Thermostable chitosanase-producing engineered yeast strain and production method of thermostable chitosanase

A technology of chitosan enzyme and production method, applied in the field of microbial genetic engineering, can solve the problems of insufficient thermal stability to meet the requirements of industrialization, reduced enzyme activity, etc.

Active Publication Date: 2011-05-11
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Now commercialized chitosanase is still not enough to adapt to the requirements of industrialization in terms of thermal stability, for example, in many industrial applications, chitosanase is often used under hig

Method used

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  • Thermostable chitosanase-producing engineered yeast strain and production method of thermostable chitosanase
  • Thermostable chitosanase-producing engineered yeast strain and production method of thermostable chitosanase
  • Thermostable chitosanase-producing engineered yeast strain and production method of thermostable chitosanase

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1: Synthesis of a full-length gene encoding chitosanase and construction of Pichia pastoris genetically engineered bacteria using nested PCR method.

[0042] 1. Synthesize the full-length gene encoding chitosanase by nested PCR method. From the amino acid sequence of chitosanase reported from Aspergillus fumigatus (GenBank TM accession number AY190324), the gene encoding chitosanase was optimized according to the codon bias of Pichia pastoris. The optimized sequence is shown in Sequence List 1, and the primers (see Table 1) were designed. Except that hbcsn-1 is 26bp and hbcsn-24 is 55bp, the length of each primer is 50, and there is a gap of about 20bp between two adjacent primers. Overlapping sequences with Tm values ​​of 58-62°C. Then the full-length chitosanase gene was synthesized by nested PCR method.

[0043] Table 1 Primers for the synthesis of full-length chitosanase gene by nested PCR

[0044]

[0045] hbcsn-20

5'AGTGAAACCAATAAACAAA...

Embodiment 2

[0051] Example 2: Pichia pastoris genetically engineered strain HBCSN was cultured in a shake flask to prepare chitosanase.

[0052] The genetically engineered bacterium HBCSN is colonized into the BMGY medium, cultured at 28°C-30°C for 46-48 hours, centrifuged, and then the bacteria are transferred into BMM for induced expression. 100% methanol was added to the medium every 24 hours to a final methanol concentration of 1%. After six days of induction, the fermentation liquid was centrifuged, and the supernatant was the crude enzyme liquid containing chitosanase. Samples were taken every 24 hours to detect protein expression by SDS-PAGE, and the results were as follows image 3 shown.

Embodiment 3

[0053] Example 3: Preparation of Chitosanase by Scale-Up Culture of Pichia pastoris Genetic Engineering Bacteria HBCSN in a Fermenter.

[0054] 1. Seed preparation is as follows: a single colony is placed in BMGY medium, and cultured at 28°C-30°C for 46-48 hours.

[0055] 2. Inoculation: Add the seed medium into the fermenter at a ratio of 5% (volume of the seed medium / volume of the initial medium in the fermenter). The initial medium in the fermentor was BMGY.

[0056] 3. The whole fermentation process is divided into two stages:

[0057] A. The first stage is the long biomass stage, that is, the cell growth stage. The fermentation parameters are: temperature 28°C, pH 5.8, ventilation rate 0.5vvm, stirring speed 250-700 rpm, dissolved oxygen 30%. After 10-14 hours from the start of inoculation, the glycerol in the fermenter was consumed, and at this time, a 50% glycerol (W / V) mixture was added (that is, 12 mL of PTM1 was contained in every liter of 50% glycerol), when the ...

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Abstract

The invention provides a recombinant thermostable chitosanase-producing engineered Pichia pastoris GS115/HBCSN and a production method of thermostable chitosanase. The invention optimizes Aspergillus fumigatus chitosanase gene (GenBank accession number AY190324) based on the codon bias of Pichia pastoris, and the optimized nucleic acid sequence shows a 70% homology to the original nucleic acid sequence. The chitosanase produced by the recombinant Pichia pastoris can be secreted out of cells in a soluble form, with expression level of 3 mg/mL, and enzyme activity of 25,000U/mL. The chitosanase after glycosylation modification in Pichia pastoris has excellent thermal stability, which is proven by that the residual activities after treating at 80DEG C for 20 min, treating at 90DEG C for 40 min and treating at 100DEG C for 20 min are 56%, 87% and 87% respectively. The inventive thermostable chitosanase is especially suitable for large-scale industrial degradation of chitosan and environmental pollution reduction.

Description

technical field [0001] The invention relates to the construction of a high-temperature-resistant chitosanase yeast genetically engineered bacterium and a method for producing the high-temperature-resistant chitosanase. It belongs to the field of microbial genetic engineering. Background technique [0002] Chitosanase (Chitosanase, EC.3.2.1.132) is a specific enzyme for degrading chitosan, which is widely present in a variety of microorganisms. ), fungi (Penicillium, AspergiUus, Rhizopus, Basidiomyce), viruses (Chlorella virus PBCV-1, CVK-2) and plant tissues are found chitosanase, but mainly in bacteria and fungi. [0003] In recent years, domestic research on chitosanase has mainly focused on strain screening, optimization of strain fermentation conditions, enzymatic properties and separation and purification. In the existing commercialized chitosanase, due to the low enzyme production ability of the original strain, the purification rate and yield are not high, so the ou...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N1/19C12N9/42C12N15/81C12R1/84
Inventor 马立新余晓岚陈小梅
Owner HUBEI UNIV
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