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Method for preparing low-temperature neutral lipase by using Geotrichum candidum

A neutral lipase and production method technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problem of low yield of isolated and purified enzymes, and achieve the effect of excellent low-temperature activity

Inactive Publication Date: 2010-02-03
THE INST OF MICROBIOLOGY XINJIANG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chromatographic separation method has a low yield of separating and purifying the enzyme, generally between 15% and 40%, and the purity is above 60%.

Method used

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  • Method for preparing low-temperature neutral lipase by using Geotrichum candidum
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  • Method for preparing low-temperature neutral lipase by using Geotrichum candidum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Example 1: Screening of low-temperature lipase-producing bacteria

[0097] Using the olive oil-bromocresol purple indicator plate method well known to those skilled in the art, 12 strains of lipase-producing bacteria were screened from the oily permafrost of the oil and fat chemical plant in Changji City, Xinjiang, and then transferred to the rhodamine B plate for further identification and detection, and finally inserted into the enzyme-producing medium for lipase activity determination. The experimental results showed that the ratio of the yellow color circle formed by Geotrichum candidum ch-3 on the olive oil-bromocresol purple indicator plate to the diameter of the colony was larger, and the lipase activity was higher. See attached figure 1 A can see that Geotrichum candidum (Geotrichum candidum) ch-3 forms an orange fluorescent circle on the rhodamine B plate; figure 1 B shows the situation of Geotrichum candidum ch-3 forming a yellow color circle on the oil as...

Embodiment 2

[0099] Embodiment 2: the identification of producing enzyme strain

[0100] The colony diameter of the strain G.candidum ch-3 cultured on PDA and wort medium at 20°C for 2-3 days is 30-40 mm, white powdery, and the center of the colony protrudes and has radiation. When the colony surface is disturbed, it becomes a yeast-like viscous. Observed under a microscope, it has mycofilaments, some have bifurcated branches, and more or less septa. The mycelium is 3-7um wide, and the reproduction method is fission. The formed arthrospores are single or connected in chains, and the spores are oblong, square, oval or round, with blunt rounded ends. Most of the arthrospores are 3~6×6~12um. According to the above morphological characteristics, it was preliminarily determined that the strain ch-3 belonged to the genus Geotrichum.

[0101] In order to further determine the taxonomic position of the strain G. candidum ch-3, we determined its 18SrRNA gene sequence. The genomic DNA of the st...

Embodiment 3

[0103] Embodiment 3: low temperature neutral lipase activity assay method

[0104] According to the standard GB / T 1803-93 (2002) promulgated by the Ministry of Light Industry of the People's Republic of China, general test methods for industrial enzyme preparations, the temperature was set at 35°C during the test.

[0105] A4.1 Definition

[0106] 1g of solid enzyme powder (or 1mL of liquid enzyme), under certain temperature and pH conditions, hydrolyzes fat to produce 1umoL of fatty acid per minute, which is one international unit of lipase, expressed in U / g (or U / mL).

[0107] A4.2 Principle

[0108] Under certain conditions, lipase can hydrolyze triglycerides into fatty acids, diglycerides, monoglycerides and glycerol. The released fatty acids can be neutralized and titrated with standard alkali solution, and the reaction end point can be indicated by pH meter or phenolphthalein indicator solution. , according to the amount of alkali consumed, calculate its enzyme activit...

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Abstract

The invention discloses a method for preparing low-temperature neutral lipase by using Geotrichum candidum (CGMCC.No.197). The methods of aqueous two-phase extraction and SephadexG-75 gel filtration are used for separating and purifying the fermentation liquid of the Geotrichum candidum. The bacteria provided by the invention are low-temperature neutral lipase producing bacteria which have the advantages that commercial application prospect is good, the lipase producing temperature is stable, the sources of the raw materials are wide, and the production cost is low. The expression product prepared by the invention is novel low-temperature neutral lipase, wherein the optimal action temperature is 35 DEG C, and the optimal pH is 7.0. The novel low-temperature neutral lipase can be widely used in the fields of food, chemical industry, medicinal products and the like.

Description

[0001] The present invention application is a divisional application of the invention and creation titled "a low-temperature neutral lipase and Geotrichum candidum producing the enzyme" filed by the applicant on April 13, 2007, and the filing date of the original application is April 2007 On the 13th, the application number was 200710100487.0, and the name of the invention was "a low-temperature neutral lipase and Geotrichum candidum producing the enzyme". technical field [0002] The invention relates to the field of microbial fermentation and its product enzymes, in particular to a Geotrichum candidum strain producing low-temperature neutral lipase isolated from an alpine region and a novel low-temperature neutral lipase produced by the strain. Background technique [0003] Low-temperature microorganisms are excellent materials for studying biological adaptability under low-temperature conditions, and are also the best sources for obtaining low-temperature enzymes with high...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12R1/645
Inventor 付建红姚斌石玉瑚孟昆欧阳平凯袁铁铮古丽尼莎
Owner THE INST OF MICROBIOLOGY XINJIANG ACADEMY OF AGRI SCI
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