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Recombinant pichia pastoris for heterogenous efficient expression of lipase and application of recombinant pichia pastoris

A lipase, heterologous technology, applied in the biological field, can solve the problems of secretion efficiency, high enzyme activity, etc.

Active Publication Date: 2017-08-22
XUZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the recombinant Pichia pastoris has achieved the heterologous secretion of Rhizomucor miehei lipase, and the secreted recombinant enzyme activity is relatively high, but its secretion efficiency is ultimately a weakness that affects its popularization and application

Method used

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  • Recombinant pichia pastoris for heterogenous efficient expression of lipase and application of recombinant pichia pastoris
  • Recombinant pichia pastoris for heterogenous efficient expression of lipase and application of recombinant pichia pastoris
  • Recombinant pichia pastoris for heterogenous efficient expression of lipase and application of recombinant pichia pastoris

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Experimental program
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Effect test

Embodiment 1

[0025] The preparation of embodiment 1 Rhizomucor miehei cDNA

[0026] 1.1 Extraction of Rhizomucor militaris total RNA

[0027] (1) Take an appropriate amount of Rhizomucor miehei hyphae, absorb the water with filter paper, grind with liquid nitrogen, add 1ml Trizol reagent (Invitrogen), vibrate with an oscillator for 5 minutes, and let stand at room temperature for 1 minute;

[0028] (2) Add 0.2ml chloroform, shake for 15s, and let it stand for 2min;

[0029] (3) 4°C, 12000rpm, 15min;

[0030] (4) Aspirate the supernatant, add an equal volume of isopropanol, and precipitate at -20°C for 30 minutes;

[0031] (5) 4°C, 12000rpm, 15min;

[0032] (6) Pour off the supernatant, wash the precipitate with 1ml 75% ethanol, 7500rpm, 4°C, 5min;

[0033] (7) Repeat (6) step once;

[0034] (8) Pour off the supernatant and dry for 10 minutes;

[0035] (9) Add appropriate amount of DEPC water to dissolve to obtain total RNA;

[0036] 1.2 Preparation of the first strand of Rhizomucor ...

Embodiment 22

[0041] Example 2 Construction of 2 copies of pPICZα A-2prorml plasmid

[0042] 2.1 Primer design

[0043] According to the sequence of the rml gene in GenBank (GenBank accession number is A02536.1), the following pair of primers were designed and synthesized:

[0044] FW(P1): 5'—CG GAATTC GTGCCAATCAAGAG—3' (SEQ ID NO.2)

[0045] REV (P2): 5'-TAG TCTAGAGTACAGAGGCCTGTG-3' (SEQ ID NO.3)

[0046] EcoR I and Not I restriction sites are designed at both ends of P1 and P2 respectively (see the italicized and underlined part in the above sequence)

[0047] 2.2 PCR amplification of Rhizomucor miehei lipase pro-rml containing leader peptide

[0048] Using P1 and P2 primers, Rhizomucor miehei (Boel E, Huge-Jensen B, Christensen M, Thim L, Fiil N: Rhizomucor miehei triglyceride lipase is synthesized as aprecursor.Lipids 1988,23(7):701-706. ) cDNA as a template, the PCR reaction system is:

[0049]

[0050] The reaction conditions are: 95°C for 5min, 5°C for 40s, 60°C for 40s, ...

Embodiment 3

[0057] Example 3 Screening of Pichia pastoris recombinant strains with 4 copies of pro-rml gene

[0058] 3.1 Preparation of Pichia pastoris X-33 (purchased by Invitrogen) electroporation competent cells

[0059] (1) Pick a fresh single colony and put it in 5ml YPD liquid medium, cultivate it at 30°C and 250rpm for 12-14h;

[0060] (2) Inoculate 0.1% of the inoculum into a 2L Erlenmeyer flask containing 500ml of YPD medium, cultivate at 30°C and 250rpm for 12-14h to make OD 600 =1.3-1.5;

[0061] (3) Centrifuge at 1500 rpm for 5 minutes at 4°C to collect the cells;

[0062] (4) Wash the cells twice with 500-250ml ice-cold sterile water;

[0063] (5) Wash the cells once with 20ml of ice-cold 1M sorbitol solution;

[0064] (6) Resuspend the cells with 1ml of ice-cold 1M sorbitol solution to a final volume of about 1.5ml, and dispense 80μl into small centrifuge tubes;

[0065] 3.2 Electric shock transformation of Pichia pastoris yeast cells

[0066] (1) Mix about 10 μl of th...

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Abstract

The invention discloses recombinant pichia pastoris for heterogenous efficient expression of lipase and application of the recombinant pichia pastoris. The recombinant pichia pastoris is obtained by converting plasmid HAC1-pPIC3.5K of an overexpression HAC1 gene in recombinant pichia pastoris X-33 which contains a pro-rml gene of a copy number 4 and is capable of expressing Pro-RML. When the strain is subjected to 96 hours of flask shaking fermentation, the extracellular enzyme activity is up to 1078U / mL at most, and the enzyme activity secretion efficiency is up to 47U / OD600. 120 hours of fermentation is needed when pichia pastoris which is disclosed by patent CN103361327A and has a rhizomucor mieheilipase copy number of 2 meets the highest extracellular enzyme activity and enzyme secretion efficiency, the extracellular enzyme activity is 1038U / mL at most, and the enzyme activity secretion efficiency is only 25U / OD600. By adopting the recombinant pichia pastoris, expression of rhizomucor mieheilipase is effectively promoted, on the premise that the highest extracellular enzyme activity is not influenced, the secretion efficiency of the rhizomucor mieheilipase is increased by 1.9 times, and the fermentation time is shortened by 24 hours.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a strain of recombinant Pichia pastoris expressing lipase with heterologous high-efficiency and application thereof. Background technique [0002] Lipase (E C3.1.1.3) is a class of enzymes that can hydrolyze triacylglycerol. It can catalyze a variety of chemical reactions such as: hydrolysis, esterification, transesterification, aminolysis, etc., and has mild and specific reaction conditions. Strong, non-polluting and other advantages are widely used in food, medicine, cosmetics, petroleum and other industries, known as the third largest industrial enzymes. [0003] Although lipase has a wide range of uses, the low yield of the starting strain limits its application. Therefore, heterologous protein expression is currently the main strategy for industrial protein production. However, there are many factors that affect the expression of heterologous proteins, such as host selection, gen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/81C12N1/19C12R1/84
CPCC12N9/20C12N15/815C12Y301/01003
Inventor 黄金金王一洲孙梦雪郑维发赵艳霞
Owner XUZHOU NORMAL UNIVERSITY
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