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Genetic engineering xylanase and preparation and application of genetic engineering xylanase

A kind of genetic engineering, xylanase technology, applied in genetic engineering, application, glycosylase and other directions, to achieve the effect of convenient purification, small molecular weight and improved thermal stability

Active Publication Date: 2015-01-28
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The sequence, structure, and function of natural xylanases are all limited by natural evolution, and they are often not in an optimal functional state when applied in industrial pathways

Method used

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  • Genetic engineering xylanase and preparation and application of genetic engineering xylanase
  • Genetic engineering xylanase and preparation and application of genetic engineering xylanase
  • Genetic engineering xylanase and preparation and application of genetic engineering xylanase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The cultivation and preservation of embodiment 1, C.bescii DSM 6725 bacterial strain

[0042] According to the formula of DSMZ, configure anaerobic medium (anaerocellum medium), pack it into anaerobic test tubes, each tube is 5ml, after the medium is closed, use a vacuum pump to dry the air in the test tube, and fill it with a certain amount of nitrogen and carbon dioxide Mixed gas (80% N 2 and 20% CO 2 ), dissolve the Caldicellulosiruptor bescii DSM 6725 purchased from DSMZ with 1ml of culture medium in an anaerobic box, inoculate it into a 5ml test tube according to 1% of the inoculum, mix well, and culture it statically at 75°C for several days until the bacteria start to grow . Then transfer to a Erlenmeyer flask containing 100ml of culture medium and culture at 75°C for several days, and store the cells at -80°C.

Embodiment 2

[0043] Embodiment 2, the extraction of genomic DNA

[0044] Take the C.bescii DSM 6725 cultured in a 5ml small test tube, and use the Bacteria Genomic Mini Preparation Kit from Beijing Puboxin Biotechnology Co., Ltd. to extract the bacterial genome, and put the resulting genomic DNA solution at 4°C Refrigerator for spare.

Embodiment 3

[0045] Embodiment 3, the cloning of target gene

[0046] Using the C.bescii DSM 6725CbX (Gene ID: 222454987) gene sequence (ie, SEQ ID NO: 1) reported by GenBank as a template, two specific primers were designed with Primer Premier 5.0, and the target gene was obtained by PCR. Primers are as follows:

[0047] P1:5'-TGCG CCATGG ATATGAGGTTTAAAAAGT-3' (underlined is Nco I restriction site)

[0048] P2:5'-AGCGTG AAGCTT TCATTGTATTAACAAAT-3' (underlined is the restriction site of Hind III)

[0049] The target gene and vector were subjected to Nco I and Hind III double enzyme digestion treatment, the system is as follows:

[0050]

[0051] The purified digested product was ligated by T4 ligase according to the molar ratio of the target gene to the vector of 10:1, and the ligated product was transformed into Escherichia coli BL21(DE3)-CodonPlus-RIL. During the construction of the recombinant plasmid, the complete target gene is cloned, and the His-tag on the vector is not in...

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Abstract

The invention discloses genetic engineering xylanase which is a mutant, wherein a signal peptide is truncated at the tail end N of xylanase CbX, and meanwhile, efficient expression of a xylanase gene in a heterologous host cell is achieved. According to the genetic engineering xylanase disclosed by the invention, xylanase is modified, a carbohydrate combined structural domain at the terminal C is truncated and a disulfide bond is introduced at the tail end C, so that the molecular weight of the constructed xylanase protein mutant is smaller, the thermal stability is improved, and the specific vitality is further greatly improved. Due to the adoption of the manner, the xylanase and the mutant of the xylanase are of more potential and are applied to industrial production.

Description

technical field [0001] The invention belongs to the field of genetic engineering of enzymes, and relates to a xylanase, in particular to a highly thermostable endo-xylanase and a genetically engineered bacterium for producing the highly thermostable endo-xylanase. Background technique [0002] Xylanase (Xylanase, EC.3.2.1.8) is the most critical enzyme in the xylan degrading enzyme system, and is often used in aspects such as feed, food, beverage, pharmacy, paper industry and bioenergy (Beg, Q.K.Kapoor , M. Mahajan, L. Hoondal, G.S (2001) Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol. 56(3-4):326-338). Especially in the paper industry, xylanase hydrolyzes the hemicellulose coagulated on the surface of the fiber during the pre-bleaching of the pulp and deinking of the secondary fiber, so as to increase the diffusion speed of the bleaching agent in the pulp, improve the bleaching efficiency, and reduce the The amount of chlorine us...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56
CPCC12N9/2482C12Y302/01008
Inventor 杨广宇冯雁安娇于瑶王慧楠张少博
Owner SHANGHAI JIAO TONG UNIV
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