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

A kind of endo-type β-mannan hydrolase man01929 and its method and application of mutation into glycosyltransferase

A technology of mannanase and mutant enzymes, applied in the direction of transferase, hydrolase, glycosylase, etc., can solve the problems of little mechanism research, and achieve the effect of stable physical and chemical properties and wide pH tolerance

Active Publication Date: 2021-01-19
JINAN ACTIVE BIOTECHNOLOGY CO LTD
View PDF6 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Second, β-mannanase itself has a flexible structure and can recognize and bind a variety of substrates, but there are few mechanistic studies on which glycosyl binding sites are specifically involved in the selective recognition and action process of substrates
[0008] It has been reported in the literature above that this ability to transglycosylate is related to tryptophan (Trp) at the +2 subsite of β-mannanase, but whether amino acid residues at other subsites are involved in this regulatory mechanism, and Whether other types of amino acid residues are involved has not been reported

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
  • A kind of endo-type β-mannan hydrolase man01929 and its method and application of mutation into glycosyltransferase
  • A kind of endo-type β-mannan hydrolase man01929 and its method and application of mutation into glycosyltransferase
  • A kind of endo-type β-mannan hydrolase man01929 and its method and application of mutation into glycosyltransferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Extraction of Genomic DNA from Pyrobacterium pyrobacter MY04 Strain

[0088] Pyrochrome bacterium MY04 was inoculated into liquid medium YT04, and cultured with shaking at 28°C and 200 rpm until its absorbance at 600 nm (OD 600 ) is 1.2; take 10mL of the cultured bacteria solution, and centrifuge at 4°C for 15min under the condition of 12,000×g (g, the gravitational constant of the earth) to collect the bacterial precipitate; Suspend the bacteria, centrifuge at 12,000x g, 4°C for 15 minutes, and collect the bacteria pellet.

[0089] The above-mentioned liquid medium YT04 has the following components per liter:

[0090] Tryptone 10g, yeast extract 5.0g, sodium chloride 30g, dissolved in water and adjusted to 1L, pH 7.2.

[0091] Add 6.0mL of lysozyme buffer solution to each tube to obtain about 7.0mL of bacterial solution, and add 280μL of lysozyme solution with a concentration of 20mg / mL to make the final concentration of lysozyme 800μg / mL; Place in an ice-water bath...

Embodiment 2

[0093] Genome Scanning and Sequence Analysis of Pyrobacterium pyrobacter MY04 Strain

[0094] Genomic DNA prepared in Example 1 was scanned and sequenced by Shanghai Meiji Biotechnology Co., Ltd. using pyrosequencing technology. The DNA sequencing results were analyzed with the online software of NCBI (National Center for Biotechnology Information, http: / / www.ncbi.nlm.nih.gov / ) website. The analysis software used on the NCBI website is Open Reading Frame Finder (ORF Finder, http: / / www.ncbi.nlm.nih.gov / gorf / gorf.html) and Basic Local Alignment Search Tool (BLAST, http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0095] The results analyzed by the above-mentioned biological software show that the genomic DNA of the Pyrobacterium pyrobacterium MY04 strain carries a coding gene man01929 of β-mannanase, the coding region of the gene man01929 is 2799bp long, and the nucleotide sequence is shown in SEQ ID NO.1 . The β-mannanase Man01929 encoded by the gene man01929 contains a total of ...

Embodiment 3

[0098] Recombinant expression of gene man01929 in Escherichia coli BL21 (DE3) bacterial strain:

[0099] Using the genomic DNA prepared in Example 1 as a template, PCR amplification was performed. The primer sequences are as follows:

[0100] forward primer for man01929 amplification

[0101] Man01929-F: 5'-gcg CATATG GCACTTTTTGCTCATGC-3';

[0102] reverse primer for man01929 amplification

[0103] Man01929-F: 5'-gcg CTCGAG TTGCTTGTAGATTCTCCTAAC-3';

[0104] The underlined mark of the forward primer is the specificity site of the restriction endonuclease Nde I, and the underlined mark of the reverse primer is the specificity site of the restriction endonuclease Xho I.

[0105] The high-fidelity DNA polymerase Prime STAR HS DNA Polymerase used was purchased from China Dalian Bao Biological Company, and the PCR reaction reagents used were operated according to the product instructions provided by the company.

[0106] PCR reaction system:

[0107] 2×Primer star GC buff...

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

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
Login to View More

Abstract

The present invention relates to an endo-type β-mannanase Man01929 and a method for mutating it into a glycosyltransferase and its application. The amino acid sequence of the endo-type β-mannanase Man01929 is shown in SEQ ID NO.2 shown; the nucleotide sequence encoding Man01929 is shown in SEQ ID NO.1. The present invention conducts site-directed mutagenesis of the enzyme, and the results show that the 118th, 119th, 124th, and 323rd positions of the amino acid sequence play an important role in the process of Man01929's substrate selectivity in the recognition, binding and degradation of galactomannan . This has reference value for promoting the research on the substrate selectivity mechanism of GH5 family β-mannanase. In addition, the present invention also obtains the mutant D124Y with transglycosylation ability, which provides an important reference for revealing the conversion mechanism of the catalytic mechanism of hydrolase into glycosyltransferase.

Description

technical field [0001] The invention relates to an endo-type β-mannan hydrolase Man01929 and a method for mutating it into a glycosyltransferase and its application, which belongs to the field of biotechnology and relates to protein improvement technology. Background technique [0002] Mannan is a kind of polysaccharide with complex structure, which can be divided into four types according to the structure of sugar units: pure mannan, glucomannan, galactomannan ) and galactoglucomannan (galactoglucomannan). Due to the complex structure of mannan, the synergy of various enzymes is required for complete hydrolysis, such as β-mannanase (EC 3.2.1.78), β-mannosidase (EC 3.2.1.25), β-glucosidase (EC 3.2.1.21), acetylmannan esterase (EC 3.1.1.6), alpha-galactosidase (EC 3.2.1.22). β-mannanase is a kind of glycosyl hydrolase (Glycoside Hydrolase, GH), which plays a key role in the hydrolysis process. It can hydrolyze the β-1,4 glycosidic bonds inside the main chain of mannan to pr...

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 Patents(China)
IPC IPC(8): C12N15/56C12N9/24C12N9/10C12P19/14C12P19/00
CPCC12N9/1048C12N9/2494C12P19/00C12P19/14C12Y302/01078
Inventor 韩文君程媛媛宓延红古静燕李新卫洁
Owner JINAN ACTIVE BIOTECHNOLOGY CO LTD
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