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Pichia pastoris strain capable of efficiently heterologously expressing Penicillium cyclopium var.albus lipase

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: 2016-08-17
秦皇岛惠恩生物技术有限公司
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0009] 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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  • Pichia pastoris strain capable of efficiently heterologously expressing Penicillium cyclopium var.albus lipase
  • Pichia pastoris strain capable of efficiently heterologously expressing Penicillium cyclopium var.albus lipase
  • Pichia pastoris strain capable of efficiently heterologously expressing Penicillium cyclopium var.albus lipase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The preparation of embodiment 1 Penicillium arcuum cDNA

[0032] 1.1 Extraction of total RNA from Penicillium albicans

[0033] (1) Take an appropriate amount of Penicillium albicans mycelium, absorb the water with filter paper, grind with liquid nitrogen, add 1mL Trizol reagent (Invitrogen), shake with an oscillator for 1min, and let stand at room temperature for 5min;

[0034] (2) Add 0.2mL chloroform, shake for 15s, and let stand for 3-5min;

[0035] (3) 4°C, 12000rpm, 15min;

[0036] (4) Aspirate the supernatant, add an equal volume of isopropanol, and precipitate at -20°C for 30 minutes;

[0037] (5) 4°C, 12000rpm, 15min;

[0038] (6) Pour off the supernatant, wash the precipitate with 1mL 75% DEPC-ethanol, 12000rpm, 4°C, 5min;

[0039] (7) Repeat (6) step once;

[0040] (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;

[004...

Embodiment 2

[0048] Example 2 The primer design of the Penicillium albus lipase gene pro-alip containing leader peptide

[0049] 2.1 Primer design

[0050] 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:

[0051] pro-alip-F (P1): 5'-CCCG GAATTC GCACCTATTTTGGAGTCGA-3'

[0052] pro-alip-R (P2): 5'-ATAAT GCGGCCGC GCTCAGATAGCCAC-3'

[0053] 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)

[0054] 2.2 PCR amplification of Penicillium albus lipase pro-alip containing leader peptide

[0055] Using P1 and P2 primers, Penicillium cyclopium (Zhang H M, Wu M C, Guo J, et al. Cloning and Sequence Analysis of Complete Gene Encodingan Alkaline Lipase from Penicillium cyclopium. Applied Biochemistry and Microbiology. 2011, 47 (6): 586-593.) cDNA is a template, and the PCR reaction system i...

Embodiment 3

[0065] Example 3 Secretion and expression of Penicillium albicans lipase gene pro-alip containing leader peptide in Pichia pastoris X-33

[0066] 3.1 Preparation of Pichia pastoris X-33 (purchased by Invitrogen) electroporation competent cells and their electroporation transformation

[0067] (1) Pick a fresh single colony in 5mL YPD liquid medium and culture it at 28°C and 200rpm for 12-14h;

[0068] (2) Inoculate 0.1% of the inoculum into a 2L Erlenmeyer flask containing 500mL of YPD medium, cultivate at 28°C and 200rpm for 12-14h, and make the OD 600 =1.3~1.5;

[0069] (3) Centrifuge at 12,000 rpm for 5 min at 4°C to collect the cells;

[0070] (4) Wash the cells twice with 500-250 mL ice-cold sterile water;

[0071] (5) Wash the cells once with 20 mL of ice-cold 1M sorbitol solution;

[0072] (6) Resuspend the cells in 1 mL of ice-cold 1 M sorbitol solution to a final volume of about 1.5 mL, and dispense 80 μL into small centrifuge tubes.

[0073] 3.2 Electric shock t...

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Abstract

The invention provides a Pichia pastoris strain capable of efficiently heterologously expressing Penicillium cyclopium var.albus lipase. The Penicillium cyclopium var.albus lipase comprising precursor peptides (7 amino acids) is heterologously expressed in a Pichia pastoris gene engineering strain, thereby enhancing the activity of the exogenous protein lipase and improving part of enzymological properties of the exogenous protein lipase. According to the enzyme-producing culture medium provided by the invention, the YNB components in the BMMY culture medium are optimized, thereby lowering the cost of the culture medium by up to 70%. The Pichia pastoris strain provides reliable experimental references for industrialized application of the enzyme and especially catalysis of hydrolysis of the substrate Haematococcus pluvialis astaxanthin ester to generate the astaxanthin monomer.

Description

[0001] The patent application of the present invention is a divisional application. The name of the parent case is "Pichia pastoris and enzyme-producing medium for efficient heterologous expression of Penicillium arcuum lipase", the application number is: 201310296638X, and the application date is: July 2013 16th. technical field [0002] 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 [0003] 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 hyd...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/81C12N1/19C12P23/00C12R1/84
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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