Heat-resistant mannosidase gene as well as expression protein and application thereof

A mannosidase and heat-resistant technology, applied in the field of bioengineering, can solve the problems of low enzymatic hydrolysis efficiency, cost problems, and hard structure of agricultural and sideline products, and achieve the effect of great superiority, strong heat resistance, and single product

Active Publication Date: 2022-04-05
HUAIYIN INSTITUTE OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing production of mannose oligosaccharides mainly relies on the use of various enzyme preparations combined with hydrolysis substrates such as konjac flour, yeast, coconut meal and other agricultural and sideline products, but there are many problems: 1. The raw materials of konjac flour are expensive, and the practical application is greatly limited
3. Coconut meal agricultural and sideline products have a hard structure and low enzymatic hydrolysis efficiency. The raw materials need to be pretreated, which causes certain cost problems and environmental protection problems
However, similar methods are rarely used in the production of mannooligosaccharides because mannosidases rarely exhibit transglycosidic properties.

Method used

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  • Heat-resistant mannosidase gene as well as expression protein and application thereof
  • Heat-resistant mannosidase gene as well as expression protein and application thereof
  • Heat-resistant mannosidase gene as well as expression protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The preparation of embodiment 1 mannosidase gene

[0030] Using the amino acid sequence of Pseudothermotoga thermarum DSM 5069 mannosidase (NCBI number: AEH51527.1) as a template, the non-conserved site of mannosidase was obtained as a potential transformation site through database comparison; the three-dimensional site after saturation mutation was simulated by computer structure, use AutoDock to simulate the interaction between mannose and protein, and select the optimal modification scheme. The modified sites are shown in Table 1. The modified amino acid sequence was codon-optimized according to the codon preference of Escherichia coli, and the gene was artificially synthesized. The gene sequence is shown in SEQ NO:1.

[0031]

[0032]

[0033] Table 1 Amino acid modification sites

Embodiment 2

[0034] Construction and verification of embodiment 2 recombinant cloning, expression vector pET-28a-PtMan

[0035] The purified PCR product (prepared in Example 1) and pET-28a (Novagen) were double-digested with NdeI and XhoI respectively, and agarose electrophoresis was used to recover the enzyme-cut PCR and large vector fragments. Add 1 μL of 10X Ligase Buffer and 1 μL of Ligase to the target fragment recovered by tapping the gel and connect it overnight at 16°C. Escherichia coli DH5α was transformed with the product of the ligation reaction, and then spread on a petri dish containing 100 μg / mL Kana (Kanapenicillin), and incubated at 37° C. for 10-15 hours.

[0036] Pick multiple single colonies from the transformation plate, and use BIOMIGA's plasmid mini-extraction kit to extract the plasmid. The obtained plasmid was verified by double enzyme digestion and the obtained recombinant plasmid was sequenced. Sequencing results showed that the cloned target fragment (3033bp in...

Embodiment 3

[0037] Embodiment 3 Expression and purification of recombinant mannosidase

[0038] The recombinant clone and expression vector pET-28a-PtMan (prepared in Example 2) were heat-shock transformed into the host bacterium E.coliBL21 (DE3) (Novagen) to obtain the recombinant bacterium containing the recombinant plasmid. A single colony of recombinant bacteria was inoculated into 5 mL of Luria-Bertani broth (LB) medium containing 100 μg / mL kanapenicillin, and cultured at 37° C. with shaking at 200 rpm for 4 h. Inoculate the above 4mL bacterial solution into a 2000mL shake flask containing 800mL of culture medium, shake at 37°C at 200rpm, and when the absorbance reaches 0.4-0.6, add 800μL of 0.1M IPTG, and incubate at 22°C at 150rpm Induce expression for 15h. The culture solution was centrifuged at 6000 rpm for 10 min at 4°C with a high-speed refrigerated centrifuge to collect the bacteria. Wash with 50mL ultrapure water and centrifuge at 6000rpm at 4°C for 10min, recover the bacte...

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Abstract

The invention discloses a heat-resistant mannosidase gene as well as an expression protein and application thereof, and mannosidase better meeting requirements is obtained by carrying out directional modification on enzyme through related technical means of protein engineering. The mannosidase has the characteristics of better high temperature resistance and high activity under a slightly acidic pH (Potential of Hydrogen) condition. The enzyme activity is the highest under the conditions that the temperature is 75 DEG C and the pH value is 6.0, and the specific enzyme activity reaches 8.2 mu mol/mg min; the mannosidase has relatively high enzyme activity at the temperature of 70 to 75 DEG C and the pH value of 6 to 6.5. The heat resistance of the mannosidase is extremely high, and the enzyme activity can be kept above 80% after the mannosidase is incubated for 1 hour in an environment with the temperature of 70-75 DEG C and the pH value of 4.5-7. Compared with the mannosidase before modification, the mannosidase has the transglycosylation capability, mannodisaccharide and mannotriose with the total sugar content of about 10% can be converted after 2 days under the conditions that the mannose concentration is 60% (w/w), the pH value is 6, and the temperature is 55 DEG C. Compared with the mannodisaccharide and mannotriose produced by an existing enzymolysis method, the mannosidase has the advantages that the product is relatively single and pure, the implementation is simple and convenient, and the mannosidase has greater superiority.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and relates to a mannosidase, in particular to a heat-resistant mannosidase gene, its expressed protein and its application. Background technique [0002] Mannose oligosaccharides are oligosaccharides linked by D-mannose through β-1,4 glycosidic bonds, and the degree of polymerization is between 2-10. As a kind of beneficial body, it can regulate human intestinal bifidobacteria and lactic acid bacteria Maintain intestinal microecological balance. The existing production of mannose oligosaccharides mainly relies on the use of various enzyme preparations combined with hydrolysis substrates such as konjac flour, yeast, coconut meal and other agricultural and sideline products, but there are many problems: 1. The raw materials of konjac flour are expensive, and the practical application is greatly limited . 2. The content of mannose in yeast cell wall is low. 3. Coconut meal agricultural and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/70C12P19/12C12P19/14C12P19/00C12N1/21C12R1/19
Inventor 时号言行聂新玲李青飞高凤王士岩李相前
Owner HUAIYIN INSTITUTE OF TECHNOLOGY
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