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Preparation and application of Pichia pastoris whole-cell catalyst expressing Candida tropicalis lipase intracellularly

A recombinant strain, typical technology, applied in the field of bioengineering

Active Publication Date: 2020-10-16
JIANGXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in previous studies, there is no report on the synthesis of 6-O-butyryl castanospermine using whole-cell biocatalysis

Method used

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  • Preparation and application of Pichia pastoris whole-cell catalyst expressing Candida tropicalis lipase intracellularly
  • Preparation and application of Pichia pastoris whole-cell catalyst expressing Candida tropicalis lipase intracellularly
  • Preparation and application of Pichia pastoris whole-cell catalyst expressing Candida tropicalis lipase intracellularly

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Preparation of Pichia pastoris whole-cell catalyst expressing Candida tropicalis lipase intracellularly:

[0022] According to the codon preference of Pichia pastoris, the lipase gene of Candida tropicalis was codon-optimized, and then the codon-optimized lipase gene of Candida tropicalis was synthesized in vitro. The gene contains KpnI and NotI restriction sites. The gene and the pPICZB plasmid were digested with KpnI and NotI respectively, and then the digested products were ligated, and the obtained recombinant plasmid was transformed into E.coli Top 10 competent cells, and recombinants containing the recombinant plasmid were screened.

[0023] The recombinant plasmid was extracted from the recombinant, and SalI linearized the recombinant plasmid at 37°C for 2 hours, and then transferred the recombinant plasmid into Pichia pastoris GS11 by electroporation, and the electroporation conditions were 1500V, 25μF, 200Ω. Add 1mL 1mol / L sorbitol immediately after the electr...

Embodiment 2

[0028] The Pichia pastoris whole-cell catalyst expressing Candida tropicalis lipase prepared in Example 1 catalyzes castanospermine and vinyl butyrate to synthesize 6-O-butyryl castanospermine:

[0029] Add 4 ml of THF, 4 micromol of castanospermine, 8 micromol of vinyl butyrate and 100 mg of Pichia whole-cell catalyst expressing Candida tropicalis lipase in a 10 ml stoppered Erlenmeyer flask. The yield of 6-O-butyryl castanospermine reached 27.2% within 60 minutes under the conditions of 30°C and 160r / min.

[0030] Codon-optimized Candida tropicalis lipase gene sequence:

[0031] Jiangxi Normal University

[0032] Preparation and Application of Pichia pastoris Whole Cell Catalyst Expressing Candida tropicalis Lipase Intracellularly

[0033] 1

[0034] Patent In Version 3.5

[0035] 1

[0036] 1364

[0037] DNA

[0038] Artificial sequence

[0039]

[0040] 1

[0041]

[0042]

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Abstract

The invention belongs to the field of bioengineering, and provides a method for preparing a Pichia whole-cell catalyst expressing Candida tropicalis lipase intracellularly: a codon-optimized Candida tropicalis lipase gene in Pichia pastoris Intracellular expression was carried out in yeast GS115, and the best recombinant strain 3-1 was obtained after primary screening and re-screening; the recombinant strain 3-1 was permeabilized with 5% cetyltrimethylammonium bromide, and frozen After drying, the whole-cell catalyst of Pichia pastoris expressing Candida tropicalis lipase in the cell is obtained. The present invention also provides an application method of the Pichia pastoris whole-cell catalyst expressing Candida tropicalis lipase in the synthesis of 6-O-butyryl castanospermine: tetrahydrofuran, castanospermine, vinyl butyrate Esters and Pichia pastoris whole-cell catalyst expressing Candida tropicalis lipase in cells were used as raw materials, and the synthesis reaction was carried out at 30°C and 160r / min. These two methods are simple and efficient, and suitable for industrial applications.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to the expression of the codon-optimized Candida tropicalis lipase gene in Pichia pastoris GS115, the optimization of expression conditions, the preparation of Pichia whole-cell catalyst and its application in the synthesis of Australian chestnut Application of spermine derivatives. Background technique [0002] Lipase (EC 3.1.1.3) belongs to the α / β hydrolase family. It can catalyze various reactions such as hydrolysis, esterification, transesterification, acidolysis, alcoholysis, and ammonolysis. It is used in food, washing, tanning, feed, It is widely used in pharmaceutical, energy and other fields. The sources of lipase mainly include plants, animals and microorganisms. Among them, microbial lipase has the advantages of diverse catalytic activity, good stability, high yield, and mass production. Therefore, microbial lipase is more widely used. It is estimated that 28 g...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12N9/20C12P17/18C12R1/84
CPCC12N9/20C12N15/815C12P17/182C12Y301/01003
Inventor 彭仁张雨佳洪远明辉辉洪彬文
Owner JIANGXI NORMAL UNIV
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