are shown in table 1.

Inoculating the recombinant GS115 strain with improved activity into a 1L triangular flask of 300mL BMGY medium, and performing shake culture at 30 ℃ and 220rpm for 48 h; after this time, the culture broth was centrifuged at 3000g for 5min, the supernatant was discarded, and the pellet was resuspended in 100mL BMMY medium containing 0.5% methanol and again placed at 30 ℃ for induction culture at 220 rpm. 0.5mL of methanol is added every 12h, so that the concentration of the methanol in the bacterial liquid is kept at 0.5%, and meanwhile, the supernatant is taken for enzyme activity detection.

TABLE 1 error-prone PCR primers and combinatorial mutation construction primers

Example 3 Activity analysis of recombinant high catalytic Activity dextranase mutants and wild type

Enzyme activity determination of beta-1, 3-1, 4-glucanase

The activity of the beta-1, 3-1, 4-glucanase was determined according to the method previously reported by Yang et al (Yang 10.1021/jf800303b) with 1% (w/v, g/mL) of oat beta-glucan as substrate. The specific method comprises the following steps: under the given conditions of pH and temperature, 1mL of reaction system comprises 100 μ L of enzyme solution and 900 μ L of substrate, the reaction is carried out for 10min, 1.5mL of DNS is added to stop the reaction, and the reaction is boiled in boiling water for 5 min. After cooling, the OD was measured at 540 nm. The beta-1, 3-1, 4-glucanase activity unit (U) is defined as the amount of enzyme required to produce 1. mu. mol glucose-equivalent reducing sugars per minute under the above conditions.

Secondly, determining the properties of the mutant and the wild type of the recombinant glucanase with high catalytic activity

1. The optimum pH of the recombinant high catalytic activity dextranase mutant and the wild type is determined as follows:

the recombinant high catalytic activity dextranase mutant purified in example 2 and the wild type (FIG. 5) were subjected to enzymatic reactions at different pH to determine their optimum pH. The substrate (oat glucan) was diluted with 0.1mol/L citrate-disodium phosphate buffer at different pH and the glucanase activity assay was performed at 60 ℃.

The results (FIG. 1) show that the optimum reaction pH value of the recombinant high catalytic activity dextranase mutant and the wild type is between 2 and 4.5, and the same action trend exists in the pH range of 1.0 to 6.5 (FIG. 2). The purpose of improving the specific activity at lower temperature without changing the optimum pH value is met.

2. The optimal temperature of the recombinant high catalytic activity dextranase mutant and the wild type is determined as follows:

the optimal temperature of the recombinant high catalytic activity dextranase mutant and the wild type is determined by performing enzymatic reaction in a 0.1mol/L citric acid-disodium hydrogen phosphate buffer solution (pH 3.5) buffer solution system at different temperatures. The optimum temperature measurement result of the enzymatic reaction (figure 3) shows that the optimum temperature of the recombinant high catalytic activity dextranase mutant and the wild type is between 50 ℃ and 65 ℃, and the difference is not obvious. But catalytic activities at 40 ℃ were 1.75 times, 2.22 times and 1.71 times of those of the wild type, respectively.

3. The heat stability of the recombinant high catalytic activity dextranase mutant and the wild type at 65 ℃ is determined as follows:

the heat stability of the high catalytic activity dextranase mutant and the wild type is gradually reduced after the mutant and the wild type are treated at 65 ℃ for a certain time, but the heat stability of all the mutants is superior to that of the wild type. By fitting curves, it can be found that the half-life of mutant BisGlu16B-D213A is prolonged by 11min compared with that of the wild-type enzyme, while the half-life of mutant BisGlu16B-D253A is prolonged to 58min, the combined mutant BisGlu16B-D213A/D253A has the best effect, and the half-life is prolonged to 60min, which is twice that of the wild-type enzyme.

4. The dynamics of the mutant and the wild type of the recombinant high catalytic activity dextranase are determined as follows:

under the optimal conditions, the kinetic parameters and specific activities of the glucanase with high catalytic activity and the wild type are respectively measured by using three substrates of oat glucan, lichenin and laminarin, and the results are shown in the table 2.

Wherein, the K of BisGlu16B-D213A is the optimum condition with oat glucan as a substrate_mAnd V_m2.44mg/mL and 7200. mu. mol/min. mg. K of BisGlu16B-D253A_mAnd V_mRespectively 2.21mg/mL and 7100. mu. mol/min. mg. K of BisGlu16B-D213A/D253A_mAnd V_m1.76mg/mL and 7600. mu. mol/min. mg, respectively. Wild type K_mAnd V_m1.51mg/mL and 4200. mu. mol/min. mg, respectively.

K of BisGlu16B-D213A under optimal conditions by using lichenin as substrate_mAnd V_m2.37mg/mL and 5900. mu. mol/min. mg. K of BisGlu16B-D253A_mAnd V_mRespectively 2.41mg/mL and 5500. mu. mol/min. mg. K of BisGlu16B-D213A/D253A_mAnd V_m1.07mg/mL and 3500. mu. mol/min. mg, respectively. Wild type K_mAnd V_mRespectively 2.19mg/mL and 3600. mu. mol/min. mg.

K of BisGlu16B-D213A under optimal conditions by using laminarin as a substrate_mAnd V_m1.12mg/mL and 4000. mu. mol/min. mg. K of BisGlu16B-D253A_mAnd V_m1.04mg/mL and 3600. mu. mol/min. mg, respectively. K of BisGlu16B-D213A/D253A_mAnd V_m1.59mg/mL and 5200. mu. mol/min. mg, respectively. Wild type K_mAnd V_mRespectively 0.87mg/mL and 3000. mu. mol/min. mg.

Compared with wild type, the three mutants have greatly improved catalytic efficiency and specific activity, oat glucan is used as a substrate, and k of the three mutants_cat/K_m1.05, 1.15 and 1.57 times of wild type respectively, and the specific activity is 1.78, 1.68 and 1.90 times; k of three mutants using lichenin as substrate_cat/K_m1.54, 1.45 and 2.09 times of wild type respectively, and the specific activity is 1.48, 1.42 and 1.08 times; when laminarin was used as the substrate, the specific activities of the three mutants were 1.35, 1.25 and 1.92 times higher than those of the wild type.

In combination, the three mutants not only greatly improve the enzyme activity under the condition of animal body temperature, but also are superior to the wild glucanase in the aspect of heat stability. The superposition advantage of the combined mutation is weaker, but the mutant BisGlu16B-D213A/D253A is better in thermal stability than BisGlu16B-D213A and BisGlu16B-D253A to a certain extent.

TABLE 2 kinetics and specific activities of BisGlu16B and its mutants on different substrates under optimal conditions

Sequence listing

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