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Codon optimized pullulanase, expression vector thereof and construction method of expression vector

A technology of pullulanase and expression vector, applied in the field of codon-optimized pullulanase and its expression vector and construction, which can solve the problem of low pH adaptability, unsuitability for large-scale industrial production, poor industrial shape of strains, etc. problem, to achieve the effect of increasing production

Active Publication Date: 2018-05-04
NANJING FOODSAFETY BIOTECH CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Pullulanase derived from acid-resistant Pullulanbacterium (B.acidpullulyticus) has good low pH adaptability and good temperature stability, and can adapt to high temperature and high temperature in starch processing, fermentation industry, and high maltose syrup production process. Acidic environment has a wide range of application prospects, but the original acid-resistant Pullulan bacteria fermentation enzyme activity is too low, the industrial shape of the strain is poor, and it is not suitable for large-scale industrial production

Method used

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  • Codon optimized pullulanase, expression vector thereof and construction method of expression vector
  • Codon optimized pullulanase, expression vector thereof and construction method of expression vector
  • Codon optimized pullulanase, expression vector thereof and construction method of expression vector

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Embodiment 1

[0029] Embodiment 1: Pullulanase gene acquisition

[0030]In order to obtain pullulanase with good activity under acidic conditions, the invention refers to the pullulanase amino acid sequence (Accession: 2WAN_A, GI: 229597615) of acid-resistant Pullulan bacteria (Bacillus acidpullulyticus), and according to the amino acid sequence (SEQ ID NO.1), based on the codon of Bacillus subtilis 168, optimize its base composition, so that it can be better expressed in Bacillus subtilis. The optimized base sequence (SEQID NO.2) is 2763bp in total, which does not contain common restriction endonuclease cutting sites, which is convenient for subsequent operations. The absence of additional promoter sequences and ribosomal recognition sites prevents the production of additional products. An mRNA stabilizing sequence (from the crystal protein cry3A mRNA stabilizing sequence (SEQ ID NO.3) of Bacillus thuringiensis) was added before the pullulanase gene. The designed pullulanase gene was synt...

Embodiment 2

[0031] Example 2: Construction of autonomously replicating expression plasmids

[0032] Self-replicating plasmids can be any shuttle plasmids that can replicate in Bacillus subtilis and E. coli, such as pWB980, pHP13, pHP13-43, pHT01, pHT43, pHT304, pMK3, pMK4, pHCMC04, pHCMC05, pMA5, pBE, etc. any of the In this embodiment, the pHT43 plasmid is taken as an example to describe the construction process of an autonomously replicating plasmid, and the principles and processes of other plasmid constructions are the same. Amplify the pullulanase gene (SEQ ID NO.2), the PCR conditions are as follows: pre-denaturation at 95°C for 5 minutes. Denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 2min; 30 cycles. Final extension at 72°C for 10 min. After the product was recovered by the PCR product recovery kit (OMEGA), it was connected to the T-vector pEASY-blunt (full type gold biology). Transform Escherichia coli DH5α competent cells according to the met...

Embodiment 3

[0035] Example 3: Selection of self-replicating plasmid promoter and signal peptide

[0036] The expression of foreign gene is closely related to its promoter sequence, and the secretion of protein is related to its signal peptide. Different foreign proteins require different promoters and signal peptides. In this case, we experimented with the effects of different promoters and signal peptides on the enzyme activity of pullulanase fermentation. Based on the plasmid pHT43-pul constructed in Example 2, the promoter and signal peptide were removed by double digestion with restriction endonucleases KpnI and BamHI to form a vector that can verify the effects of different promoters and signal peptides. In this embodiment, one of the promoters and signal peptides (amyQ promoter and amyL signal peptide) is taken as an example to describe the construction of the vector (see figure 2 , pHT43-amyL-chiA-pul), the principle and method of vector construction for other promoters and signa...

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Abstract

The invention discloses codon optimized pullulanase, an expression vector thereof and an construction method of the expression vector. On the basis of codon preference of bacillus subtilis 168 and onreference to pullulanase genes of bacillus acidpullulyticus, a base sequence is designed and expressed in bacillus subtilis with a method of autonomously replicating plasmids and integrative plasmids.A promoter, an mRNA stable sequence, signal peptide and a combination of the promoter, the mRNA stable sequence and the signal peptide of an expression cassette is selected, and a combination capableof performing efficient secretory expression in the bacillus subtilis is obtained. Industrial production is realized and meanwhile, the enzyme activity stability is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a method for improving the expression of pullulanase in Bacillus subtilis by means of codon optimization and changing promoters and signal peptides, in particular to a codon-optimized pullulanase and its Expression vector and construction method. Background technique [0002] Pullulanase is a debranching enzyme that can specifically cut the α-1,6 glycosidic bond at the branch point of amylopectin, thereby cutting off the entire side branch to form amylopectin. Among the cereal starches used in industry, the content of amylopectin accounts for about 75%-85%, and amylopectin contains about 4%-5% of α-1,6 glycosidic bonds. At present, the production of alcohol, amino acids, nucleotides, antibiotics, etc. by industrial fermentation requires the use of a large amount of starch, which is hydrolyzed by amylase and glucoamylase to generate monosaccharides or disaccharides as the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N15/70C12N15/66
CPCC12N9/2457C12N15/70C12Y302/01041
Inventor 李金良孟凡强陆兆新吕凤霞杨娟
Owner NANJING FOODSAFETY BIOTECH CO LTD
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