Site directed mutational mannose and application thereof

A mannanase and site-directed mutagenesis technology, applied in the field of mannanase, can solve problems such as destruction and lowering of use efficiency, and achieve the effects of improved trypsin resistance, wide pH stability, and improved trypsin tolerance

Inactive Publication Date: 2017-11-28
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this process, the feed enzymes will be damaged to varying degrees by the intestinal digestive enzymes of animals, which reduces the use efficiency. Enhanced β-mannanase, valuable for use of β-mannanase as a feed enzyme

Method used

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  • Site directed mutational mannose and application thereof
  • Site directed mutational mannose and application thereof
  • Site directed mutational mannose and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Synthesis of wtMAN47 and mtMAN47

[0028] The present invention takes the β-mannanase MAN47 gene (NCBI database ID: DQ286392) sequence of Armillariella tabescens as a reference, and adds 5'-GTC at the 5' end GAATTC -3' join 3'- at the 3' end CCTAGG GAC-5', ( GAATTC ) is the restriction enzyme cutting site EcoRI, ( GGATCC ) is the restriction enzyme cutting site BamHI. The wtMAN47 gene was synthesized by artificial total synthesis.

[0029] The present invention takes the β-mannanase MAN47 gene sequence (NCBI database ID: DQ286392) of Armillariella tabescens as a reference, and replaces the 197th amino acid with leucine (Leucine, Leu, L), and the 280th amino acid Substitute with Asparagine (Asparagine, Asn, N), replace the 380th amino acid with Alanine (Alanine, Ala, A), add 5'-GTC at the 5' end GAATTC -3' join 3'- at the 3' end CCTAGG GAC-5', ( GAATTC ) is the restriction enzyme cutting site EcoRI, ( GGATCC ) is the restriction enzyme cutting ...

Embodiment 2

[0031] Embodiment 2: Construction of recombinant plasmid wtMAN47 and mtMAN47 expression plasmid

[0032] Gene cloning was carried out according to conventional methods (Sambrook, et al.2001, Molecular Cloning A Laboratory Manual.Cold Spring Harbor Laboratory Press.USA), wtMAN47 and mtMAN47 obtained in Example 1 were respectively cloned into pHIL-S1 to construct expression plasmid pHIL-S1 -wtMAN47 and the expression plasmid pHIL-S1-mtMAN47, the cloned target gene was identified by enzyme digestion and sequencing.

[0033] specific methods:

[0034] The construction process of the recombinant plasmid pHIL-S1 containing mtMAN47 is as follows: EcoRI+BamHI double digestion of the plasmid pHIL-S1 and the target fragment mtMAN47, the digested products were subjected to 0.8% agarose gel electrophoresis, and the gel was cut and recovered. Ligation of plasmids pHIL-S1 and mtMAN47 was performed using T4 DNA ligase. CaCl 2 Prepare E.coli DH5α competent cells, transform DH5α competent c...

Embodiment 3

[0036] Embodiment 3: Expression of recombinant mtMAN47 and recombinant wtMAN47

[0037] Expression of recombinant mtMAN47: Digest recombinant plasmid pHIL-S1-mtMAN47 and plasmid pHIL-S1 with SacI, perform 0.8% agarose gel electrophoresis on the digested product, and recover linear recombinant plasmid pHIL-S1-mtMAN47 and Plasmid pHIL-S1. Transform Pichia yeast GS115 according to the protoplast method in the Pichia Expression Kit (Invitrogen Inc., U.S.) manual, and screen Mut + Turn. Using methanol as the sole carbon source to induce expression of recombinant bacteria (operated according to the PichiaExpression Kit manual), the SDS-PAGE electrophoresis analysis of the culture solution showed that after the transformants were induced to express, the supernatant of the culture solution showed obvious target protein bands, while The control bacteria transformed with an empty plasmid without the target gene were induced under the same conditions for 96 hours, and no target protein...

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Abstract

The invention discloses a site directed mutational mannose. The site directed mutational mannose is a mutant generated by manufacturing a plurality of amino acid substitutions in beta-mannose MAN47 derived from Armillariella tabescens, the amino acid sequence of the beta-mannose MAN47 is SEQ IDNO. 1, and the amino acid substitutions comprise the No. 197, the No. 280 and the No. 380 substitutions. The beta-mannose MAN47 mutant has the advantages of being high in temperature resistance, relatively wide in PH stability, and capable of improving the tolerance to trypsin.

Description

technical field [0001] The present invention relates to a site-directed mutation of mannanase, in particular to a mutant of β-mannanase MAN47 derived from Armillariellatabescens. Background technique [0002] β-mannanase (EC 3.2.1.78) is a general term for a class of enzymes that can hydrolyze β-1,4-glycosidic bonds on mannan and promote its hydrolysis to generate small oligomannose. According to the substrate specificity and action characteristics of the enzyme, β-mannanase can be divided into two types: exo-β-mannanase and endo-β-mannanase. Among them, endo-β-mannanase is the main enzyme that degrades β-mannan, and it mainly hydrolyzes mannan and isomannan (galactomannan, glucomannan, galactoglucomannan ) in the β-1,4-D-mannosidic bond. β-mannanases have strict bond specificity for substrates. [0003] β-mannanase widely exists in nature, in the seeds of animals (marine animals), sprouting plants (such as tomato, coffee bean, guar bean, konjac, square bean, etc.) and mi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56A23K20/189A23L33/10
CPCA23K20/189A23L33/10C12N9/2494C12Y302/01078
Inventor 姚冬生虞晓丹吕晓慧胡凤娟谢春芳刘大岭
Owner JINAN UNIVERSITY
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