Method for preparing lipase of antarctic candida

A technology of Candida Antarctica and lipase, which is applied in the field of nuclear preparation in the field of molecular biotechnology, can solve the problems of low expression of lipase B, high culture cost, and undiscovered problems, and achieves increased expression, reduced cost, and broad The effect of industrialization prospects

Inactive Publication Date: 2010-12-08
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although CALB was heterologously expressed in Aspergillus oryzae in 1995 (Journal of Botany.1995, 73:869), it overcomes the high cost of fermentation and culture of wild-type Candida antarctica and the low expression of lipase B to a certain extent. However, it has not been found that there are reports of documents closely related to the subject of the present invention

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Sequence information and homology analysis of CALB protein gene

[0026] According to the amino acid sequence of Candida antarctica lipase CALB protein, the present invention synthesized the Candida antarctica lipase CALB gene according to the Pichia pastoris preferred codon comparison table. The full length is 957bp, which is the open reading frame. For the detailed sequence, see SEQ ID NO.1. Based on this, it is deduced that except for a stop codon, it encodes a total of 318 amino acid residues, with a molecular weight of 33.3KD and an isoelectric point (pI) of 5.26. See SEQ ID NO.2 for the detailed sequence.

[0027] The artificially synthesized CALB gene sequence and its encoded protein were designed according to the preferred codons of Pichia pastoris, and were nucleated in the Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBankCDS translations+PDB+SwissProt+Superdate+PIR databases using the BLAST program Nucleic acid and protein homology search, it was ...

Embodiment 2

[0072] Production of the CALB gene synthesized in Example 1 in Pichia pastoris

[0073] Construction of Pichia pastoris expression vector

[0074] Under the premise of ensuring the correct reading frame, E. coli containing the pPICZαA plasmid vector of the synthetic gene CALB was inoculated into LB medium containing Zeocin antibiotics, and cultured overnight on a constant temperature shaker at 37°C. Provided methods for extracting plasmids.

[0075] Then it was transferred into Pichia pastoris (such as GS115, or KM71) by electric shock method (refer to "Molecular Cloning", Sambrook et al., 1989).

Embodiment 3

[0077] Induced expression of CALB gene in Pichia pastoris cells and identification of expressed products

[0078] Induced expression of CALB gene in Pichia pastoris cells

[0079] 1. Pick out the recombinant yeast cells containing the CALB gene obtained in Example 2 with a sterile toothpick, inoculate them in 25ml of BMGY medium, and culture them at 28°C with shaking at 250rpm until the optical density value is OD 600 =2-6 (generally 16-18 hours), the cells are in the logarithmic growth phase.

[0080] 2. The cultured cells were centrifuged at 1500-3000g for 5min at room temperature, and the supernatant was discarded.

[0081] 3. Suspend the bacteria in BMMY medium to make the OD of the bacteria solution 600 =1.0, the bacterial solution was kept shaking at 28°C and 250 rpm, and pure methanol was added every 24 hours to a final concentration of 0.5%, so as to ensure normal induction by supplementing volatilized methanol.

[0082] 4. According to the time points (0, 6, 12, 24...

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Abstract

This invention provides a nucleotide sequence of Candida Antarctica lipase B (CALB) gene. In this invention, CALB gene is artificially synthesized according to Pichia pastoris preferred codon. The obtained CALB nucleotide sequence can code the protein with the same function as wild-type CALB protein, and can hybridize with the 1st-957th nucleotide sequence of SEQ ID No.1 under moderate stringencyconditions. CALB gene can effectively express lipase in Pichia pastoris, while the lipase expression level can reach 21.6 mg / L. This invention can be used in lipase mass production.

Description

technical field [0001] The invention relates to a method for preparing nuclei in the field of molecular biology technology, in particular to a method for preparing lipase from Candida antarctica. Background technique [0002] Lipase (EC.3.1.1.3) is a very important class of enzymes in biocatalytic reactions, and has been widely used in many fields such as oil chemistry, detergent, food industry and fine chemical product preparation. Among many lipases, CALB (Candida antarctica lipase B, Candida antarctica lipase B) is the most widely used, and it has strong catalytic activity for both water-insoluble and water-soluble substances. Research results in recent years have shown that CALB and Novozym 435 (CALB adsorbed and immobilized on macroporous acrylic resin) have shown better catalytic performance than other lipases in esterification, hydrolysis, transesterification and other types of reactions. The good catalytic performance of CALB has attracted the attention of the whole...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C07H21/04C12N9/20C12N15/81C12N1/19C12R1/84
Inventor 唐克轩姚红艳
Owner SHANGHAI JIAO TONG UNIV
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