JP7449605B2 - GH10 family high temperature resistant xylanase mutants and their use - Google Patents

Description

The present invention belongs to the field of biotechnology, and specifically relates to a group of high temperature resistant xylanase mutants of the GH10 family and methods for their construction and uses.

Conventional feeds are generally made from corn, bran, wheat, rice, or the like as main raw materials, and contain large amounts of non-starch polysaccharides, including cellulose, hemicellulose, and pectin. Cellulose is a large polymer obtained by linking D-glucopyranos with β-1,4 glycosidic bonds, and is also the main component of plant cell walls. Hemicellulose generally refers to other sugar chain substances other than pectin and cellulose components in plant cell walls in nature, and mainly includes xylan, galactomannan and galactoglucomannan. The structure of xylan is very complex; the main chain consists of xylopyranose linked by β-1,4-D-xyloside bonds, the degree of polymerization is between 150 and 200, and the side chains contains multiple types of substituents or groups of substituents, common substituents include arabinose, gluconic acid, galactose, ferulic acid, acetic acid and coumaric acid (Kulkarni et al., 1999, Liab et al., 2000, Squina et al., 2009). However, non-ruminants lack the enzymes that break down non-starch polysaccharides, so it is very difficult for them to digest hemicellulose by itself, easily forming a gruel-like lump that can cause problems in the animal's intestinal tract. Affect the internal environment. On the one hand, it leads to a change in the type of intestinal microbial population of the animal. On the other hand, the gruel-like texture affects the animal's feed absorption, significantly reducing the feed utilization rate and increasing the cost of feed feeding.

By adding appropriate xylanase to the feed, the cell walls of plant cells can be crushed, causing the hydrolysis of hemicellulose, releasing the cell contents, and realizing the purpose of reducing the viscosity of the gruel-like meal. Significantly increases feed utilization and reduces feed costs. Hydrolysis products of xylan are oligosaccharides, of which xylobiose 1) prevents tooth decay, 2) lowers blood fat, blood pressure, and blood sugar, 3) promotes mineral absorption, and 4) suppresses bacteria. It also has excellent health care functions, such as growing bifidobacteria, which has the effect of increasing immunity and improving vitamin metabolism.

However, in the industrial preparation of feed, the working environment temperature is relatively high, and the high-pressure pelletizing process of feed is accompanied by instantaneous high temperatures. It is required to have excellent thermal stability even in environments such as Although xylanases are widely distributed in nature, there are not many xylanases that are resistant to high temperatures. Among them, xylanase derived from microorganisms is the material most commonly used in current practical use and research. However, many of them are incompatible with thermostability and catalytic activity, and they are often unable to express xylanase in large amounts due to the characteristics of natural microorganisms.

Considering the difficulty of screening xylanases of natural origin and their adverse properties, rational, semi-rational or random targeted evolution, mutation, segmental recombination, hybrid construction and activity of natural enzymes. Research using methods such as site mutation technology and constructing high expression process strains are currently the main methods to obtain superior xylanases through modification.

The present invention provides a group of high temperature resistant xylanase mutants of the GH10 family and uses thereof. This group of mutants is derived from the xylanase HwXyl10a of the fungus Hortaea werneckii, and through homologous sequence comparison and structural analysis, a rational design for the thermal stability of xylanase HwXyl10A was developed for use in industrial production. and target improvement.

A group of high temperature resistant xylanase mutants of the GH10 family, the mutants being HwXyl10A-G363R, HwXyl10A-T324V, HwXyl10A-N318W and HwXyl10A-N318W/G363R/T324V mutants, The nucleotide sequence is SEQ ID NO. The nucleotide sequence of HwXyl10A-T324V is shown in SEQ ID NO. The nucleotide sequence of HwXyl10A-N318W is shown in SEQ ID NO. The nucleotide sequence of HwXyl10A-N318W/G363R/T324V is shown in SEQ ID NO. 4.

The amino acid sequence of HwXyl10A-G363R is shown in SEQ ID NO. The amino acid sequence of HwXyl10A-T324V is shown in SEQ ID NO. The amino acid sequence of HwXyl10A-N318W is shown in SEQ ID NO. The amino acid sequence of HwXyl10A-N318W/G363R/T324V is shown in SEQ ID NO. 8.

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

A recombinant strain containing the above recombinant vector.

Use of the above-mentioned high temperature resistant xylanase mutants of the GH10 family in the preparation of feed additives.

Use of the above recombinant strain in the preparation of feed additives.

The method for constructing high temperature resistant xylanase mutants is to select HwXyl10A as a starting material, design single site mutations through multi-sequence comparison and structural analysis, and identify key amino acids that may affect its thermostability. a step of preliminary screening of sites, a step of designing target mutation primers for the key sites obtained by screening, a step of combining each mutation site by employing an over-lap PCR method, and a step of combining each mutation site with xylanase mutation. Cloning the sequence fragment of the mutant between the EcoR I and Not I restriction enzyme recognition sites of the expression vector pPIC9r to obtain a recombinant plasmid; Transforming into a GS115 receptor state and inducing expression to obtain recombinant bacteria, culturing the recombinant bacteria, inducing them to express xylanase mutants, and recovering xylanase mutants. and purification.

The present invention provides a group of xylanase mutants with excellent thermostability and suitable for feed addition. The optimal pH value of this group of xylanase mutants is 3.5 to 5.5, which does not change much compared to the wild type, and the pH stability is similar to that of the wild type, with a pH of 2 to 9. Both maintain high enzyme activity within a certain range. The optimal temperature for this group of xylanase mutants is 75°C, and the thermostability at 75°C is improved to different extents compared to the wild enzyme, mainly for the mutant HwXyl10A-G363R. The half-life of HwXyl10A-T324V was extended by 20 min compared to the wild type, the half-life of HwXyl10A-N318W was extended by 24 min compared to the wild type, and the half-life of HwXyl10A-N318W/G363R/T324V was extended by 24 min compared to the wild type. This is reflected in the fact that the half-life period is extended by 38 min compared to the wild type. Feed pelleting involves an instantaneous high-temperature process (80-90℃, 3-5s), which increases the thermal stability of the enzyme, making it more advantageous for the enzyme-resistant high-temperature pelleting process, and finally Avoid activation. Xylanase, which has relatively high enzyme activity under acidic pH environment and animal body temperature conditions and is stable under high temperature conditions, is considered to be a feed additive enzyme with excellent properties in the feed industry, and is suitable for applications. The prospects are very wide.

Protein purification of high temperature resistant xylanase mutants and wild type. Optimal pH of high temperature xylanase mutants and wild type. pH stability of high temperature xylanase mutants and wild type. Optimal temperature for high temperature tolerant xylanase mutants and wild type. Thermostability of high temperature xylanase enzyme mutants and wild type at 75°C. _T50 values of high temperature resistant xylanase enzyme mutants and wild type.

Hereinafter, the present invention will be described in conjunction with the accompanying drawings and specific examples.

1. Strain and vector: Expression host Pichia pastoris GS115, expression plasmid vector pPIC9r were prepared in the laboratory.

2. Enzymes and other biochemical reagents: Pfu enzyme was purchased from TransGen Biotech, endonuclease was purchased from Fermentas company, ligase was purchased from Promaga company, Beechwood xylan was purchased from , are purchased from Sigma company, and all others are purchased from Analytical Reagents Made in China (all are purchased from Guo Pharmaceutical Group).

3. Medium:
(1) LB 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% glycerin (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).

Example 1 Cloning of a gene encoding a high temperature resistant xylanase mutant Using the high temperature resistant xylanase gene HwXyl10A derived from the GH10 family as a parent, mutation primers were designed in the loop region of xylanase, and an over-lap PCR method was employed. Gene encoding a xylanase mutant with high catalytic efficiency SEQ ID NO. 1 (HwXyl10a-G363R), SEQ ID NO. 2 (HwXyl10a-T324V), SEQ ID NO. 3 (HwXyl10a-N318W), SEQ ID NO. 4 (HwXyl10a-N318W/G363R/T324V) and referred to the literature (You, et al., 2016) for mutation and cloning methods.

The primer sequences used are shown in Table 1.

Example 2 Preparation of high temperature resistant xylanase mutants The expression vector pPIC9r was double digested (EcoR I+Not I), and the gene encoding the high temperature resistant xylanase mutant was double digested (EcoR I+Not I), and after digestion The gene fragment encoding the mature high temperature resistant xylanase mutant was ligated to the expression vector pPIC9r to obtain a recombinant plasmid containing the high temperature resistant xylanase mutant gene, which was transformed into Pichia pastoris GS115 to produce a recombinant yeast. The bacterial strain was obtained.

The GS115 strain containing the recombinant plasmid was inoculated into a 1L Erlenmeyer flask containing 300mL of BMGY medium, placed in a shaker at 30°C and 220rpm, and cultured for 48h.Then, the 3000g culture solution was centrifuged for 5min, the supernatant was discarded, and the precipitate was precipitated. The cells were resuspended in 100 mL of BMMY medium containing 0.5% methanol, and culture was induced again at 30° C. and 220 rpm. To maintain the methanol concentration in the bacterial solution at 0.5%, 0.5 mL of methanol was replenished every 12 hours, and the supernatant was collected and used for enzyme activity detection.

Example 3 Activity analysis of high temperature resistant xylanase mutant and wild type 1. DNS method: The specific method was as follows. Under specific pH and temperature conditions, 1 mL of reaction system contained 100 μL of enzyme solution and 900 μL of substrate, reacted for 10 min, added 1.5 mL of DNS to stop the reaction, and boiled in boiling water for 5 min. . After cooling, the OD value was measured at 540 nm. One unit (U) of enzyme activity was defined as the amount of enzyme required to decompose xylan and produce 1 μmol of reducing sugar per minute under predetermined conditions.

2. Measurement of properties of recombinant high temperature resistant xylanase mutant and wild type 1. Measurement of optimal pH of recombinant high temperature resistant xylanase mutant and wild type was as follows.

The recombinant high-temperature-resistant xylanase mutant purified in Example 2 and the wild type were subjected to enzymatic reactions at different pHs, and their optimal pHs were determined. The substrate (Beachwood xylan) was diluted with 0.1 mol/L citric acid-disodium hydrogen phosphate buffer solutions of different pH, and the xylanase activity was measured at 75°C.

As a result (Fig. 2), the optimal reaction pH values for the recombinant high temperature resistant xylanase mutant and the wild type are 3.5 to 5.5, and similar within the range of pH 2.0 to 9.0. It had a tendency of action (Fig. 3). The objective of increasing the thermostability of the enzyme without changing the optimum pH was met.

2. Determination of the optimal temperature of the recombinant high temperature resistant xylanase mutant and the wild type was as follows.

Determination of the optimal temperature of recombinant high-temperature resistant xylanase mutants and the wild type was performed using a buffer system of 0.1 mol/L citric acid-disodium hydrogen phosphate buffer (pH 3.5) and at different temperatures. The purpose was to perform an enzymatic reaction. As a result of measuring the optimal temperature for enzyme reaction (FIG. 4), the optimal temperature of the recombinant high temperature resistant xylanase mutant and the wild type was 70 to 80°C, and the difference between them was not clear.

3. Measurement of thermostability at 75°C of the recombinant high temperature resistant xylanase mutant and the wild type was as follows.

The high-temperature-tolerant xylanase mutants and wild type were treated at 75°C for a certain period of time, respectively, and the concentrations of all mutants and wild type at the time of treatment were 100 μg/mL and the volumes were 100 μL, and different After sampling at the time point, the cells were immediately placed on ice, and the enzyme activity was measured at 75° C. and pH 4.0 to evaluate the thermostability of the mutant and wild type.

The thermostability of all mutants was better than that of the wild type, and after treatment at 70°C for 26 min, the residual enzymatic activity of wild type xylanase was reduced to 50% (Figure 5), and after treatment for 38 min. When continued, the residual enzyme activity of mutant G363R was reduced to 50%, but under the same conditions, the half-life of mutant T324V was 46 min, which was 20 min longer than that of the wild type. Among all the mutants, the combination mutant N318W/G363R/T324V showed the best performance in terms of thermostability, reaching 64 minutes.

4. Measurement of T50 values of the recombinant high temperature resistant xylanase mutant and the wild type was as follows.

The thermostability at 75°C could indicate the thermostability of the enzyme, and the T50 value could also reflect the thermostability of the enzyme. After treating four mutants and the wild type at different temperatures (60°C to 85°C) for 30 min, fitting results showed that the T50 value of the wild type enzyme was 74°C, but that of G363R was 76. The T50 value of T324V reached 77.5°C, and the T50 value of N318W was even higher, reaching 78.5°C. The highest T50 value was for the combination mutant N318W/G363R/T324V at 80°C.

Example 4 Kinetic analysis of high temperature resistant xylanase mutants and wild type The kinetic parameters and specific activities of four xylanase mutants and wild type were measured using Beachwood xylan substrate under optimal conditions, and the results were are shown in Table 2, respectively.

The Km values of the four xylanase mutants and the wild type are 0.88 mg/mL, 1.03 mg/mL, 0.96 mg/mL, 1.27 mg/mL and 1.04 mg/mL, respectively, but 4 The kcat values of the xylanase mutants and the wild type of the species are 2100s-1, 2300s-1, 1900s-1, 2700s-1 and 2400s-1, respectively, and the catalytic efficiency of the four xylanase mutants and the wild type (kcat/Km) were 2400 mL/s·mg, 2200 mL/s·mg, 2000 mL/s·mg, 2100 mL/s·mg, and 1700 mL/s·mg, respectively. The specific activities of the four xylanase mutants have different degrees of improvement compared to the wild-type enzyme, with G363R increasing by 1.32 times compared to the wild type, and T324V increasing by 1 times compared to the wild type. The increase in specific activity was the most obvious, with N318W increasing by 1.5 times compared to the wild type, while the combination mutation increased by 1.21 times compared to the wild type. .

Overall, the enzyme activity of the four mutants is not lost after mutation, and their thermostability is significantly increased compared to the wild type, allowing them to withstand the instantaneous high temperatures during feed pelleting. . In addition, if the enzymatic activity is not lost and the stability is increased, it means that this group of high temperature resistant xylanase mutants can remain active for a longer time and be able to catalyze the hydrolysis of hemicellulose, making it suitable for industrial applications. It was a good material.

Claims (6)

A nucleic acid encoding a high temperature resistant xylanase mutant protein of the GH10 family, said mutant protein being H wXyl10A-N318W or HwXyl10A-N318W/G363R/T324V mutant protein ,
The nucleotide sequence of the nucleic acid encoding the H wXyl10A-N318W mutant protein is SEQ ID NO. The nucleotide sequence of the nucleic acid shown in SEQ ID NO.3 and encoding said HwXyl10A-N318W/G363R/T324V mutant protein is SEQ ID NO. 4. A nucleic acid encoding a high temperature resistant xylanase mutant protein of the GH10 family characterized by the following. A high temperature resistant xylanase mutant protein of the GH10 family,
The mutant protein is HwXyl10A-N318W or HwXyl10A-N318W/G363R/T324V mutant protein,
The amino acid sequence of the H wXyl10A-N318W mutant protein is SEQ ID NO. The amino acid sequence of the HwXyl10A-N318W/G363R/T324V mutant protein is shown in SEQ ID NO. 8. A high temperature resistant xylanase mutant protein of the GH10 family characterized by the following: A recombinant vector comprising any one of the nucleotide sequences shown in claim 1. A recombinant strain comprising the recombinant vector according to claim 3. Use of a nucleic acid encoding a high temperature resistant xylanase mutant protein of the GH10 family according to claim 1 or a high temperature resistant xylanase mutant protein of the GH10 family according to claim 2 in the preparation of a feed additive. Use of the recombinant strain according to claim 4 in the preparation of feed additives.

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