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Method for improving thermal stability of aspergillus niger xylanase through N-glycosylation modification

A technology of thermostability and xylanase, applied in the field of genetic engineering, can solve problems such as poor thermostability

Pending Publication Date: 2021-10-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the most studied xylanases are derived from families 10 and 11. Among them, the xylanases of family 11 show a wide range of pH tolerance and substrate specificity, but their poor thermal stability is an urgent issue. problem to be solved

Method used

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  • Method for improving thermal stability of aspergillus niger xylanase through N-glycosylation modification
  • Method for improving thermal stability of aspergillus niger xylanase through N-glycosylation modification
  • Method for improving thermal stability of aspergillus niger xylanase through N-glycosylation modification

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Experimental program
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Effect test

Embodiment 1

[0032] The preparation of embodiment 1 xylanase mutant

[0033] (1) Construction of recombinant Aspergillus niger A.niger / xynA / hyg

[0034] Using the genome of Aspergillus niger (A. niger) as a template, the expression cassette of xylanase xynA was amplified by PCR by designing upstream and downstream primers F1 and R1 (Table 2). Using the pMD19 vector as a template, the pMD19 vector fragment was amplified by primers F2 and R2, and the xylanase xynA expression cassette and the pMD19 vector fragment were subjected to agarose gel electrophoresis, and the products were recovered by gelation. The recovered xylanase The xynA expression cassette and the pMD19 vector fragment were cloned and ligated in one step to obtain the recombinant vector pMD19-xynA, which was verified by sequencing, and a recombinant recombinant expressing the wild-type xylanase gene xynA (nucleotide sequence shown in SEQ ID NO.1) was successfully constructed Plasmid pMD19-xynA.

[0035] After the recombinant...

Embodiment 2

[0043] Example 2 Expression, purification and enzymatic property determination of XynA and mutants

[0044] The recombinant Aspergillus niger A.niger / xynA / hyg and A.niger / G53T / hyg, A.niger / A55N / D57S / hyg, A.niger / T59N / hyg, A.niger / S61N / hyg constructed in Example 1 , A.niger / T65N / hyg and A.niger / A55N / D57S / T59N / hyg were respectively inoculated in PDA liquid medium, and cultivated overnight at 28°C and 180rpm to obtain seed liquid, and the seed liquid was mixed with a volume ratio of 10 The amount of % was inoculated in 30mL of fresh PDA liquid medium. After culturing for 36 hours at 28°C and 180rpm, the culture solution was collected, and the culture solution was centrifuged at 12000rpm to collect the supernatant, and the supernatant was filtered through a 0.22μm filter membrane Use HisTrap TM FF purification, desalting column Sephadex G25 desalting to obtain purified wild-type enzyme xynA and mutant enzymes G53T, A55N / D57S, T59N, S61N, T65N and A55N / D57S / T59N, and determine th...

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Abstract

The invention discloses a method for improving thermal stability of aspergillus niger xylanase through N-glycosylation modification, and belongs to the technical field of genetic engineering. Through enzymatic property determination, the half-life period t1 / 250 DEG C of the mutant enzyme T59N is prolonged to 340 min, the enzyme activity of the mutant enzyme A55N / D57S / T59N is not reduced after the mutant enzyme A55N / D57S / T59N is incubated for 500 min at 50 DEG C, and only the loss of the enzyme activity is 25.6% after the mutant enzyme A55N / D57S / T59N is incubated for 1260 min. The specific enzyme activity of the mutant enzyme A55N / D57S / T59N is 290.5 U / mg and is improved by 70.4% compared with that of xynA, the mutant enzyme is very stable when the pH value is 2.0-9.0, 70% or above of enzyme activity can still be kept after incubation for 1 h, and the method has a wide application prospect.

Description

technical field [0001] The invention relates to a method for N-glycosylation modification to improve the thermal stability of Aspergillus niger xylanase, belonging to the technical field of genetic engineering. Background technique [0002] In food, medicine, feed and new energy, etc. Although xylanase has been studied for a long time and has a wide range of sources, its use is often limited due to problems such as low enzymatic hydrolysis efficiency and insufficient tolerance in the face of different application conditions. Xylanases are mainly distributed in families 5, 8, 10, 11, 30 and 43. At present, the most studied xylanases are derived from families 10 and 11. Among them, the xylanases of family 11 show a wide range of pH tolerance and substrate specificity, but their poor thermal stability is an urgent issue. problem to be solved. [0003] In the previous study, Wang Ling et al. compared xynA expressed in Pichia pastoris and Escherichia coli. The thermostability ...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/80C12N1/15C12R1/685
CPCC12N9/2482C12N15/80C12Y302/01008Y02E50/10
Inventor 刘松李阳阳陈坚堵国成
Owner JIANGNAN UNIV
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