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Lipase production strain and application thereof

A technology for the production of bacteria and lipase, applied in the direction of microorganisms, hydrolytic enzymes, fungi, etc., can solve the problems of reduced fermentation yield, reduced loading coefficient, and high enzyme cost

Active Publication Date: 2020-07-07
WILMAR SHANGHAI BIOTECH RES & DEV CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main problems in the current application of restriction enzyme method are the low activity of the enzyme and the high cost of the enzyme
[0010] Due to the presence of sugar, protein and metabolites in the medium that stabilize foam, under the joint action of aeration fermentation and carbon dioxide exhaled by microorganisms, the culture solution will produce foam, The generation of foam will affect stirring, dissolved oxygen and cell growth, etc. If the foam is not properly controlled and the foam overflows, it will cause a series of problems, including reducing the filling coefficient, increasing the risk of bacterial contamination, causing waste of raw materials and loss of products, and eventually leading to Lower Fermentation Yield

Method used

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  • Lipase production strain and application thereof
  • Lipase production strain and application thereof
  • Lipase production strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 : Plasmid construction

[0042] The MDGL coding gene sequence was derived from Penicillium camembertii U-150 (genebank accession number: BAA14345.1), and the codon was optimized according to the codon preference of Pichia pastoris, and the optimized gene (The specific nucleotide sequence is shown in SEQ ID NO.: 1, and the encoded amino acid sequence is shown in SEQ ID NO.: 2) Synthesized and cloned into the expression vector pPIC9K by Sangon Bioengineering Co., Ltd. to generate plasmid pPIC9K -MDGL. The PEP4 coding gene sequence is derived from Pichia pastoris (Komagataella phaffii) GS115 (genebank accession number: CP014717.1), including the upstream 1000bp and downstream 1000bp of the coding gene (the specific expression frame nucleotide sequence is shown in SEQ ID NO. : 3, its coded amino acid sequence is shown in SEQ ID NO.: 4), the gene was synthesized by Sangon Bioengineering Co., Ltd. and cloned into the expression vector pPIC9K to generate plasmid p...

Embodiment 2

[0058] Embodiment 2: the acquisition of MDGL production strain

[0059] The plasmid pPIC9K-MDGL-pep4 was digested with BglII restriction endonuclease from NEB Company to obtain the transformed fragments of MDGL and pep4, and the digested product was purified by PCR product purification kit from Axygen Company.

[0060] According to the standard transformation method of Pichia pastoris (Shixuan Wu&Geoffrey J Letchworth, Highefficiency transformation by electroporation of Pichia pastoris preserved with lithium acetate and dithiothreitol, Biotechniques, 2004,36(1):152-4), the Pichia pastoris strain was transformed by electroporation CICC32806 (purchased from China Industrial Microorganism Culture Collection Management Center), spread on MD screening plate (1.34% yeast basic nitrogen source, 1mol / L sorbitol, 1.8% agarose), and cultured at 30°C for 3 days.

[0061] Transfer the grown single colony to the MDGL screening plate (0.1mol / L citric acid buffer, pH 6.0, 1% yeast powder, 2%...

Embodiment 3

[0063] Example 3: Shake Flask Fermentation Test of MDGL Production Strains

[0064] Eight single clones (respectively named: mc1-2-MDGL-pep71, mc1-2-MDGL-pep 90, mc1-2-MDGL-pep 91, mc1-2-MDGL-pep 92, mc1-2-MDGL -pep 94, mc1-2-MDGL-pep 96, mc1-2-MDGL-pep97, mc1-2-MDGL-pep 28) were inoculated into YPD medium respectively, and cultured at 30°C and 240rpm on a shaker for 24h.

[0065] Inoculate 500 μL of culture solution into 50 mL of BMGY medium (1% yeast extract, 2% peptone, 1% glycerol, 1.34% yeast basic nitrogen source, 0.1mol / L citric acid buffer, pH 6.0), at 30°C , 240rpm shaking shaker culture overnight.

[0066] The culture solution was diluted with sterile water, and the absorbance value at 600 nm was measured (the result of the measured value between 0.2-0.8 was considered accurate). Calculate the OD per ml of bacterial solution according to the dilution factor 600 Value, take the corresponding volume of culture solution, so that the total OD of the bacteria 600The v...

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Abstract

The invention provides a lipase production strain and application thereof. Specifically, the invention provides a glycerinum mono-diacyl lipase production strain and further provides a method for preparing glycerinum mono-diacyl lipase by using the strain and application of the lipase production strain. The lipase production strain provided by the invention is stable, high in protein yield and small in foam and has great industrial production value.

Description

technical field [0001] The application belongs to the field of biotechnology, and specifically relates to a lipase production strain and its application, more specifically, to a glycerol mono-diacyl ester lipase production strain with less foam production and its application. Background technique [0002] Since researchers first discovered in 1969 that certain yeasts can use methanol as the sole carbon and energy source for growth and high-density fermentation, with the methanol-induced promoter P AOX1 The discovery of Pichia and the maturity of Pichia molecular manipulation methods, the use of Pichia to express foreign proteins has been widely used. [0003] Pichia pastoris not only has the advantages of simple operation, easy culture, fast growth, high expression level, and low cost of the prokaryotic expression system, but also has post-translational modifications of foreign proteins that the prokaryotic expression system does not have, such as glycosylation, At the same...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N9/18C12P7/62C12R1/84
CPCC12N9/18C12P7/62
Inventor 赵强徐正军宣姚吉牛其文
Owner WILMAR SHANGHAI BIOTECH RES & DEV CENT