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Preparation and application of pichia pastoris whole-cell catalyst for intracellular expression of candida tropicalis lipase

A technology of Candida tropicalis, whole cell catalyst, applied in the field of bioengineering

Active Publication Date: 2017-08-08
JIANGXI NORMAL UNIVERSITY
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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 for intracellular expression of candida tropicalis lipase
  • Preparation and application of pichia pastoris whole-cell catalyst for intracellular expression of candida tropicalis lipase
  • Preparation and application of pichia pastoris whole-cell catalyst for intracellular expression of candida tropicalis lipase

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 biological engineering and provides a preparation method of a pichia pastoris whole-cell catalyst for intracellular expression of candida tropicalis lipase. The preparation method comprises the following steps: carrying out the intracellular expression on a candida tropicalis lipase gene subjected to codon optimization in louis pasteur pichia pastoris GS115; carrying out primary screening and secondary screening to obtain an optimal recombinant strain 3-1; carrying out permeation on the recombinant strain 3-1 by utilizing 5 percent of cetyl trimethyl ammonium bromide; freezing and drying to obtain the pichia pastoris whole-cell catalyst for the intracellular expression of the candida tropicalis lipase. The invention further provides an application method of the pichia pastoris whole-cell catalyst for the intracellular expression of the candida tropicalis lipase to synthesis of 6-O-butyrylcastanospermine; the application method comprises the following steps: taking tetrahydrofuran, castanospermine, vinyl butyrate and the pichia pastoris whole-cell catalyst for the intracellular expression of the candida tropicalis lipase as raw materials and carrying out synthetic reaction under conditions that the temperature is 30 DEG C and the speed is 160r / min. The two methods are simple and efficient and are suitable for industrial application.

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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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 UNIVERSITY
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