Construction, production method and application of high-performance starch debranching enzyme chimera strain

A high-performance starch debranching enzyme technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve the problems of low enzyme activity and low saccharification efficiency, and achieve excellent performance and application effect Enhanced, good pH tolerance effect

Inactive Publication Date: 2021-11-19
南京纽田科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] For the existing pullulanase, there are disadvantages such as low saccharif

Method used

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  • Construction, production method and application of high-performance starch debranching enzyme chimera strain
  • Construction, production method and application of high-performance starch debranching enzyme chimera strain
  • Construction, production method and application of high-performance starch debranching enzyme chimera strain

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Experimental program
Comparison scheme
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Embodiment Construction

[0027] (1) Construction of pYF-tsDE plasmid

[0028] The schematic diagram of pYF-tsDE is shown in figure 1 As shown, it is prepared from pUC57-KS-erm synthesized by Nanjing Qingke Biotechnology Co., Ltd. as a raw material, and the specific method refers to CN107532155A.

[0029] (2) Construction of protease-deficient Bacillus subtilis strain

[0030] The present invention uses Bacillus subtilis as the host cell, which is directly used as the template of the PCR reaction after being processed, and the following primers are synthesized by Genscript, and are respectively used in the PCR reaction to amplify the flanking sequences Apr, Npr and SpoIIAC:

[0031] The primers for amplifying the upstream sequence of the Apr gene are:

[0032] pksb-Apr_czF1: GGTATCGATA AGCTTCCTGC AGATCTCTCA GGAGCATTTAACCT

[0033] pksb-Apr_R1: GCACCTACTG CAATAGTAAG GAACAGATTG CGCAT

[0034] The primers for amplifying the downstream sequence of the Apr gene are:

[0035] pksb-Apr_F2: ATGCGCAATC TGT...

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Abstract

The invention discloses construction, a production method and application of of a high-performance starch debranching enzyme chimera strain. A high-performance starch debranching enzyme chimera is obtained by effective chimeric combination of two parental pullulanase amino acid sequences, and the homology of the high-performance starch debranching enzyme chimera and the parental pullulanase amino acid sequences is not less than 95%; and the high-performance starch debranching enzyme chimera is obtained by carrying out chimeric combination on amino acid residues from site 1 to site 476 at the tail end of parental pullulanase of bacillus acidopullulyticus and amino acid residues from site 465 to site 976 at the tail end of parental pullulanase of bacillus deramificans. The chimeric pullulanase has a relatively high saccharification rate, also has higher heat resistance and thermal stability than the parental pullulanase, and is suitable for a saccharification reaction and starch liquefaction.

Description

technical field [0001] The invention relates to the technical field of starch debranching enzymes, in particular to the construction of high-performance starch debranching enzyme chimera strains and its production method and application. Background technique [0002] For the existing pullulanase, there are disadvantages such as low saccharification efficiency and low enzyme activity under acidic high temperature conditions. Therefore, in order to meet the needs of industrial production, it is urgent to solve the problem of saccharification efficiency of pullulanase and enzyme activity under acidic high temperature conditions (especially when the temperature reaches 60° C. and the pH value is lower than 4.5). Contents of the invention [0003] Based on the technical problems existing in the background technology, the present invention proposes the construction of a high-performance starch debranching enzyme chimera strain and its production method and application, which imp...

Claims

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Application Information

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IPC IPC(8): C12N9/44C12N15/75C12N1/21C12N9/26C12P19/16C12P19/14C12P19/02C12R1/125
CPCC12N9/2457C12N15/75C12N9/2411C12P19/16C12P19/14C12P19/02C12Y302/01041C07K2319/00
Inventor 范岩牟洋
Owner 南京纽田科技有限公司
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