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High-efficiency directed evolution method of lipase gene

A lipase gene and directed evolution technology, which is applied in the fields of directed evolution of lipase gene, directed evolution of enzyme gene, and molecular transformation, can solve problems such as complex operation steps and difficulty in screening mutants, achieve improved enzymatic properties, and avoid extracellular Genetic manipulation, the effect of facilitating high-throughput screening

Inactive Publication Date: 2010-02-10
HUBEI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invention synchronizes the construction of the mutant library and the expression of the mutants in the conventional directed evolution technology, and overcomes the disadvantages of the prior art, such as the complex operation steps of the directed evolution of lipase molecules and the difficulty in screening the mutants.

Method used

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  • High-efficiency directed evolution method of lipase gene
  • High-efficiency directed evolution method of lipase gene
  • High-efficiency directed evolution method of lipase gene

Examples

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Embodiment 1

[0026] Implementation example 1: Construction of Saccharomyces cerevisiae display vector mediated by Flop membrane protein

[0027] The protein Flo1p is a condensin-like cell wall protein encoded by the F01 gene in S. cerevisiae. According to the complete sequence of the reported F01 gene (GenBank NO.NC_01133), a primer for the coding sequence of the flocculation functional region of Flo1p was designed: FOLf-Hind III and FOLr-Bgl II (FOLf-Hind III: 5'-acataagcttatgacaatgcctcatcgctatatgtttttg-3'FOLr-Bgl II: 5'-gatagatctggtgatttgtcctgaagatgatgatgacaaa-3'), obtained by PCR amplification using the total DNA of Saccharomyces cerevisiae ATCC 60715 as template The coding sequence fragment of the flocculation functional region of Flo1p was ligated with the Saccharomyces cerevisiae expression vector pYES2 / NT after Hind III and Bg1 II double digestion with Hind III and BamH I under the action of T4DNA ligase (Bg1 II and BamHI are mutually homologous enzymes), and the expression vector p...

Embodiment 2

[0028] Implementation Example 2: Construction of Lipase Gene Saccharomyces cerevisiae Display Expression Recombinant Plasmid

[0029] 2.1. Construction of Saccharomyces cerevisiae display expression vector for lipase gene derived from Pseudomonas fluorescens

[0030] The full-length gene primers LipB52Pf (5'-aaagaattcccaacaaaaagagaggcaacagcaatg-3') and LipB52Pr (5'-aaagcgcgcgctccctccccacccttgtcgtcagg-3') were designed based on the lipase gene lipB52 (Genbank NO.AY623009) sequence, and the P.fluorescens B52 genomic DNA was used as a PCR template The lipB52 full-length gene fragment was amplified, and after purification, the fragment was digested with restriction endonucleases Eco RI and Not I, and the double-digested lipB52 gene fragment was recovered from the gel and Saccharomyces cerevisiae was digested with Eco RI and Not I. The display expression vector pLHJ042 was ligated under the action of T4DNA ligase to obtain the lipB52 gene Saccharomyces cerevisiae display expression...

Embodiment 3

[0035] Implementation Example 3: Obtaining lipase gene fragments with carrier homologous sequences and construction of expression mutation library

[0036] 3.1 Obtaining the lipase gene fragment with carrier homologous sequence

[0037] The primers pLHJ042F and pLHJ042R located at both ends of the multiple cloning site were designed according to the sequence of the expression vector pLHJ042 displayed by Saccharomyces cerevisiae. The sequence of primer pLHJ042F (5'-cactgaaccatggaccggaacttt-3') is located in the coding sequence of the Flo1p flocculation functional region, and the sequence of primer pLHJ042R (5'-ggggggagggcgtgaatgta-3') is located in the terminator CYCC1TT sequence. Using pLHJ044, pLHJ052, pLHJ053 and pLHJ042-B68 as templates and pLHJ042F and pLHJ042R as primer pairs, PCR amplification obtained fragments of lipase genes from different sources with homologous sequences of Saccharomyces cerevisiae display expression vectors.

[0038] 3.2 Construction of lipase gen...

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Abstract

The invention provides a high-efficiency directed evolution method of a lipase gene. The method comprises the following steps: (1) constructing a display expression vector of saccharomyces cerevisiae;(2) constructing display expression recombinant plasmids of the saccharomyces cerevisiae of a lipase; (3) obtaining vector-carried lipase gene fragments in a homologous sequence and constructing an expression mutation library; and (4) analyzing the display expression mutation library and mutants. The method synchronizes the construction of the mutation library and the expression of the mutants ina general directed evolution technology and overcomes the defects that directed evolution of lipase molecules has complicated operation steps, difficult mutant selection, and the like in the prior art.

Description

technical field [0001] The invention utilizes Saccharomyces cerevisiae homologous recombination combined with microbial cell surface display technology to establish an efficient lipase gene directional evolution method, relates to the technology of enzyme gene directional evolution and molecular transformation, and belongs to the field of biotechnology. Background technique [0002] Directed evolution of enzyme genes is one of the most effective methods to improve the enzymatic properties of enzymes. DNA shuffling technology in the 1990s ushered in a new era of directed molecular evolution. DNA shuffling refers to the in vitro recombination of DNA molecules, which is the sexual recombination of genes at the molecular level. By changing the original nucleotide sequence of a single gene (or gene family), new genes are created and the expression products are endowed with new functions. [0003] Although the conventional DNA shuffling technology and the subsequent improved tec...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12N15/81C12R1/865
Inventor 江正兵宋慧婷
Owner HUBEI UNIV
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