A strain of Yarrowia lipolytica mutant strain capable of highly yielding lipase, cultivation method and use of enzyme thereof

A technology of Yarrowia lipolytica and lipase lipolysis, which is applied in the field of catalyzing fatty acid methyl esterification, Yarrowia lipolytica strain, lipase or fermentation broth, and can solve the problems of amplification and high cost of lipase

Active Publication Date: 2009-05-27
领先生物农业股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cost of lipase monopolized by NOVA is high and it is not suitable for further enlargement of catalytic reactors

Method used

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  • A strain of Yarrowia lipolytica mutant strain capable of highly yielding lipase, cultivation method and use of enzyme thereof
  • A strain of Yarrowia lipolytica mutant strain capable of highly yielding lipase, cultivation method and use of enzyme thereof
  • A strain of Yarrowia lipolytica mutant strain capable of highly yielding lipase, cultivation method and use of enzyme thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083]The wild-type Yarrowia lipolytica isolated from the surrounding environment of the canteen of Nongda University was activated once by PDA solid medium at 28°C, activated twice by PDA liquid medium at 28°C, and then inoculated at 5% by weight PDA liquid culture medium was inserted in a large amount, cultivated at 28°C for 12h, and then 1ml of the bacterial solution was added to 10ml of sterilized 0.86% NaCl aqueous solution by weight to obtain the bacterial suspension of the starting strain.

[0084] The number of live bacteria of the above-mentioned starting strain bacterial suspension obtained was adjusted to 1.85×10 6 each / ml, the suspension is placed on a glass slide, and its suspension layer thickness is 0.2cm, allowing it to irradiate for 90 seconds under a 20-watt ultraviolet lamp 25cm away from the ultraviolet lamp to carry out ultraviolet mutagenesis; immediately after the ultraviolet mutagenesis ends The bacterial solution was spread on a PDA solid medium plate,...

Embodiment 2

[0089] After culturing Yarrowia lipolytica mutant strain LW1 in an eggplant bottle for 24 hours, add 50ml of sterilized distilled water to it, wash off the bacterial lawn, and inoculate it directly in a 100L fermenter. Cultivate under the fermentation conditions of 0.7 atmosphere pressure and air ventilation 1:1 (v / m). After fermenting for 63 hours, increase the rotation speed of the fermenter from 180 rpm to 240 rpm, at this time the tank pressure is increased to 1.0 atmospheric pressure, and the ventilation rate is increased from 1:1 (v / m) to 1:2 (v / m m), cultivated for 100 hours, and the enzyme activity measured by the olive oil titration method described in the specification of this application reached 8250u / ml.

Embodiment 3

[0091] Cultivate Yarrowia lipolytica mutant strain LW1 in an eggplant bottle for 24 hours, add 50ml of sterilized distilled water to it, wash off the bacterial lawn, and inoculate it directly in a 100L fermenter at 28°C, initial pH 6.5, tank pressure 0.6 Atmospheric pressure and air ventilation 1:1 (v / m) fermentation culture, when fermented to 55 hours, the growth rate of enzyme activity was slow, and 2% soybean oil by weight was added, and the rotating speed was increased from 180 rpm to 240 rpm. When cultivating for 75 hours, add 2% soybean oil by weight. At this time, the pressure of the tank is raised to 1.0 atmospheric pressure. After cultivating for 97.5 hours, the enzyme activity measured by the olive oil titration method described in the specification of this application reaches 15500u / ml.

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Abstract

The invention provides a Yarrowia lipolytica lipase high-yield strain LW1 (CGMCC No.2707) obtained through an ultraviolet mutagenesis method, a fermentation culture method, a lipase purification method, and a method for catalyzing the methyl esterification of fatty acid thereof. Yarrowia lipolytica CGMCC No.2707 is cultured for 97.5 h through fed-batch fermentation on a fermentation tank with the capacity of 100L, the lipase production level is 15,500U / ml, and the activity of produced lipase powder is 30,000U / g. The obtained lipase powder or a zymotic fluid can be directly applied to a reaction of catalyzing the methyl esterification of the fatty acid. The conversion rate for 30h is more than 90 percent when the fatty acid and methanol are added according to the equal mol ratio and the using amount of the lipase in a system is 1,000-3,000U per gram of fatty acid.

Description

【Technical field】 [0001] The invention relates to the field of biotechnology, in particular to a Yarrowia lipolytica strain with high lipase production, a fermentation culture method, and the use of the lipase obtained by fermentation or fermentation liquid in catalyzing fatty acid methyl esterification. 【Background technique】 [0002] Yarrowia lipolytica strain, also known as Yarrowia lipolytica (Yarrowia lipolytica), is a semi-ascomycete and one of the unconventional yeasts. It has been used in the production of lipase, single-cell protein, citric acid, isopropyl malic acid Wait. The lipase it produces is widely used in industry, light industry, food, medicine and other fields. [0003] Lipase can catalyze the hydrolysis of fatty acid glycerides into free fatty acids, diglycerides, monoglycerides, glycerol, and also catalyze the transesterification reaction between alcohols or esters and glycerides, and the esterification reaction between fatty acids and alcohols , has a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/16C12N9/20C12P7/62C12R1/645
Inventor 李颖王贵利关国华孟永宏李月娟徐志文
Owner 领先生物农业股份有限公司
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