Recombinant pichia pastoris for heterologously and efficiently expressing rhizomucor miehei lipase and application of recombinant pichia pastoris

A technology of Rhizomucor miehei and lipase, which is applied in the biological field and can solve the problems of the influence of secretion amount on popularization and application, high activity of recombinant enzymes, etc.

Active Publication Date: 2017-08-15
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 has a high enzyme activity, its secretion has always been a weakness that affects its popularization and application.

Method used

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  • Recombinant pichia pastoris for heterologously and efficiently expressing rhizomucor miehei lipase and application of recombinant pichia pastoris
  • Recombinant pichia pastoris for heterologously and efficiently expressing rhizomucor miehei lipase and application of recombinant pichia pastoris
  • Recombinant pichia pastoris for heterologously and efficiently expressing rhizomucor miehei lipase and application of recombinant pichia pastoris

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The preparation of embodiment 1 Rhizomucor miehei cDNA

[0029] 1.1 Extraction of Rhizomucor militaris total RNA

[0030] (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;

[0031] (2) Add 0.2ml chloroform, shake for 15s, and let it stand for 2min;

[0032] (3) 4°C, 12000rpm, 15min;

[0033] (4) Aspirate the supernatant, add an equal volume of isopropanol, and precipitate at -20°C for 30 minutes;

[0034] (5) 4°C, 12000rpm, 15min;

[0035] (6) Pour off the supernatant, wash the precipitate with 1ml 75% ethanol, 7500rpm, 4°C, 5min;

[0036] (7) Repeat (6) step once;

[0037] (8) Pour off the supernatant and dry for 10 minutes;

[0038] (9) Add appropriate amount of DEPC water to dissolve to obtain total RNA;

[0039] 1.2 Preparation of the first strand of Rhizomucor ...

Embodiment 2

[0044] Example 2 Construction of 2 copies of the pPICZα A-2prorml plasmid

[0045] 2.1 Primer design

[0046] 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:

[0047] FW(P1): 5'—CG GAATTC GTGCCAATCAAGAG—3' (SEQ ID NO.2)

[0048] REV (P2): 5'-TAG TCTAGA GTACAGAGGCCTGTG-3' (SEQ ID NO.3)

[0049] 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)

[0050] 2.2 PCR amplification of Rhizomucor miehei lipase pro-rml containing leader peptide

[0051] 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.Lipids1988,23(7):701-706.) (Preserved in this room) cDNA is used as a template, and the PCR reaction system is:

[0052]

[0053] The reaction conditions are: 95°C ...

Embodiment 3

[0061] Example 3 Screening of Pichia pastoris recombinant strains with 4 copies of pro-rml gene

[0062] 3.1 Preparation of Pichia pastoris X-33 (purchased by Invitrogen) electroporation competent cells and their electroporation transformation

[0063] (1) Pick a fresh single colony and put it in 5ml YPD liquid medium, cultivate it at 30°C and 250rpm for 12-14 hours;

[0064] (2) Inoculate 0.1% of the inoculum into a 2L Erlenmeyer flask containing 500ml of YPD medium, cultivate at 30°C and 250rpm for 12-14 hours to make OD 600 =1.3-1.5;

[0065] (3) Centrifuge at 1500 rpm for 5 minutes at 4°C to collect the cells;

[0066] (4) Wash the cells twice with 500-250ml ice-cold sterile water;

[0067] (5) Wash the cells once with 20ml of ice-cold 1M sorbitol solution;

[0068] (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;

[0069] 3.2 Electric shock transformation of Pichia pasto...

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Abstract

The invention discloses recombinant pichia pastoris for heterologously and efficiently expressing lipase and application of the recombinant pichia pastoris. The recombinant pichia pastoris is obtained through converting plasmid SEC31-pPIC3.5K overexpressing a gene SEC31 in recombinant pichia pastoris X-33, which contains a 4-copy number pro-rml gene and can express Pro-RML. According to the recombinant pichia pastoris and the application thereof, the expression of the rhizomucor miehei lipase is effectively promoted, the efficiency of secretion of the lipase is increased, the enzyme activity reaches 996U/mL after 144 hours of shake-flask fermentation, and the strain ectoenzyme activity secretion efficiency reaches 38U/OD600 and is higher than that (26U/OD600) of two recombinant pichia pastoris strains of rhizomucor miehei lipase copy numbers concerned in the patent CN103361327A.

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] Lipases (EC 3.1.1.3) are a class of enzymes that can hydrolyze triacylglycerols. It can catalyze a variety of chemical reactions such as: hydrolysis, esterification, transesterification, ammonolysis, etc. Compared with the chemical method, the reaction catalyzed by lipase has many advantages: mild reaction conditions, less by-products, no pollution, etc. It is widely used in food, medicine, cosmetics, petroleum and other industries, and is called the third largest Industrial enzymes. [0003] At present, the production of lipase mainly adopts the strategy of heterologous protein expression. However, there are many factors affecting the expression of heterologous proteins, which are mainly divided into two categories: external factors and ...

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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