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Lipase, engineering bacterium and preparing methods of the lipase and the engineering bacterium

A lipase and engineering bacteria technology, applied in the field of genetic engineering, can solve the problems of low enzyme activity and unsatisfactory economic benefits, and achieve the effects of improving unit enzyme activity, good industrial applicability, and improved expression

Inactive Publication Date: 2015-04-01
HUAZHONG UNIV OF SCI & TECH RES INST SHENZHEN +1
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0005] In view of the above defects or improvement needs of the prior art, the present invention provides a lipase, engineering bacteria and a preparation method thereof, the purpose of which is to solve the current problem of engineering bacteria production by changing the lipase codon and the genetic structure of the engineering bacteria. Lipase, the technical problem of low enzyme activity and unsatisfactory economic benefits

Method used

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  • Lipase, engineering bacterium and preparing methods of the lipase and the engineering bacterium
  • Lipase, engineering bacterium and preparing methods of the lipase and the engineering bacterium

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preparation example Construction

[0057] The preparation method of engineering bacterium provided by the invention is characterized in that, comprises the following steps:

[0058] (1) Construction of lipase gene expression vector: the Pichia pastoris expression system plasmid used for His site insertion is used as a vector, which contains the 5'AOX1 strong promoter, the α-factor signal peptide of Saccharomyces cerevisiae, and the lipase in sequence Gene fragments, the lipase gene fragments include 5'AOX1-TT fragments, His4 (histidine dehydrogenase gene), 3'AOX1, PBR322 and Ampicillin; the Pichia pastoris expression system for His site insertion Plasmid is PAO815 plasmid, its structure is as follows figure 1 shown.

[0059] (2) Constructing a molecular chaperone combination gene expression vector: using the Pichia pastoris expression system plasmid for inserting functional genes at the AOX1 site as a vector, insert two functional genes, wherein the first functional gene contains 5'AOX1 in sequence A strong p...

Embodiment 1

[0073] A lipase whose gene sequence is the gene sequence shown by SEQ ID No.1. Before and after codon optimization, the codon adaptability index changes as image 3 shown. The frequency distribution of codon usage after codon optimization is as follows: Figure 4 shown. GC content in codon usage before and after codon optimization as Figure 5 shown.

Embodiment 2

[0075] A kind of engineering bacterium, its host bacterium is GS115 type Pichia pastoris, gene structure is as follows figure 1 shown.

[0076] At its His site, the gene sequence of the lipase of Example 1 is inserted, and the copy number is 2. The α-factor signal peptide gene sequence of Saccharomyces cerevisiae is inserted immediately upstream of it.

[0077] A molecular chaperone combination gene is inserted at the AOX1 site to increase the expression of the lipase. The molecular chaperone combination gene is pGAPZAEro1p-pPICZABip, and its sequence is the gene sequence shown by SEQ ID No.2.

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Abstract

A lipase, an engineering bacterium and preparing methods of the lipase and the engineering bacterium are disclosed. The gene sequence of the lipase is a gene sequence shown as SEQ ID No.1 or an isogenous gene sequence with same functions. The host bacterium of the engineering bacterium is pichia pastoris. The gene sequence of the lipase is inserted at an His locus. The preparing method of the engineering bacterium includes: (1) constructing a lipase genetic expression vector; (2) constructing a molecular chaperone composite genetic expression vector; (3) preparing a lipase-expressed transgenic engineering bacterium; and (4) preparing the high-lipase-expression engineering bacterium. Preparation of the lipase by utilization of the engineering bacterium includes steps of: A) activating to obtain a seed solution; B) fermenting in a fermentation tank; and C) centrifuging and collecting supernate liquid. The heterologous expression of the lipase in the engineering bacterium is increased. The engineering bacterium has a secretion property, and is high in unit enzyme activity, convenient in production and good in economic benefit.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically relates to a lipase, an engineering bacterium and a preparation method thereof. Background technique [0002] Lipase (Triacylglycerol acylhydrolase, Lipase EC3.1.1.30), also known as triglyceride hydrolase, can catalyze esterification reaction, alcoholysis reaction, ammonolysis reaction, etc. It has good stability in organic solvents, and has high position selectivity and isomer selectivity. Candida rugosa, formerly known as Candida cylindracea, candida rugosa lipases (C.rugosa lipases, CRLs) are widely used in chemical, energy, food, medical and other fields, and are currently one of the most widely used commercial lipases in industry. In the original C.rugosa strain, the expression levels of several isozymes from high to low were LIP1, LIP3, LIP2, LIP5 and LIP4. [0003] It has been reported that CRL1 lipase is not high, Brocca[Design, total synthesis, and functional o...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12N1/19C12N15/81C12R1/84
CPCC12N9/20C12Y301/01003
Inventor 闫云君刘子敏王桂龙徐莉潘笃杰
Owner HUAZHONG UNIV OF SCI & TECH RES INST SHENZHEN
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