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High-catalytic-efficiency xylanase mutant as well as construction method and application thereof

A technology for xylanase mutation and catalytic efficiency, which is applied in the field of high catalytic efficiency xylanase mutants and their construction, and can solve the problems of difficult acquisition of target strains, low frequency of beneficial mutations, and large workload of artificial mutagenesis

Active Publication Date: 2019-12-13
江苏宝源新能源科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mutagenesis is divided into natural mutation and artificial mutagenesis. The probability of success of natural mutation is very small, and the workload of artificial mutagenesis is relatively large and the frequency of beneficial mutation is still low. The direction and nature of mutation are difficult to control
The blindness of the screening is large, and it is not easy to obtain the target strain

Method used

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  • High-catalytic-efficiency xylanase mutant as well as construction method and application thereof
  • High-catalytic-efficiency xylanase mutant as well as construction method and application thereof
  • High-catalytic-efficiency xylanase mutant as well as construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Cloning of the coding gene of the high catalytic efficiency xylanase mutant of embodiment 1

[0054] The GH10 family high-temperature xylanase gene Xyl10C derived from Bispora sp.MEY-1 is used as the male parent, and the GH10 family xylanase gene XylE derived from Penicillium canescens is used as the female parent, in the active loop region of xylanase Design region replacement primers, and use the method of over-lap PCR to amplify the high catalytic efficiency xylanase mutant coding gene SEQ ID NO.1 (XylE-M3, 1023bp), SEQ ID NO.2 (XylE-M6, 1029bp ) and SEQ ID NO.3 (XylE-M9, 1047bp), mutation method and cloning method reference (You, et al., 2016).

[0055] The primer sequences used are shown in Table 1:

[0056] Table 1 List of primer sequences used

[0057]

Embodiment 2

[0058] The preparation of embodiment 2 high catalytic efficiency xylanase mutants

[0059] The expression vector pPIC9r was subjected to double enzyme digestion (EcoR I+Not I), and at the same time, the gene encoding the high catalytic efficiency xylanase mutant was double enzyme digested (EcoR I+Not I), and the cut coded mature high catalytic efficiency xylanase The gene fragment of the glycanase mutant (removing the signal peptide fragment) was connected with the expression vector pPIC9r to obtain a recombinant plasmid containing the high catalytic efficiency xylanase mutant gene and transform it into Pichia pastoris GS115 to obtain the recombinant yeast strain GS115 / XylE- M3, GS115 / XylE-M6 and GS115 / XylE-M9.

[0060] Take the GS115 strain containing the recombinant plasmid, inoculate it in a 1L Erlenmeyer flask with 300mL of BMGY medium, and culture it on a shaking table at 30°C and 220rpm for 48h; then centrifuge the culture solution at 3000g for 5min, discard the supernat...

Embodiment 3

[0062] Example 3 Activity Analysis of Recombinant High Catalytic Efficiency Xylanase Mutant and Wild Type

[0063] 1. DNS method: The specific method is as follows: under the given pH and temperature conditions, 1mL of the reaction system includes 100μL of appropriate diluted enzyme solution, 900μL of substrate, react for 10min, add 1.5mL of 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 produce 1 μmol reducing sugar per minute under given conditions.

[0064] 2. Determination of the properties of recombinant high catalytic efficiency xylanase mutants and wild type

[0065] 1. The optimal pH determination method of recombinant high catalytic efficiency xylanase mutant and wild type is as follows:

[0066] The recombinant high catalytic efficiency xylanase mutant of embodiment 2 purification and wild type are carried out enzymat...

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Abstract

The invention discloses a high-catalytic-efficiency xylanase mutant as well as a construction method and an application thereof, which relate to the field of gene engineering and genetic engineering.The method comprises the following steps: GH10 family high-temperature xylanase derived from Bispora sp. MEY-1 is taken as a male parent, gH10 family xylanase XylE derived from Penicillium canescens is used as a female parent, a molecular biological technology is adopted to replace a segment corresponding to the female parent with a segment in a male parent, and then segment combination and expression are carried out. Under the modification condition, the specific activity and the thermal stability of the xylanase mutant are remarkably improved compared with those of a wild type (before mutation). According to the method, the catalytic efficiency and temperature tolerance of xylanase are greatly improved, and conditions are created for the method in the field of industrial production.

Description

technical field [0001] The invention belongs to the field of genetic engineering and genetic engineering, and specifically relates to a xylanase mutant with high catalytic efficiency and its construction method and application. Background technique [0002] Cellulose, hemicellulose, and lignin are the main components of plant cell walls, accounting for about 50% of the world's biomass (Wong et al., 1988). Hemicellulose mainly exists on the surface of the cell wall, cellulose is wrapped in hemicellulose, and hemicellulose is covalently linked with lignin to form a network structure. Xylan is the main component of hemicellulose, a renewable resource second only to cellulose in nature, accounting for almost one-third of the renewable organic carbon content of the earth. The structure of xylan is complex, and its main chain is connected by xylopyranose by β-D-1,4-xylosidic bonds, and has various substituents (Collins et al., 2005). [0003] Xylanase can degrade the β-1,4-glyco...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/81C12P19/14C12P19/12C12P19/04C12P19/02C12P19/00
CPCC12N9/2482C12Y302/01008C12N15/815C12P19/14C12P19/12C12P19/04C12P19/02C12P19/00
Inventor 游帅王俊谢晨朱林琳盛晟吴福安
Owner 江苏宝源新能源科技有限公司
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