Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Lipase mutant with improved enzyme activity and regioselectivity and application of mutant

A lipase and mutant technology, applied in the field of genetic engineering, can solve the problems of poor position selectivity, high cost, and low lipase activity

Active Publication Date: 2018-10-12
无锡优普克生物科技有限公司
View PDF1 Cites 9 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, most of the existing lipases are commercial lipases, which have the problem of high cost, and the lipases have the problems of low activity and poor position selectivity. It is necessary to further improve the activity and position selectivity to increase the yield of HMFS

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
  • Lipase mutant with improved enzyme activity and regioselectivity and application of mutant
  • Lipase mutant with improved enzyme activity and regioselectivity and application of mutant
  • Lipase mutant with improved enzyme activity and regioselectivity and application of mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: Construction of lipase mutant

[0023] Through the analysis of the interaction between the lipase leader peptide and the main protein structure, the key sites are respectively determined: E3, V5, D11 that interact with the lid, M8, T9, T9 that interact with the active center or are relatively close. L12, P17, L10 interacts with the lid and the active center, and T22 interacts with the main protein but is far from the active center. Mutations were designed based on the interaction between lipase leader peptide amino acids and the host protein. Primers were designed as follows:

[0024] Table 1 Leader peptide mutation primers

[0025]

[0026] Using a point mutation kit ( Lightning Site Directed Mutagenesis Kit, Stratagene, Agilent technologies, La Jolla, CA, USA) used the MBP-proRCL plasmid (Sha Chong, Ph.D. dissertation, Jiangnan University, 2015) as a template for full-plasmid PCR, and heat shock transformed Escherichia coli JM109 competent, const...

Embodiment 2

[0027] Example 2: Expression and purification of lipase mutants

[0028] Expression and purification method of mutants E3A, V5A, M8A, T9A, L10A, D11A, L12A, P17G, T22D:

[0029] (1) Optimization of expression and purification conditions: Pick a single colony of Escherichia coli BL21trxB(DE3) carrying the recombinant plasmid of the mutant gene on LB solid medium, and inoculate 500 μL of LB liquid medium (containing 100 μg·mL -1 Ampicillin antibiotic and 50 μg·mL -1 Kanamycin antibiotic) 37°C, 200rpm for 4-6h. Take 10 μL and inoculate 10 mL of liquid LB medium, and culture overnight at 37°C and 200 rpm. Transfer all 10mL of liquid LB medium to 200mL of liquid LB medium and cultivate to OD at 37°C and 200rpm 600 When the temperature reaches 0.6-0.8, take 20mL and add 1mM IPTG to 17°C, 29°C, and 37°C, respectively, and induce overnight, 6h, and 4h respectively. 1 L of fermentation broth was collected, and the cells were collected by centrifugation at 6000 rpm for 30 min, and t...

Embodiment 3

[0032] Example 3: Construction, expression and kinetic analysis of RCL leader peptide mutants

[0033] Pick a single colony of Escherichia coli BL21trxB(DE3) containing the recombinant plasmid on LB solid medium, and inoculate 500 μL LB liquid medium (containing 100 μg·mL -1 Ampicillin antibiotic and 50 μg·mL -1 Kanamycin antibiotic) 37°C, 200rpm for 4-6h. Take 50 μL and inoculate 50 mL of liquid LB medium, and culture overnight at 37°C and 200 rpm. Transfer all 50mL of liquid LB medium to a 2L shake flask containing 950mL of liquid LB medium and culture to OD at 37°C and 200rpm 600 After reaching 0.6-0.8, add 1mM IPTG to induce, and induce culture overnight at 17°C. SDS-PAGE was used to detect the whole cell protein and supernatant protein of each mutant.

[0034] The specific activity of the wild-type enzyme is 156.15 U / mg, and the relative activity of the wild-type is set as 100%. like figure 1 As shown, the specific enzyme activities of the wild enzyme WT and T9A are...

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 discloses a lipase mutant with improved enzyme activity and regioselectivity and application of the mutant and belongs to the technical field of gene engineering. According to the lipasemutant, wild-type lipase is mutated, the enzyme activity of mutants M8A, L10A, D11A, L12A and P17G is increased by 1.1-2.4 times compared with the enzyme activity of wild-type lipase, and the 1,3-regioselectivity of the mutants M8A, L10A and T9A is increased by 4.75-5 times compared with the regioselectivity of wild-type lipase. The enzyme activity and regioselectivity are important parameters ofthe lipase mutant in structural lipid catalytic application. The lipase mutant is applied to a catalytic reaction of human milk fat substitutes and has an obvious effect.

Description

technical field [0001] The invention relates to a lipase mutant with improved enzyme activity and position selectivity and application thereof, especially a lipase mutant with improved enzyme activity and position selectivity designed based on "interface activity" molecules, which belongs to the technical field of genetic engineering. Background technique [0002] Triacylglycerols (TAGs) are formed by the condensation of one glycerol molecule and three fatty acid molecules, accounting for more than 95% of edible oil components. Fatty acid composition, carbon chain length and saturation in TAGs affect its physicochemical and nutritional properties. Therefore, research on the structure, physicochemical properties and their impact on human health has always been the focus of oil scientists. Studies have shown that the acylation position distribution of fatty acids in TAGs has a very important impact on the oxidation stability, melting point, freezing point and nutritional value...

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
IPC IPC(8): C12N9/20C12N15/55A23D9/02A23L29/00
CPCA23D9/02A23L29/06C12N9/20C12Y301/01003
Inventor 喻晓蔚徐岩
Owner 无锡优普克生物科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com