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A Pichia pastoris for highly efficient heterologous expression of white Penicillium cyclopium lipase and an enzyme production medium

An arc penicillium and lipase technology, applied in the directions of enzymes, enzymes, fermentation, etc., can solve the problems of expensive medium for enzyme-producing strains, limited industrial application, low enzyme activity level, etc. Catalytic reaction temperature range and the effect of improving expression efficiency

Inactive Publication Date: 2013-11-13
秦皇岛惠恩生物技术有限公司
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Although the previous studies have proved that the enzyme has specificity and has been tried to be applied to the process of preparing astaxanthin monomer, the cost of the enzyme is too high due to the expensive medium of the enzyme-producing strain and the low level of enzyme activity, which limits its use. Industrial application

Method used

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  • A Pichia pastoris for highly efficient heterologous expression of white Penicillium cyclopium lipase and an enzyme production medium
  • A Pichia pastoris for highly efficient heterologous expression of white Penicillium cyclopium lipase and an enzyme production medium
  • A Pichia pastoris for highly efficient heterologous expression of white Penicillium cyclopium lipase and an enzyme production medium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The preparation of embodiment 1 Penicillium arcuum cDNA

[0031] 1.1 Extraction of total RNA from Penicillium albicans

[0032] (1) Take an appropriate amount of Penicillium albicans mycelia, blot dry with filter paper, grind with liquid nitrogen, add 1mL Trizol reagent (Invitrogen), oscillate for 1min, and let stand at room temperature for 5min;

[0033] (2) Add 0.2mL chloroform, shake for 15s, and let stand for 3-5min;

[0034] (3) 4°C, 12000rpm, 15min;

[0035] (4) Aspirate the supernatant, add an equal volume of isopropanol, and precipitate at -20°C for 30 minutes;

[0036] (5) 4°C, 12000rpm, 15min;

[0037] (6) Pour off the supernatant, wash the precipitate with 1mL 75% DEPC-ethanol, 12000rpm, 4°C, 5min;

[0038] (7) Repeat step (6) once;

[0039] (8) Pour off the supernatant and centrifuge at 12000rpm, 4°C for 2min. Use the tip of the pipette to absorb the residual liquid, and dry it in ice for 10 minutes; dry for 10 minutes;

[0040] (9) Add appropriate am...

Embodiment 2

[0047] Example 2 The primer design of the Penicillium albus lipase gene pro-alip containing leader peptide

[0048] 2.1 Primer design

[0049] According to the sequence of the alip gene in GenBank (GenBank accession number is AF274320.1), the following pair of primers were designed and synthesized:

[0050] pro-alip-F (P1): 5'-CCCG GAATTC GCACCTATTTTGGAGTCGA-3'

[0051] pro-alip-R (P2): 5'-ATAAT GCGGCCGC GCTCAGATAGCCAC-3'

[0052] 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)

[0053] 2.2 PCR amplification of Penicillium albus lipase pro-alip containing leader peptide

[0054] Using P1 and P2 primers, using Penicillium alba (ZhangHM, WuMC, GuoJ, et al. Cloning and Sequence Analysis of Complete Gene Encodingan Alkaline Lipase from Penicilliumcyclopium. Applied Biochemistry and Microbiology. 2011, 47(6): 586-593.) cDNA as a template, the PCR reaction system is:

[0055] ...

Embodiment 3

[0064] Example 3 Secretion and expression of Penicillium albicans lipase gene pro-alip containing leader peptide in Pichia pastoris X-33

[0065] 3.1 Preparation of Pichia pastoris X-33 (purchased by Invitrogen) electroporation competent cells and their electroporation transformation

[0066] (1) Pick a fresh single colony in 5mLYPD liquid medium, and culture it at 28°C, 200rpm for 12-14h;

[0067] (2) Inoculate 0.1% inoculum into a 2L Erlenmeyer flask containing 500mL YPD medium, culture at 28°C, 200rpm for 12-14h, and make it OD 600 =1.3~1.5;

[0068] (3) Centrifuge at 12,000 rpm for 5 minutes at 4°C to collect cells;

[0069] (4) Wash the cells twice with 500-250 mL ice-cold sterile water;

[0070] (5) Wash the cells once with 20 mL of ice-cold 1M sorbitol solution;

[0071] (6) Resuspend the cells with 1 mL of ice-cold 1M sorbitol solution to a final volume of about 1.5 mL, and dispense 80 μL into small centrifuge tubes.

[0072] 3.2 Electric shock transformation of P...

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Abstract

The present invention provides a Pichia pastoris for highly efficient heterologous expression of white Penicillium cyclopium lipase and an enzyme production medium. According to the present invention, the white Penicillium cyclopium lipase containing a self leader peptide (7 amino acids) is heterologously expressed in a genetically engineered Pichia pastoris, increasing the activity of exogenous protein lipase, and improving a part of enzymatic properties of the exogenous protein lipase. According to the enzyme production medium of the invention, through optimizing the YNB component of a BMMY medium, the medium cost is reduced by 70%. According to the invention, reliable experimental basis is provided for the industrial application of the enzyme, especially for the catalyzed hydrolysis of substrate astaxanthin ester from Haematococcus pluvialis to produce astaxanthin monomers.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a bacterial strain that expresses a lipase efficiently heterologously, and the present invention also relates to a method for expressing the lipase efficiently heterologously, the gene encoding the lipase and the application of the enzyme , and a lipase heterologous expression medium. Background technique [0002] Lipase (Lipase E.C.3.1.1.3) is a class of hydrolytic enzymes that can hydrolyze triglycerides to produce free fatty acids and glycerol with different chain lengths. As an important industrial enzyme, lipase can catalyze a series of reactions such as hydrolysis, ester synthesis, transesterification, transesterification, etc., so it is widely used in traditional industrial fields such as food, leather, feed, washing, medicine, and oil chemical industry . [0003] One of the most notable features of lipase is that it is different from the catalytic properties of most...

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
CPCC12N9/20C12P23/00C12Y301/01003
Inventor 李颖杨震黄金金关国华陈芝
Owner 秦皇岛惠恩生物技术有限公司
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