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Neutral cellulase optimized gene as well as preparation method and application thereof

A neutral cellulase and gene technology, applied in the field of genetic engineering, can solve the problems of high price, few types of neutral cellulase, and little research on the production of neutral cellulase fermentation, and achieves enhanced enzyme activity, The effect of increased secretion expression

Pending Publication Date: 2022-03-25
WUHAN SUNHY BIOLOGICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the invention is to overcome the problems that neutral cellulase species are few in the prior art, the price is higher, and there are still few researches on the fermentation production of neutral cellulase at home and abroad

Method used

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  • Neutral cellulase optimized gene as well as preparation method and application thereof
  • Neutral cellulase optimized gene as well as preparation method and application thereof
  • Neutral cellulase optimized gene as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Cloning and sequence analysis of the target gene

[0032] The H.insolens Y1 used in this example was purchased from China General Microorganism Culture Collection and Management Center, CGMCC No.4573.

[0033] H. insolens Y1 was inoculated in the fermentation medium, cultured with shaking at 200 rpm at 42°C for 4 days, the cells were collected, total RNA was extracted and reverse-transcribed. Using total cDNA as a template, using primers

[0034] HiZXF: 5'-ATGCGTTCCTCCCCCCCTCC-3'

[0035] and

[0036] HiZXR: 5'-TTACAGGCACTGATGGTACC-3'

[0037] Amplify the HiZX gene encoding glycoside hydrolase, connect the target fragment into the cloning vector pEASY-Blunt, and verify it by sequencing.

[0038] The obtained sequence is compared with the homology of the full-length gene through the Blast online server; the signal peptide sequence of the protein is predicted through SigalP5.0 (https: / / services.healthtech.dtu.dk / service.php?SignalP-5.0) .

[0039] Sequenc...

Embodiment 2

[0046] Embodiment 2: Optimization of neutral cellulase gene

[0047] The sequence of the neutral cellulase wild-type gene HiZX cloned in H. insolens Y1 was optimized. The optimization process was mainly based on the following principles: 1) the amino acid sequence encoded by the cellulase gene HiZX was not changed; 2) according to the expression host Codon usage bias to minimize the probability of rare codon tandem; 3) According to the codon bias in Pichia pastoris, change the distribution range of its CAI value; 4) Control the GC content and distribution area in the gene sequence; 5) Avoid Emergence of high GC and continuous AT regions; 6) Optimization of the stem-loop structure (energy) of mRNA to extend the half-life of mRNA.

[0048] Through optimization design, we obtained three sequences HiZX-m1, HiZX-m2 and HiZX-m3, and their gene sequences are shown in sequence as SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4.

[0049] SEQ ID NO.2:

[0050] GCTGATGGTAAGAGTACGAGATACTGGGAT...

Embodiment 3

[0059] Embodiment 3: Construction of recombinant vector and recombinant strain

[0060] 1. Construction of recombinant vector

[0061] The HiZX gene and its mutant genes HiZX-m1, HiZX-m2 and HiZX-m3, which have removed their own signal peptide coding sequence, were recovered and purified after double enzyme digestion with EcoRI and NotI, and then connected into the expression vector pPIC9K cut by the same enzyme to construct Recombinant vectors pPIC9K-HiZX, pPIC9K-HiZX-m1, pPIC9K-HiZX-m2 and pPIC9K-HiZX-m3.

[0062] 2. Construction of recombinant Pichia pastoris

[0063] The constructed neutral cellulase expression plasmids pPIC9K-HiZX, pPIC9K-HiZX-m1, pPIC9K-HiZX-m2 and pPIC9K-HiZX-m3 were transformed into competent cells of Pichia pastoris GS115 to integrate them into the chromosome of Pichia pastoris , small-scale fermentation to screen transformants with neutral cellulase activity, the specific operations are as follows:

[0064] 1. Preparation of Pichia Competent Cells...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly provides a neutral cellulase optimized gene as well as a preparation method and application thereof. On the premise of not changing the amino acid sequence of the neutral cellulase, factors such as codon preference, codon adaptation index, deletion of an unstable sequence, an mRNA secondary structure and the like are comprehensively considered, the HiZX gene for coding the neutral cellulase is subjected to multi-strategy transformation, and finally an optimized gene sequence HiZX-m1 for coding the neutral cellulase is screened out. When the optimized gene HiZX-m1 is transferred into pichia pastoris for expression, the secretory expression quantity of the neutral cellulase is remarkably improved, and the enzyme activity is remarkably enhanced.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a neutral cellulase optimization gene HiZX-m1 and a production method and application thereof. Background technique [0002] Cellulase refers to the general term of a group of enzymes that can hydrolyze the β-1,4 glucosidic bonds of cellulose and turn cellulose into cellobiose and glucose. Cellulase is an inducible compound enzyme system composed of three main components: endoglucanase, exoglucanase and β-glucosidase. According to the difference in the optimum pH value, cellulase can be divided into Acid cellulose, neutral cellulase, alkaline cellulase. Neutral cellulase has broad application prospects in textile, food, feed and pharmaceutical industries, especially in the textile field. In terms of textile finishing, neutral cellulase has the advantage of high-contrast appearance compared with acid cellulase. Neutral cellulase can make the fabric have ...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/81C12N15/67C12N1/19C12R1/84
CPCC12N9/2437C12N15/815C12N2800/22Y02E50/10
Inventor 黄火清周樱詹志春张成杰辜玲芳刘文悦
Owner WUHAN SUNHY BIOLOGICAL
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