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Method for improving thermal stability of GH10 xylanase through N-terminal replacement

A xylanase and heat-resistant technology, applied in the field of bioengineering, can solve the problems of difficult thermal stability, poor thermal stability, restricting the application of xylanase, etc., and achieve the effect of improving the optimum temperature and thermal stability

Inactive Publication Date: 2013-03-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, most of the xylanases produced in the industry are directly fermented by the original bacteria, and their optimum reaction temperature is mostly around 45-55°C, and their thermal stability is poor, which greatly restricts the use of xylanases in feed, Applications in high-temperature environments in papermaking and other industries, such as Alves-Prado et al. isolated a strain of high-yield xylanase bacteria Lysinibacillussp.strain P5B1 from the soil in the Cerrado region of Brazil. was 55°C; Menon et al. screened and isolated a salt-tolerant xylanase-producing Bacillus pumilus GESF1 from the sediments of the experimental salt farm of the Indian Institute of Marine Chemical Engineering, and separated and purified the xylanase. According to the study of chemical properties, the optimum temperature of the enzyme is 40°C. Although these xylanases have good enzymatic properties, they are difficult to be used in high-temperature manufacturing industries due to thermal stability problems.

Method used

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  • Method for improving thermal stability of GH10 xylanase through N-terminal replacement
  • Method for improving thermal stability of GH10 xylanase through N-terminal replacement
  • Method for improving thermal stability of GH10 xylanase through N-terminal replacement

Examples

Experimental program
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Effect test

Embodiment 1

[0016] Example 1 Construction of mutant enzyme

[0017] The T vector (pUCm-T-Aus xyn10A) containing the Aus Xyn10A gene was extracted, and the mature peptide primer Xyn10A-F1 and the primer XynCHNR1 of the Aus Xyn10A gene were used as a template for the first round of PCR. The reaction conditions were 94°C for 5 min; 2 cycles, 94°C 30s, 45°C 30s, 72°C 70s; 28 cycles of 94°C 30s, 55°C 30s, 72°C 30s; 72°C 10min; 10°C storage; then pUCm-T-Ausxyn10A as template, Use large primer PCR to use Xyn10A-R1 (5'-GCGGCCGCCTAGAGAGCATTTGCGATAG-3') and the first round of PCR products as primers for the second round of PCR. The reaction conditions are: 94℃ for 5min; 2 cycles, 94℃ for 30s, 45℃ 30s, 72℃70s; 28 cycles of 94℃30s, 55℃30s, 72℃70s; 72℃10min; 10℃ storage. Two rounds of PCR amplification products were analyzed by 1% agarose gel electrophoresis, the gel was tapped and the target band was recovered and ligated with pUCm-T vector (pUCm-T-ATx10AM), transformed into JM109, and sent to Shanghai...

Embodiment 2

[0018] Example 2 Construction of an expression plasmid containing the gene encoding the mature peptide of ATx10AM

[0019] Use EcoR I and Not I to double-enzyme digestion of the target gene recovered by tapping and pPIC9K. The digestion time is 4 hours. The digested product recovered from tapping is ligated overnight (>12h) under the action of T4DNA ligase to obtain the recombinant plasmid pPIC9K. -ATx10AM( figure 1 ), and sequence the recombinant expression plasmid.

Embodiment 3

[0020] Example 3 Construction, expression, product purification and activity determination of GS115 / ATx10AM

[0021] The pPIC9K-ATx10AM was linearized with Sal I, electrotransformed and screened according to the Pichia expression manual, and the high-copy Pichia recombinant GS115 / ATx10AM was obtained. The engineered bacteria was induced with 0.5% methanol for 72 hours, and the recombinant xylanase activity in the fermentation broth measured by DNS method reached 521 IU / mL. The supernatant of the centrifugation is the crude recombinant xylanase enzyme solution, which is concentrated by an ultrafiltration membrane with a molecular weight cut-off of 10kDa, and then purified by DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-75 gel filtration chromatography. SDS-PAGE detected a single band, and showed that the molecular weight of recombinant xylanase was 42kDa. The optimal temperature of the recombinant xylanase is 55°C, which is 10°C higher than the original enz...

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Abstract

The invention aims at providing a method for thermal stability modification of GH10 xylanase Aus Xyn10A and efficient expression and purification of recombinant mutant enzyme. According to the protein sequence comparison result between Aus Xyn10A (from aspergillus usamii E001) and heat-resistant xylanase (from thermoascus aurantiacus 751K6A), the sequence of the N-terminal area of the heat-resistant enzyme is replaced by a corresponding sequence of Aus Xyn10A by a genetic engineering method. The obtained mutant enzyme is named ATx10AM. The experimental result indicates that the optimal temperature and thermal stability of the enzyme after mutation are obviously improved; and as a heat-resistant enzyme preparation, the xylanase has relatively great industrial production potential and economic value.

Description

Technical field [0001] The invention relates to a GH10 xylanase modification and a highly efficient expression method of a mutant enzyme, and belongs to the technical field of biological engineering. Background technique [0002] Xylanase is a general term for a class of enzymes that can degrade xylan into xylo-oligosaccharides and xylose. In the narrow sense, xylanase specifically refers to β-1,4-xylanase (endo-β-1,4-xylanase, EC 3.2.1.8). β-1,4-endoxylanase can act on the xylosidic bond from the inside of the main chain and is one of the most important enzymes in the degradation of xylan. Most of the cloned xylanases currently belong to the F / 10 and G / 11 families. Compared with the 11th family, the 10th family of xylanase has low substrate specificity, fast hydrolysis rate, and low degree of polymerization of the hydrolysate. Great research and application value. Xylanase has broad application prospects in the fields of papermaking, food, energy, feed and environment, especi...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/42C12N15/10C12N15/81C12R1/66C12R1/84
Inventor 邬敏辰汪俊卿殷欣李剑芳
Owner JIANGNAN UNIV
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