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A kind of alkaline pectinase secretion enhanced bacterial strain and its application

A pectinase and enhanced technology, applied in the field of genetic engineering, can solve the problems of restricting the industrial production of alkaline pectinase, and the yield of alkaline pectinase cannot be further improved.

Active Publication Date: 2019-09-17
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

A comprehensive comparison of different hosts that can express alkaline pectinase shows that the protein expressed by Pichia pastoris is easy to purify and has high yield, but the overexpression of heterologous protein will lead to growth stress and cause the unfolded protein effect (UPR), resulting in alkaline pectinase The yield can not be further improved, which limits the industrialized production of alkaline pectinase

Method used

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  • A kind of alkaline pectinase secretion enhanced bacterial strain and its application
  • A kind of alkaline pectinase secretion enhanced bacterial strain and its application
  • A kind of alkaline pectinase secretion enhanced bacterial strain and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: Construction and identification of recombinant bacteria

[0025] Extract Pichia pastoris GS115 RNA, reverse transcribe it into cDNA, use the cDNA as a template, design primers, and obtain UPR-related genes (HAC1, ERO1, SLY1, BMH2, GCN4, UBC1, HRD1, SSO2, SEC53, BIP, PDI , GSH2), and clone it into the expression vector pGAPZA to obtain the recombinant plasmid pGAPZA-X (see attached figure 1 , X represents HAC1, ERO1, SLY1, BMH2, GCN4, UBC1, HRD1, SSO2, SEC53, BIP, PDI or GSH2), the recombinant vector was transformed into Pichiapastoris GS115-pPIC9K-PGL, and the co-expression recombinant strain PichiapastorisGS115-pPIC9K was obtained after screening and identification -PGL / pGAPZA-X.

[0026] The Pichia pastoris GS115-pPIC9K-PGL expressing the recombinant alkaline pectinase is that the alkaline pectinase gene whose nucleotide sequence is shown in SEQ ID NO.1 is connected to the expression vector pPIC9K, and then transformed to Pichia pastoris host strain GS...

Embodiment 2

[0031] Example 2: Enzyme activity assay and protein electrophoresis of co-expressed genetically engineered strains

[0032] Cultivation method: After seed activation, the strain was inoculated into the basic fermentation medium YPD, cultured at 30°C and 220rpm for 14h, then transferred to the optimized growth medium BMGY and cultured at 30°C and 220rpm for 24h, and then the strain Transfer to the induction medium BMMY at 23°C, 220rpm and add 1.5% methanol every 24h to induce the expression of alkaline pectinase.

[0033] The enzyme activity measurement conditions are: the fermentation broth is centrifuged at 8000rpm for 10 minutes, extracellular PGL is contained in the fermentation supernatant, and a certain amount is taken for detection. PGL reaction system: glycine-NaOH buffer containing 0.2% polygalacturonic acid (substrate) (0.2mol L -1 , 0.44mmol·L -1 CaCl 2 , pH9.4) 2mL, the sample to be tested was 20μL, and the inactive enzyme solution was used as the blank control. ...

Embodiment 3

[0035] Embodiment 3: the purification of alkaline pectinase

[0036] Centrifuge the recombinant bacterial fermentation broth at 8000r / min for 20min, take the supernatant, add ammonium sulfate for gradient salting-out, collect 30-50% ammonium sulfate precipitated part by low-temperature centrifugation, and dissolve the salted-out precipitated enzyme in glycine-sodium hydroxide buffer Solution (pH7.5), dialyzed with 20mmol / L glycine-sodium hydroxide buffer solution for 24h. The supernatant obtained by centrifugation was further separated and purified by cation exchange chromatography.

[0037] Compared with the starting strain Pichia pastoris GS115-pPIC9K-PGL before the co-expression of UPR-related genes, the enzyme activity was 301.32 U / ml after 96 hours of shake-flask induced fermentation, and the secretion-enhanced recombinant strain Pichia pastoris GS115-PGL / pGAPZA-HAC1 enzyme in the present invention The activity of 373.94U / ml increased by 24.1%, the activity of Pichia pas...

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Abstract

The invention discloses an alkaline pectinase secretion-enhanced strain and an application thereof, and belongs to the technical field of genetic engineering. Through a gene recombination technology, UPR related genes of pichia pastoris are cloned and linked to a pichia pastoris expression vector pPGAZA and are converted to a Pichia pastoris GS115-pPIC9K-PGL strain, so that a plurality of strains, namely GS115-PGL / pGAPZA-HAC1, GS115-PGL / pGAPZA-ERO1, GS115-PGL / pGAPZA-UBC1 and the like, which are stronger in secretion and expression of alkaline pectinase than an original strain, are obtained, the enzyme activities of the strains, before the application of the method, are improved by 24.1%, 35.5% and 22.3%, and with the significant improvement, a good foundation is laid for the large-scale production of the alkaline pectinase. The alkaline pectinase disclosed by the invention can catalyze the pyrolysis of an alpha-1,4 glucosidic bond of polygalacturonic acid by virtue of a trans-elimination effect under an alkaline condition;and the alkaline pectinase can be widely applied to such industries as food, textile, paper-making and the like.

Description

technical field [0001] The invention relates to an alkaline pectinase secretion-enhanced bacterial strain and application thereof, belonging to the technical field of genetic engineering. Background technique [0002] Pectinase is a complex enzyme that breaks down pectin polymers into unsaturated oligogalacturonic acids. The enzyme is widely distributed and found in some parasitic nematodes, plants and microorganisms. Pectinase is widely used and has a history of industrial application for more than 40 years. Pectinases are divided into acid pectinases and alkaline pectinases PGL according to the optimum reaction pH. Among them, acid pectinase is mainly used in clarification of fruit juice and wine, extraction of fruit and vegetable juice, fruit peeling and so on. PGL applications are mainly used in textile, food, paper industry and environmental fields. The application of enzymatic method to the above-mentioned field-related reactions has the advantages of environmental...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/60C12N9/88C12R1/84
Inventor 刘松陈双全陈坚堵国成
Owner JIANGNAN UNIV
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