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Method for enhancing secretion of glucose oxidase by coexpression of UPR (unfolded protein response) key genes and downstream target genes

A glucose oxidase and key gene technology, applied in the direction of microorganism-based methods, oxidoreductase, biochemical equipment and methods, etc., can solve the problems of high cost, complicated separation and extraction, etc.

Active Publication Date: 2015-04-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are still difficulties in large-scale production of highly active GOD
During the production of GOD by fermentation, a large amount of miscellaneous proteins are produced, and the separation and extraction are complicated and costly

Method used

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  • Method for enhancing secretion of glucose oxidase by coexpression of UPR (unfolded protein response) key genes and downstream target genes
  • Method for enhancing secretion of glucose oxidase by coexpression of UPR (unfolded protein response) key genes and downstream target genes
  • Method for enhancing secretion of glucose oxidase by coexpression of UPR (unfolded protein response) key genes and downstream target genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Construction and Identification of Embodiment 1 Recombinant Bacteria

[0038] 1) Design primers to amplify Haclp, Pdi1, Ero1, Lhs1, Kar2, Sil1 genes using Pichia pastoris and Saccharomyces cerevisiae reverse transcription cDNA as template;

[0039] Hac-F: 5'-A G TTCGAA ATGCCCGTAGATTCTTCTC-3'

[0040] Hac-R: 5'-AAATAT GCGGCCGC CTATTCCTGGAAGAATACAAAGTC-3'

[0041] Pdi-F: 5'-CG GGATCC ATGCAATTCAACTGGGATATTAAAACTGTG-3'

[0042] Pdi-R: 5'-AAATAT GCGGCCGC TTAAAGCTCGTCGTGAGCGTC-3'

[0043] Ero-F: 5'-CG GGATCC ATGAGATTAAGAACCGCCAT-3'

[0044] Ero-R: 5'-AAATAT GCGGCCGC TTATTGTATATCTAGCTTAT-3'

[0045] Lhs-F: 5'-CG GGATCC ATGCGAAACGTTTTAAGGCTTTT-3'

[0046] Lhs-R: 5'-AAATAT GCGGCCGC CTATAATTCATCATGCAAAATGTCT-3'

[0047] Kar-F: 5'-CG GGATCC ATGCTGTCGTTAAAACCATCTT-3'

[0048] Kar-R: 5'-AAATAT GCGGCCGC TATCTACAACTCATCATGAT-3'

[0049] Sil-F: 5'-CG GGATCC ATGGTCCGGATTCTTCCCAT-3'

[0050] Sil-R: 5'-AAATAT GCGGCCGC TCAGAGTTCATCTCTGAAATTT-3'

[0051] Add t...

Embodiment 2

[0059] Example 2 RT-PCR determination of key secretion genes of secretion-enhanced strains and the influence on the physiological activities of the strains.

[0060] Medium: YPD medium (1L) for seed and slant medium: tryptone 20g, yeast extract 10g, glucose 20g; slant medium with agar 20g; basic fermentation medium is BMGY medium (1L): tryptone 20g, yeast extract 10g, glycerol 10mL, YNB 13.4g, 100mM phosphate buffer (pH 6.0); induction medium is BMMY medium (1L): tryptone 20g, yeast extract 10g, methanol 8mL, YNB 13.4 g, 100mM phosphate buffer (pH 6.0);

[0061] Cultivation method: Cultivate to OD at 30°C and 200rpm 600 The seeds between 1.6 and 1.7 are transferred to the basic fermentation medium with an inoculum of 2%, and cultivated at 30°C and 200rpm;

[0062] Induction conditions: when cultured in BMGY to an OD value of 1.2-1.5, the yeast cells were transferred into BMMY medium to induce protein production.

[0063] One day after the induction of the secretion-enhanced...

Embodiment 3

[0077] Fermentation of embodiment 3 secretion-enhanced bacterial strain on 3L fermentor

[0078] Pichia pastoris GS 115-pPIC9K-GOD / pPICZ-Hac1 / pPIC3.5K-Pdi1 was used as the starting strain.

[0079] The cultured YPD bacterial solution was aseptically inserted into a 3L fully automatic fermenter (LiFlus GMBioTRON, Korea) with an inoculum size of 10%. The initial conditions are: liquid volume 800-1000mL, initial stirring speed 500r / min, ventilation volume 2.5vvm, 30% phosphoric acid solution and 25% concentrated ammonia water to control the pH to 5.5, and the culture temperature during the growth period is 30°C , using DO-stat control associated with stirring to maintain DO at 30%, and set the maximum stirring speed to 950r / min.

[0080] When glycerol is exhausted (DO rises rapidly), and DO>60%, start to add 50% glycerol medium in different exponential feeding ways. When glycerol is exhausted again, DO rebounds again, and when DO>60%, induction begins. Reduce the induction tem...

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Abstract

The invention discloses a method for enhancing secretion of glucose oxidase by coexpression of UPR (unfolded protein response) key genes and downstream target genes, and belongs to the technical field of genetic engineering. The method comprises the steps of respectively cloning and connecting pichia pastoris UPR key gene Haclp and the downstream target genes Pdi1, Erol, Lhs1, Kar2 and Sil1 to pichia pastoris expression carriers pPICZ alpha and pPIC3.5K, transforming into a Pichia pastoris GS115-pPIC9K-GOD strain of which the preservation number is CCTCC NO: M 2012266, and obtaining a strain with enhanced secretion expression of the glucose oxidase compared with the original strain by screening and identifying. The enzyme activity of the glucose oxidase expressed by the strain on a 3L of fermentation tank is 585U / mL. Therefore, good foundation is established for large-scale production of the glucose oxidase.

Description

technical field [0001] A method for co-expressing UPR key genes and downstream target genes to enhance the secretion and expression of glucose oxidase belongs to the technical field of genetic engineering. Background technique [0002] Glucose oxidase is one of the most important tool enzymes in the biological field. Since Updike and Hicks immobilized GOD on the surface of Clark oxygen electrode and applied it to blood glucose measurement in 1967, GOD has been widely used in food, feed, medicine and many other related fields. [0003] In the food industry, the presence of oxygen causes many chemical reactions that are not conducive to product quality and creates conditions for the growth of many microorganisms. At present, many countries have widely used GOD as a safe antioxidant for workers in various foods and food processing techniques. Although it has various uses, the functions of GOD mainly lie in four aspects: removing glucose, removing oxygen, forming hydrogen pero...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/81C12N1/19C12N9/04C12R1/84C12R1/865
Inventor 陈坚顾磊张娟堵国成沈伊娜闻一凡李梦洁李婷丁雪
Owner JIANGNAN UNIV
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