Marine low-temperature alkaline lipase Bohai Sea-9145 crystal and preparation method thereof

A lipase, low-temperature technology, applied in the field of marine microorganisms, can solve the problems of undiscovered, and achieve the effects of short fermentation period, high low-temperature catalytic activity, and wide source of raw materials

Active Publication Date: 2014-03-19
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Through literature search, do not find the public literature report identical with marine lipase Bohai Sea-9145 crystal structure of the present invention

Method used

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  • Marine low-temperature alkaline lipase Bohai Sea-9145 crystal and preparation method thereof
  • Marine low-temperature alkaline lipase Bohai Sea-9145 crystal and preparation method thereof
  • Marine low-temperature alkaline lipase Bohai Sea-9145 crystal and preparation method thereof

Examples

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Effect test

Embodiment 1

[0028] Example 1——Preparation of marine low-temperature alkaline lipase Bohai Sea-9145 crystals

[0029] 1) Preparation of crystallization (growth) buffer: Prepare a 0.1M sodium acetate buffer with a pH of 4.6, which contains 20% by mass of ammonium sulfate as a precipitant.

[0030] 2) Preparation of marine low-temperature alkaline lipase Bohai Sea-9145 aqueous solution

[0031] The marine low-temperature alkaline lipase Bohai Sea-9145 with an enzyme activity of 4000U / mL was prepared as a 10mg / ml aqueous solution.

[0032] 3) Preparation of crystallization solution:

[0033] 1 μL of crystallization buffer prepared in the above step 1, 1 μL of 20 mg / ml marine yeast low-temperature alkaline lipase Bohai Sea-9145 aqueous solution; using the hanging drop method, place the above crystallization solution on a plastic cover glass, and then drop the crystallization solution Put it upside down on a 24-well crystallization plate, seal the gap in the middle with Vaseline, culture the ...

Embodiment 2、3

[0034] Embodiment 2, 3—Preparation of marine low-temperature alkaline lipase Bohai Sea-9145 crystal

[0035] The preparation procedure of embodiment 2,3 is identical with embodiment 1, and difference is: wherein prepare respectively 0.2M, 0.3M, pH is the sodium acetate buffer solution of 4.6, and the mass percent content containing precipitating agent ammonium sulfate is respectively 25% 30% ; The prepared marine low-temperature alkaline lipase Bohai Sea-9145 aqueous solution was 15, 20mg / ml respectively.

Embodiment 4

[0036] Example 4——Structural analysis of marine low-temperature alkaline lipase Bohai Sea-9145 crystal

[0037] First, use software such as HKL2000 to conduct X-ray diffraction on the crystal of marine low-temperature alkaline lipase Bohai Sea-9145, collect the obtained diffraction data for processing, and obtain a complete data file; secondly, use software such as Phaser and Molrep in the CCP4 program package, Using molecular replacement (MR, Molecular Replacement) method, Yarrowia lipolytica lipase (PDBid: 3O0D), thermophilic (Humicola) humic lipase (PDBid: 1DT3) and fungal lipase (PDBid: 1TIA) were used as Search the model and obtain the fine three-dimensional structure of marine low-temperature alkaline lipase Bohai Sea-9145 ( figure 2 , 3 ).

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Abstract

The invention relates to a marine low-temperature alkaline lipase Bohai Sea-9145 crystal and a preparation method thereof. The crystal is in a short prismatic shape the space structure of which is a fine three dimensional structure, and structurally comprises 371 amino acids and 8 alpha helical structures and 9 beta folded structures. The crystal structurally comprises crystal package elements which meet the conditions a=b=128.87 angstrom, C=171.77 angstrom and alpha=beta=gamma=90 degrees; the protein space group of crystals is I4, the position of the cover domain of the crystals is serine (S112)-phenylalanine (F122). Serine (S195), aspartic acid (A258) and histidine (H318) form a catalytic triplet. Tyrosine (T110) and leucine (L193) form an oxygen anion cavity region. The crystal is an important biological catalyst, has a series of advantages of high low temperature catalytic activity, stable property under alkaline condition, good compatibility with metal ions and surfactants and the like, and can be widely applied to the low-temperature catalytic fields of daily use chemicals, food processing and medicines.

Description

technical field [0001] The invention relates to the field of marine microorganisms, in particular to a novel marine low-temperature alkaline lipase Bohai Sea-9145 crystal and a preparation method thereof. Background technique [0002] Lipase is an ancient enzyme with a history of hundreds of years. With the rise of technologies such as cell engineering, genetic engineering, and protein engineering in recent years, its application range in the fields of bioenergy, food processing, and medical engineering has been continuously expanded and rejuvenated. new vitality. At present, more than 4,000 lipase genes have been reported in the National Center for Biological Information (NCBI) (including lipase genes from many microorganisms such as Pseudomonas, yeast, Staphylococcus, Streptomyces, and Bacillus subtilis). There are about 200 structures, and the number is still growing rapidly. [0003] The development and application of lipase depend on the substrate specificity of the e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20
CPCC12N9/20C12Y301/01003
Inventor 盛军孙谧郝建华徐甲坤王跃军
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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