Mutant enzyme of type I pullulanase and its preparation method and application

A pullulanase, mutant technology, applied in microorganism-based methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of harsh growth conditions, harsh culture conditions, unfavorable sampling and screening, etc., Achieve high enzymatic activity and enhanced thermal stability

Active Publication Date: 2021-11-23
INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The microbial sources of heat-resistant pullulanase are mostly thermophilic or extreme thermophilic microorganisms, which have harsh growth conditions and harsh culture conditions, which are not conducive to sampling and screening

Method used

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  • Mutant enzyme of type I pullulanase and its preparation method and application
  • Mutant enzyme of type I pullulanase and its preparation method and application
  • Mutant enzyme of type I pullulanase and its preparation method and application

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Embodiment Construction

[0035] The present invention will be further described in detail below in conjunction with the accompanying drawings, so that those skilled in the art can implement it with reference to the description.

[0036] It should be noted that the experimental methods described in the following embodiments, unless otherwise specified, are conventional methods, and the reagents and materials, unless otherwise specified, can be obtained from commercial sources.

[0037] 1. Raw material preparation

[0038] Biological materials: as shown in Table 1

[0039] Table 1 Biomaterials

[0040]

[0041] Construction method of pET-22b(+)-pulF:

[0042] The accession number and microbial source were WP_011994577.1 (Fervidobacterium nodosum Rt17-B1) as the original enzyme. For the convenience of expression, this pullulanase was named PulF.

[0043] After removing the signal peptide coding sequence from the gene sequence of pullulanase PulF, the gene fragment derived from Fervidobacterium nodo...

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Abstract

The invention discloses a mutant enzyme of type I pullulanase, and the amino acid sequence of the mutant enzyme is shown in SEQ ID No.1. The present invention also discloses the nucleotide sequence of the gene encoding the mutant enzyme as shown in SEQ ID No.2. The invention also discloses a method for preparing mutant enzymes, including performing homology modeling according to the amino acid sequence of type I pullulanase, selecting mutation sites and designing pairs of mutation primers based on sequence comparison analysis and protein structure analysis; Using the vector carrying type I pullulanase gene as a template, carry out site-directed mutagenesis respectively to obtain mutant plasmids; replicate and express the corresponding mutant enzymes in engineering bacteria transformed with the mutant plasmids. The mutant enzyme of the invention has better heat resistance and specific enzyme activity.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and enzyme engineering. More specifically, the present invention relates to a mutant enzyme of type I pullulanase and its preparation method and application. Background technique [0002] At present, many pullulanases have been excavated, and even pullulanases have been studied at the genetic level. However, very few of them can be used in industrial production. At present, the most commercialized pullulanase is derived from Bacillus acidopullulyticus. The main application method of pullulanase is used in the saccharification process of starch. The industrial application conditions are pH4.5-5.5, temperature 55-65℃ or even higher, and the duration of action is as long as 48-60h (NISHA and SATYANARAYANA, 2016 ). Most of the reported type I pullulanases are unable to adapt to this industrial condition. Some enzymes have poor heat resistance, and some enzymes have very low enzyme acti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/44C12N15/56C12N15/70C12N1/21C12P19/16C12P19/04C12R1/19
CPCC12N9/2457C12N15/70C12P19/04C12P19/16C12Y302/01041
Inventor 逄晓阳吕加平杨洋芦晶张书文李伟勋朱青徐琛
Owner INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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