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GH10 family high-temperature-resistant xylanase mutants and application thereof

A technology of xylanase mutation and high temperature resistance, which is applied in the field of bioengineering, can solve the problems of inability to express xylanase in large quantities, cannot take into account thermal stability and catalytic activity, and achieve excellent thermal stability and improved thermal stability , the effect of high enzyme activity

Active Publication Date: 2021-09-28
JIANGSU UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of them cannot take into account thermal stability and catalytic activity, and due to the characteristics of natural microorganisms, they often cannot express xylanase in large quantities

Method used

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  • GH10 family high-temperature-resistant xylanase mutants and application thereof
  • GH10 family high-temperature-resistant xylanase mutants and application thereof
  • GH10 family high-temperature-resistant xylanase mutants and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Cloning of embodiment 1 thermostable xylanase mutant coding gene

[0032] Using the high-temperature-resistant xylanase gene HwXyl10A from the GH10 family as the parent, design mutant primers at the loop region of xylanase, and use over-lap PCR to amplify the gene encoding a mutant xylanase 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), mutation method and Cloning method reference (You, et al., 2016).

[0033] The primer sequences used are shown in Table 1:

[0034] Table 1 Primer Synthesis List

[0035]

Embodiment 2

[0036] The preparation of embodiment 2 thermostable xylanase mutants

[0037] The expression vector pPIC9r was subjected to double enzyme digestion (EcoR I+Not I), and at the same time, the gene encoding the high-temperature-resistant xylanase mutant was double-enzyme-digested (EcoR I+Not I), and then the enzyme-digested gene encoding the mature high-temperature-resistant xylanase The gene fragment of the mutant glycanase is connected with the expression vector pPIC9r to obtain a recombinant plasmid containing the gene of the mutant xylanase resistant to high temperature and transform it into Pichia pastoris GS115 to obtain a recombinant yeast strain.

[0038]Take the GS115 strain containing the recombinant plasmid, inoculate it in a 1L Erlenmeyer flask with 300mL of BMGY medium, place it at 30°C, and culture it on a shaker at 220rpm for 48h; then centrifuge the culture solution at 3000g for 5min, discard the supernatant, and use 100mL of 0.5% methanol for precipitation. The B...

Embodiment 3

[0039] Example 3 Activity Analysis of Thermostable Xylanase Mutant and Wild Type

[0040] 1. DNS method: the specific method is as follows: under the given pH and temperature conditions, 1mL reaction system includes 100μL enzyme solution, 900μL substrate, react for 10min, add 1.5mL DNS to terminate the reaction, and boil for 5min. After cooling, the OD value was measured at 540 nm. One enzyme activity unit (U) is defined as the amount of enzyme required to decompose xylan to generate 1 μmol reducing sugar per minute under given conditions.

[0041] 2. Determination of the properties of recombinant thermostable xylanase mutants and wild type

[0042] 1. The optimal pH of recombinant thermostable xylanase mutant and wild type is determined as follows:

[0043] The recombinant thermostable xylanase mutant purified in Example 2 and the wild type were subjected to enzymatic reactions at different pHs to determine their optimum pH. The substrate (beech wood xylan) was diluted wit...

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Abstract

The invention provides GH10 family high-temperature-resistant xylanase mutants and application thereof. The mutants are HwXyl10A-G363R, HwXyl10A-T324V, HwXyl10A-N318W and HwXyl10A-N318W / G363R / T324V mutants; and the half-life periods of the four mutants at 75 DEG C are prolonged by 12min, 20min, 24min and 38min respectively compared with those of wild mutants. The xylanase mutants have high catalytic activity and excellent thermal stability at animal body temperature and have great application potential in feed addition and biomass degradation.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a group of GH10 family thermostable xylanase mutants and applications thereof. Background technique [0002] The existing feed generally uses corn, bran, wheat or rice as the main raw material, which contains a considerable amount of non-starch polysaccharides, including cellulose, hemicellulose, pectin and the like. Cellulose is a macromolecular polymer composed of D-glucopyranose linked by β-1,4 glycosidic bonds, and it is also the main component of plant cell walls. Hemicellulose generally refers to other polysaccharides other than pectin and cellulose components in natural plant cell walls, mainly including xylan, galactomannan and galactoglucomannan. The structure of xylan is very complex, the main chain is composed of xylopyranose connected by β-1,4-D-xylosidic bonds, the degree of polymerization is between 150-200, and the side chain contains various forms of sub...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/81C12N1/19A23K20/189C12R1/84
CPCC12N9/248C12N15/815A23K20/189C12N2800/102
Inventor 游帅陈忠立庄愉储呈平葛研王俊
Owner JIANGSU UNIV OF SCI & TECH
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