Food-grade safe saccharomycetes for producing chondroitin sulfate lyase ABCI and application of food-grade safe saccharomycetes

A technology of chondroitin sulfate and lyase, which is applied in the field of bioengineering and can solve the problems of limited application and the safety of chondroitin sulfate cannot be guaranteed.

Active Publication Date: 2021-11-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, at present, chondroitin sulfate lyase is mostly expressed in Escherichia coli, and the safety of the chondroitin sulfate thus prepared cannot be guaranteed, which limits its application in many fields

Method used

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  • Food-grade safe saccharomycetes for producing chondroitin sulfate lyase ABCI and application of food-grade safe saccharomycetes
  • Food-grade safe saccharomycetes for producing chondroitin sulfate lyase ABCI and application of food-grade safe saccharomycetes
  • Food-grade safe saccharomycetes for producing chondroitin sulfate lyase ABCI and application of food-grade safe saccharomycetes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Construction of secreted and expressed recombinant Pichia pastoris GS115 / GAPZαB-ABCI

[0034]According to the chondroitin sulfate lyase derived from Proteus vulgaris, the coding gene ABCI was optimized through yeast codon preference to obtain the codon sequence shown in SEQ ID NO.1. The codon-optimized ABCI sequence was amplified with primers ABCI-F / R, and the vector GAPZαB was digested with EcoRI / KpnI, the amplified ABCI fragment and the digested vector were recovered separately, and obtained by one-step cloning Recombinant plasmid / GAPZαB-ABCI. The plasmid was linearized with fast cutting enzyme AvrII and then transformed into Pichia pastoris GS115 to obtain recombinant strain GS115 / GAPZαB-ABCI.

[0035] Streak the recombinant strain on the plate, pick a single colony and inoculate it in 5mL YPD liquid medium, cultivate it at 30°C and 220rpm for 16h, then transfer it to 50mL YPD medium according to 10% (v / v) inoculation amount, and place it in 30 Cultivate...

Embodiment 2

[0036] Example 2: P GAP Promoter mutations increase the expression of ABCI

[0037] Since the initial secreted expression of ABCI was not high, the expression of the protein was further improved through promoter engineering. In order to facilitate the screening, ABCI needs to be expressed intracellularly, and at the same time, the fluorescent protein eGFP is connected after the ABCI gene, and the expression level of the protein is indicated by the intensity of fluorescence.

[0038] Use primer 2ABCI-F / R to amplify the codon-optimized ABCI sequence, and eGFP-F / R to amplify the eGFP gene fragment. The vector GAPZB is digested with the fast-cutting enzyme EcoRI / KpnI, and the amplified ABCI fragment and eGFP are recovered respectively. The fragment and the digested vector were assembled into a recombinant plasmid GAPZB-ABCI-eGFP by means of multi-fragment assembly. Further, using error-prone PCR, the promoter P on the recombinant plasmid GAPZ-ABCI GAP Carry out random mutation,...

Embodiment 3

[0039] Embodiment 3: the fermentation culture of recombinant Pichia pastoris S3' in 3-L tank

[0040] With the above-mentioned mutated promoter sequence SEQ ID NO.2, replace the promoter on the secretory expression vector GAPZαB to obtain the plasmid GAPmZαB, construct the GAPmZαB-ABCI plasmid in the same manner as in Example 1, and use the fast cutting enzyme AvrII to linearize the plasmid After transformation into Pichia pastoris GS115, the secreted and expressed recombinant strain S3' was obtained. Pick the recombinant Pichia pastoris S3' strain, inoculate it in 50mL YPD liquid medium, cultivate it at 30°C and 220rpm for 20h, then transfer it to a 3-L fermenter for fermentation at 10% (v / v). The liquid volume is 900mL, the fermentation medium is BSM medium, the initial rotation speed is 200rpm, the fermentation temperature is 30°C, the pH is 5, and the coupling between dissolved oxygen and rotation speed is set to keep the dissolved oxygen greater than 30%. When the strain...

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Abstract

The invention discloses food-grade safe saccharomycetes for producing chondroitin sulfate lyase ABCI and an application of the food-grade safe saccharomycetes, and belongs to the technical field of biological engineering. According to a method, chondroitin sulfate lyase ABCI is expressed through food-grade safe strain pichia pastoris in a heterologous integration manner, two intracellular and extracellular production modes can be realized, and no antibiotics are added in the culture process. A promoter capable of effectively improving the enzyme activity expression quantity of the chondroitin sulfate lyase ABCI is obtained by screening the promoters, so that the secretory expression enzyme activity of recombinant pichia pastoris can reach 2840U/L under the condition of a fermentation tank, a certain foundation is laid for efficiently producing and preparing the chondroitin sulfate lyase by food-grade microorganisms, and the food-grade safe saccharomycetes is suitable for industrial production and application.

Description

technical field [0001] The invention relates to a food-grade safe yeast for producing chondroitin sulfate lyase ABCI and an application thereof, belonging to the technical field of bioengineering. Background technique [0002] Chondroitin sulfate (CS) is a linear polysaccharide composed of D-glucuronic acid and N-acetylgalactosamine alternately linked, its molecular weight is between 7-50kDa, mainly in the form of proteoglycan Distributed in cartilage tissue. CS has important biological functions. Its skeleton chondroitin sulfate is formed by sulfation modification of chondroitin through sulfotransferase. According to the different positions of sulfation modification, chondroitin sulfate can be divided into the following four types: CSA (4-O-sulfation), CSC (6-O-sulfation), CSD (2,6-di-O-sulfation) and CSE (4,6-di-O-sulfation). Due to its excellent biocompatibility, CS has a wide range of applications, such as medical health, health care products, food and cosmetics. Cho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N9/88C12P19/26C12R1/84
CPCC12N9/88C12P19/26C12Y402/0202
Inventor 康振金学荣陈坚堵国成李江华
Owner JIANGNAN UNIV
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