Neutral cellulase mutant and application thereof

A technology of cellulase and mutants, applied in application, fiber treatment, biochemical fiber treatment, etc., can solve the problems of few types of neutral cellulase, low catalytic efficiency, short product storage period, etc., and achieve fabric strength loss Small, good effect, stable batch difference

Active Publication Date: 2015-11-11
QINGDAO VLAND BIOTECH GRP
View PDF2 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there are few types of neutral cellulase currently available on the market, and there are generally disadvantages such as low catalytic efficiency, short product storage period, and rapid loss of enzyme activity. Therefore, it is urgent to develop a product with high stability and high catalytic efficiency per unit protein. Neutral cellulase with little loss of fabric strength to meet the needs of industrial production

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Neutral cellulase mutant and application thereof
  • Neutral cellulase mutant and application thereof
  • Neutral cellulase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Obtaining of cellulase mutants

[0021] In order to improve the tolerance and stability of wild-type cellulase NCE4 (the amino acid sequence is SEQIDNO: 1, and its encoding nucleotide sequence is SEQIDNO: 2) to temperature, the applicant has carried out a large number of amino acid sites of the enzyme Point mutation screening, and extensive substitution screening of the zymogen's CBD-binding domain sequence. During the screening process, the applicant found that: some mutations have no effect on the heat resistance of cellulase, while some mutations can even lead to worse heat resistance, and some mutations can significantly improve the heat resistance of cellulase , but the enzyme activity level is very low, not suitable for industrial production. Finally, the applicant screened a combination of mutation sites and CBD binding domain sequences that can significantly improve the heat resistance and thermostability of cellulase without affecting its enzyme ...

Embodiment 2

[0026] Expression of embodiment 2 cellulase mutants

[0027] 2.1 Construction of expression vector

[0028] The synthesized cellulase mutant gene sequence and pTG vector were digested with restriction endonucleases KpnI and XbaI (Fermentas), respectively, and the digested products were purified using a gel purification kit, and T4DNA ligase (Fermentas) was used to The above two digested products were ligated and transformed into Escherichia coli Trans5α (Transgen), selected with ampicillin, and the clones were sequenced (Invitrogen). After the sequencing is correct, the recombinant vector pTG-NCE4-D containing the cellulase mutant gene is obtained.

[0029] The recombinant vector pTG-NCE4 containing the wild-type cellulase NCE4 gene was constructed by the same method as above.

[0030] 2.2 Construction and screening of recombinant strains

[0031] (1) Protoplast preparation

[0032] Inoculate Trichoderma reesei (Trichodermareesei) SCHD4 mycelia on a PDA plate, and culture ...

Embodiment 3

[0043] Embodiment 3 fermentation verification and enzyme activity assay

[0044] Trichoderma reesei NCE4-D (TrichodermareeseiNCE4-D) obtained by the above-mentioned construction and Trichodermareesei NCE4 (TrichodermareeseiNCE4) were respectively inoculated in MM fermentation medium (1.5% glucose, 1.7% lactose, 2.5% corn steep liquor , 0.44% (NH 4 ) 2 SO 4 , 0.09% MgSO 4 , 2% KH 2 PO 4 , 0.04% CaCl 2 , 0.018% Tween-80, 0.018% trace elements, 0.018% polypropylene glycol-2000), cultivated at 28°C for 48 hours, then cultivated at 25°C for 48 hours, centrifuged to get the supernatant. The enzyme activities were measured respectively.

[0045] (1) Enzyme activity assay method

[0046] Under the conditions of 50°C and pH value of 4.8 (neutral is pH 6.0), the amount of enzyme needed to degrade and release 1 μmol of reducing sugar from a sodium hydroxymethylcellulose solution with a concentration of 5 mg / ml per minute is one enzyme The unit of activity is U, and the reducing ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention aims to provide a neutral cellulase mutant. The amino acid sequence of the neutral cellulase mutant is shown in SEQ ID NO: 5. The optimum action temperature of the cellulase mutant is 60 DEG C, the optimum action pH of the cellulase mutant is 6.5, and an enzyme activity level above 80% can be kept within the pH range from 6.0 to 7.0; however, the optimum action pH of wild cellulase is 5.5, and enzyme activity above 80% is kept within the pH range from 5.0 to 6.5. Compared with the wild cellulose, the pH range of the cellulase mutant is more suitable for the neutrality condition, and therefore the cellulase mutant has a broader application range. In addition, the heat resistance and stability of the cellulase mutant are significantly improved, and residual activity above 90% still can be kept after the cellulase mutant is stored for 35 days at 42 DEG C; however, a residual rate lower than 60% of the wild cellulase only can be kept under the condition.

Description

technical field [0001] The invention belongs to the technical field of functional gene modification, and specifically relates to a neutral cellulase mutant and application thereof. Background technique [0002] Cellulase, a compound enzyme, is a complex enzyme system composed of a variety of hydrolytic enzymes. It is customary to divide cellulase into three categories: endoglucanase, exoglucanase and β-glucose sidase. Cellulase is one of the most widely used enzymes in industry. It is generally used in textile industry, detergent industry, pulp and paper industry, feed and food industry (including baking), and for hydrolysis of lignocellulosic materials. For example in bioethanol production etc. [0003] Cellulases can be classified according to their primary sequences into various glycosyl hydrolases families, which is supported by the analysis of the three-dimensional structures of some members of the families (Henrissat 1991, Henrissat and Bairoch 1993, 1996). For exam...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N1/15D06M16/00C12R1/885
CPCC12N9/2437D06M16/003
Inventor 刘艳萍张青李瑞许韦李宾许丽红黄亦钧王华明
Owner QINGDAO VLAND BIOTECH GRP
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap