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Method for producing lipase by recombinant Muts type methylotrophic pichia pastoris

Inactive Publication Date: 2012-11-14
ZHENGZHOU HUIZE BIOCHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mut + Mut-type strains have strong ability to metabolize methanol, and in most cases, the expression level of foreign proteins is higher than that of Mut s The type strain is high, so the recombinant methanolotrophic Pichia pastoris has always used Mut + However, when this type of strain is fermented, the consumption of methanol is large and the requirements for dissolved oxygen are high, which in turn leads to large consumption of energy such as electric energy and cooling water. Not only is the cost high, the equipment requirements are high, and it is not easy to control
At the same time, methanol is volatile and flammable, and it is potentially dangerous to store in large quantities.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0017] Preparation of medium and culture medium:

[0018] 1) Seed medium

[0019] Yeast powder 10g / ml, peptone 20g / ml, glycerin 10g / ml, 100mM pH6.0 phosphate buffer 100ml / L.

[0020] 2) Fermentation medium BSM

[0021] 85%H 3 PO 4 26.7ml / L, CaSO 4 0.93g / L,K 2 SO 4 18.2g / L, MgSO 4 ·7H 2 O14.9g / L, KOH4.13g / L, glycerin 40g / L, PTM 1 4.00ml / L, 25% ammonia water to adjust pH5-6.

[0022] 3) PTMs 1

[0023] CuSO 4 ·5H 2 O6g / L, NaI0.08g / L, MnSO 4 ·H 2 O3g / L, Na 2 MoO 4 2H 2 O0.2g / L, H 3 BO 3 0.02g / L, CoCl 2 0.5g / L, ZnCl 2 20g / L, FeSO 4 ·7H 2 O65g / L, H 2 SO 4 5ml / L, biotin 0.2g / L.

[0024] 4) Supplement growth solution

[0025] Contains 12ml / LPTM 1 50% glycerin.

[0026] 5) Induction medium

[0027] Contains 12ml / L PTM 1 of 100% methanol.

Embodiment 1

[0029] This embodiment reorganizes Mut s Type methanolotrophic Pichia pastoris produces the method for lipase, concrete steps are as follows:

[0030] 1) Pick the recombinant Mut s A single colony of methanolotrophic Pichia pastoris GS115 was inoculated in the seed medium, cultured at 28°C and 200 rpm for 20 hours, and then inoculated in the sterilized fermentation medium BSM, with an inoculation amount of 5%;

[0031] 2) Fermentation with ventilation and stirring. During the fermentation process, the dissolved oxygen is controlled at 40%, 25% industrial ammonia water is automatically added to adjust the pH to 6, and the temperature is controlled at 28°C;

[0032] 3) When the glycerol in the fermentation medium is exhausted, add 12ml / L PTM 1 50% glycerol, continue to maintain dissolved oxygen at 35%;

[0033] 4) When the wet weight of the cells reaches more than 350g / L, stop adding glycerol and maintain a starvation state;

[0034] 5) When the glycerin is exhausted, add 12...

Embodiment 2

[0037] This embodiment reorganizes Mut s Type methanolotrophic Pichia pastoris produces the method for lipase, concrete steps are as follows:

[0038] 1) Pick the recombinant Mut s A single colony of methanolotrophic Pichia pastoris GS115 was inoculated in the seed medium, cultured at 28°C and 200 rpm for 20 hours, and then inoculated in the sterilized fermentation medium BSM, with an inoculation amount of 10%;

[0039] 2) Fermentation with aeration and stirring. During the fermentation process, the dissolved oxygen is controlled at 30%, 25% industrial ammonia water is automatically added to adjust the pH to 5, and the temperature is controlled at 28°C;

[0040] 3) When the glycerol in the fermentation medium is exhausted, add 12ml / L PTM 1 50% glycerol, continue to maintain dissolved oxygen at 30%;

[0041] 4) When the wet weight of the cells reaches more than 350g / L, stop adding glycerol and maintain a starvation state;

[0042] 5) When the glycerin is exhausted, add 12ml...

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PUM

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Abstract

The invention discloses a method for producing lipase by recombinant Muts type methylotrophic pichia pastoris. The method specifically comprises the following steps: inoculating recombinant Muts type methylotrophic pichia pastoris GS115 to a seed culture medium to culture; then inoculating to a fermenting culture medium BSM to ferment; adding an additional medium culture solution till glycerol of the fermenting culture medium is used up; stopping adding the additional medium culture solution when the cell wet weight reaches more than 350g / L; feeding an induction culture solution till glycerol is used up, wherein the feeding amount is 1-3ml / L per h, the culture temperature is controlled at 22-26 DEG C, and induction culture is carried out; and centrifuging to obtain lipase-containing supernate. In the method, the lipase is produced by using the recombinant Muts type methylotrophic pichia pastoris without an AOX1 gene, the strain consumes less methanol and requires less dissolved oxygen, thus the power load can be alleviated, the energy consumption is small, the raw material cost is saved, the method is convenient to control and is beneficial for industrial production.

Description

technical field [0001] The present invention relates to a kind of recombinant Mut s The invention discloses a method for producing lipase by methanolotrophic Pichia pastoris, belonging to the technical field of microorganisms. Background technique [0002] Lipase is a class of enzymes with various catalytic capabilities, which can catalyze the hydrolysis, alcoholysis, esterification, transesterification and reverse synthesis of triacylglycerides and other water-insoluble esters. In addition, it also exhibits some other enzyme activities, such as phospholipase, lysophospholipase, cholesterol esterase, acyl peptide hydrolase activity, etc., with high chemoselectivity and stereoisomerism. The different activities of lipase depend on the characteristics of the reaction system, such as promoting ester hydrolysis at the oil-water interface, and enzymatic synthesis and transesterification in the organic phase. It is widely used in food, textile, leather, spices, cosmetics, deterge...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12R1/84
Inventor 司富强蔡翔
Owner ZHENGZHOU HUIZE BIOCHEM TECH