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Recombinant pichia pastoris for heterogenous high level expression of lipase

A lipase and recombinant vector technology, applied in recombinant DNA technology, fermentation, hydrolase and other directions, can solve the problems of high enzyme cost and low enzyme activity, achieve high expression efficiency and reduce separation and purification costs.

Inactive Publication Date: 2013-10-23
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although the previous studies have proved that the enzyme has specificity and has been tried to be applied to the preparation of biodiesel, but due to the low enzyme activity, the cost of the enzyme is too high, which limits its application

Method used

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  • Recombinant pichia pastoris for heterogenous high level expression of lipase
  • Recombinant pichia pastoris for heterogenous high level expression of lipase
  • Recombinant pichia pastoris for heterogenous high level expression of lipase

Examples

Experimental program
Comparison scheme
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 (Rhizomucor miehei) mycelium, filter paper to dry up the water, grind with liquid nitrogen, add 1ml Trizol reagent (Invitrogen), oscillate for 5 minutes, and let stand at room temperature for 1 minute;

[0028] (2) Add 0.2ml chloroform, shake for 15s, and let 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 step (6) 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 2

[0041] Example 2 The acquisition of the Rhizomucor miehei lipase gene pro-rml containing the leader peptide and the construction of the expression plasmid

[0042] 2.1 Primer design

[0043] According to the sequence of 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'

[0045] REV (P2): 5'-TAG TCTAGA GTACAGAGGCCTGTG—3'

[0046] EcoRI and NotI 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, using the above-mentioned Rhizomucor miehei cDNA as a template, the PCR reaction system is:

[0049]

[0050] The reaction conditions are: 95°C for 5 minutes; 5°C for 40s, 60°C for 40s, 72°C for 1 min, 30 cycles; 72°C for 10 minutes; 4°C for 2 minutes. ...

Embodiment 3

[0061] Example 3 Secretory expression of Rhizomucor miehei lipase gene pro-rml containing leader peptide in Pichia pastoris X-33

[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, and culture it at 30°C, 250rpm for 12-14 hours;

[0064] (2) Inoculate 0.1% of the inoculum into a 2L Erlenmeyer flask containing 500ml of YPD medium, and culture at 30°C, 250rpm for 12-14 hours to make OD600=1.3-1.5;

[0065] (3) Centrifuge at 1500 rpm for 5 minutes at 4°C to collect 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 transf...

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Abstract

The invention discloses recombinant pichia pastoris for heterogenous high level secretory expression of a rhizomucor miehei lipase. The recombinant pichia pastoris is obtained by introducing a self leader peptide gene for encoding the rhizomucor miehei lipase by employing a molecular biology technology, then optimizing a copy number of an objective gene, and constructing a vector which can be expressed in pasteur pichia pastoris. Researches show that: the introduction of the self leader peptide greatly increases the secretion amount of the objective lipase, a pichia pastoris recombinant of a lipase gene (pro-rml) containing two copies has highest enzyme activity, and the lipase Pro-RML has higher activity of hydrolysis of tri-acylglycerol.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a bacterial strain for heterologously expressing Rhizomucor miehei lipase. Background technique [0002] Lipase (EC3.1.1.3) is a class of enzymes that can hydrolyze triacylglycerol. Because it can catalyze various reactions such as hydrolysis, esterification, transesterification, ammonolysis and reverse synthesis of esters, it can be applied in Food, medicine, cosmetics, biodiesel and many other aspects of production and life. Known as the third largest industrial enzyme. [0003] Compared with chemical methods, enzymatic methods will gradually replace chemical methods with high energy consumption and high pollution because of their mild reaction conditions, less by-products, and environmental friendliness. [0004] The application of lipase mainly includes, in the food industry, by applying lipase to decompose the short-chain fatty acids and monoglycerides released from oil to imp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/81C12N1/19C12P7/64C12R1/84C12R1/645
CPCY02E50/13Y02E50/10
Inventor 李颖黄金金杨震关国华崔迪
Owner CHINA AGRI UNIV
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