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A kind of marine low-temperature alkaline lipase bohai Sea-9145 crystal and preparation method thereof

A lipase and alkaline technology, which is applied in the field of marine low-temperature alkaline lipase BohaiSea-9145 crystal and its preparation, can solve undiscovered problems and achieve the effects of short fermentation cycle, good compatibility and high low-temperature catalytic activity

Active Publication Date: 2017-02-15
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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  • A kind of marine low-temperature alkaline lipase bohai Sea-9145 crystal and preparation method thereof
  • A kind of marine low-temperature alkaline lipase bohai Sea-9145 crystal and preparation method thereof
  • A kind of marine low-temperature alkaline lipase bohai Sea-9145 crystal and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1——Preparation of marine low-temperature alkaline lipase Bohai Sea-9145 crystals

[0030] 1) Preparation of crystallization (growth) buffer: prepare a 0.1 M sodium acetate buffer with a pH of 4.6, which contains 20% by weight of the precipitating agent ammonium sulfate.

[0031] 2) Preparation of marine low-temperature alkaline lipase Bohai Sea-9145 aqueous solution

[0032] The marine low-temperature alkaline lipase Bohai Sea-9145 with an enzyme activity of 4000U / mL was prepared into 10mg / ml aqueous solution respectively.

[0033] 3) Preparation of crystallization solution:

[0034] 1 μL of crystallization buffer and 1 μL of 20 mg / ml marine yeast low-temperature alkaline lipase aqueous solution prepared by the above steps; using the hanging drop method, place the above crystallization solution on a plastic cover glass, and then invert the crystallization solution at 24 On the well crystallization plate, the gap in the middle was sealed with Vaseline, and the ...

Embodiment 2

[0035] Embodiment 2,3-Preparation of marine low-temperature alkaline lipase Bohai Sea-9145 crystal

[0036] The preparation steps of Examples 2 and 3 are the same as those of Example 1, the difference is that 0.2M, 0.3M, pH 4.6 sodium acetate buffer solution is prepared, and the mass percentage of ammonium sulfate containing precipitant is 25% and 30% respectively; The prepared marine low-temperature alkaline lipase Bohai Sea-9145 aqueous solutions were 15 and 20 mg / ml respectively.

Embodiment 4

[0037] Example 4——Structural analysis of marine low-temperature alkaline lipase Bohai Sea-9145 crystal

[0038] First, use software such as HKL2000 to process the diffraction data collected in the previous step to obtain a complete data file; secondly, use software such as Phaser and Molrep in the CCP4 program package, and use the molecular replacement (MR, Molecular Replacement) method to obtain a complete data file. Yarrowia lipolytica lipase (PDBid: 3O0D), thermophilic (Humicola) humic lipase (PDBid: 1DT3) and fungal lipase (PDBid: 1TIA) were used as search models to obtain marine low-temperature alkaline lipase Bohai Sea The fine three-dimensional structure of -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 has a short prism shape, and its spatial structure is a fine three-dimensional structure. The structure contains 371 amino acids, 8 α-helical structures and 9 β-fold structures; the structure has α=β=γ=90°unit cell , the protein space group of the crystal is I4, and the position of the lid domain is serine (S112)-phenylalanine (F122). Serine (S192), aspartic acid (D258) and histidine (H318) constitute its "catalytic triad", and tyrosine (Y110) and leucine (L193) constitute its oxyanion cavity. The crystal is an important class of biocatalysts, with a series of advantages such as high catalytic activity at low temperature, stable properties under alkaline conditions, and good compatibility with metal ions and surfactants, and can be widely used in daily chemicals, food processing and Medicine and other low-temperature catalysis fields.

Description

technical field [0001] The invention relates to the field of marine microorganisms, in particular to a 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 enzyme ...

Claims

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Application Information

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Patent Type & Authority Patents(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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