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Lactone hydrolase mutant and application thereof

A technology of mutants and hydrolytic enzymes, applied in the fields of genetic engineering technology and biocatalysis, can solve problems such as low activity, poor stability, and difficulties in immobilizing cells

Pending Publication Date: 2021-01-05
江西兄弟医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aspergillus oryzae engineered bacteria express lacton hydrolase intracellularly, and Cephalosporium secrete and express lacton hydrolase outside the cell, but the enzymatic activity of the two genetically engineered bacteria producing lacton hydrolase is not significantly improved, and even outperforms the wild Fusarium oxysporum low activity
In addition, the microorganisms involved in producing lactone hydrolase mentioned above are all filamentous fungi, and the thalline presents hyphae in various forms. Compared with single-celled microorganisms, filamentous fungi are prepared into immobilized cells ( Catalyst) difficult, poor stability

Method used

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  • Lactone hydrolase mutant and application thereof
  • Lactone hydrolase mutant and application thereof
  • Lactone hydrolase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: Construction of wild-type lactone hydrolase expressing strain

[0081] For the derived lactone hydrolase LTO, according to the reported coding gene acid sequence SEQ ID NO: 1, Wuhan Jinkairui Bioengineering Co., Ltd. was entrusted to carry out the synthesis of the whole gene sequence, subcloned into the pGM-T vector, and transformed into E. coli E. .coli DH5α host to obtain the recombinant plasmid pGM-T-LT0 transformant containing the lactone hydrolase gene.

[0082] Design cloning primers:

[0083] Forward primer F1: 5'- CATATG CCTTCCACCATTTCTG-3’

[0084] Forward primer R1: 5'- CTCGAG CTAGTCATACAACTTG-3’

[0085] The pGM-T-LT0 plasmid was extracted as the gene template, and the gene encoding LTO was amplified by PCR with F1 and R1 forward and reverse primers, and the amplified fragment was about 1.2 kb. 50 μl PCR reaction system: 1 μl each of primers F1 and R1, 3 μl plasmid template, 25 μl 2×Premix PrimeSTAR HS, 10 μl sterilized distilled water. PCR...

Embodiment 2

[0093] Example 2: Obtaining mutants of lactone hydrolase with excellent performance by directed evolution

[0094] Design mutation primers:

[0095] Forward primer F2: 5'-TAATACGACTCACTATAGGG-3'

[0096] Reverse primer R2: 5'-TCGCTTGGAACATTATTAAC-3'

[0097] Using the pET-28-LTO plasmid containing the gene sequence of SEQ ID NO: 1 as a template, and using F2 and R2 as forward and reverse primers, a random mutation library was constructed by error-prone PCR technology. Error-prone PCR system 50 μl: 25 μl 2×TaqPCR MaterMix, 1 μl each of primers F2 and R2, 1 μl pET-28-LT0 plasmid, 0.5 μl dGTP (10 mM), MnCl 2 (10mM) 1 μl, sterile water 20.5 μl. PCR conditions: 95°C for 5 min; 30 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 1.5 min; last at 72°C for 10 min.

[0098] The PCR product was detected by agarose gel electrophoresis, and the amplified product was about 1200bp. The error-prone PCR gene products were recovered by a gel recovery kit, and used as large primers for who...

Embodiment 3

[0108] Example 3: Construction of yeast engineering bacteria and secretion and expression of lactonohydrolase mutants

[0109] Using pPICZαA as the shuttle vector and Pichia pastoris as the expression host GS115, a yeast engineering bacterium producing lactone hydrolase mutant was constructed to realize the extracellular secretory expression of the lactone hydrolase mutant.

[0110] Design cloning primers:

[0111] Forward primer F3: 5'- GAATT CGCCTTCCACCATTTCTG-3’

[0112] Forward primer R3: 5'- GCGGCCGC CTAGTCATACAACTTG-3’

[0113] The recombinant plasmid contained in the lactone hydrolase mutant strain TB4 obtained in Example 2 is pET-28-LT-23, and the lactone hydrolase mutant amino acid sequence of its expression is SEQ ID NO: 48, and the corresponding gene sequence is SEQ ID NO: 47.

[0114] The plasmid pET-28-LT-23 of the mutant strain strain TB4 was extracted by a plasmid extraction kit, and the plasmid pET-28-LT-23 of the mutant strain strain TB4 was taken as th...

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PUM

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Abstract

The invention discloses a lactone hydrolase mutant in the fields of genetic engineering technology and biocatalysis, and application of the lactone hydrolase mutant as a biocatalyst to resolution of D, L-pantolactone to prepare optically pure D-pantoic acid. The lactone hydrolase mutant is obtained by carrying out single mutation or combined mutation on 1-14 amino acid sites in an amino acid sequence SEQ NO: 2 by taking fusarium oxysporum lactone hydrolase as a parent enzyme. The hydrolysis activity of the lactone hydrolase mutant on D, L-pantolactone is improved by 2.3-128 times compared withthat of parent enzyme. The high-activity lactone hydrolase provided by the invention can be used for large-scale production of preparing optically pure D-pantoic acid by biologically splitting D, L-pantolactone.

Description

technical field [0001] The invention belongs to the fields of genetic engineering technology and biological catalysis, and in particular relates to a lactone hydrolase mutant and its application. Background technique [0002] D-pantoate is a key chiral intermediate in the manufacture of D-pantothenate calcium, D-panthenol and D-pantethamine. Calcium D-pantothenate is widely used in feed, medicine and food industries, especially as an important feed additive, it has become the vitamin whose demand is second only to niacin. D-panthenol is used as a skin medication to prevent skin infections and is effective in the treatment of skin wounds, skin abrasions and burns. D-pantidine can be used as a hypolipidemic drug in the pharmaceutical industry, which can reduce total cholesterol and triglyceride in serum and increase high-density lipoprotein cholesterol in serum. [0003] The preparation methods of D-pantoate include chemical resolution, physical resolution and biological enz...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12N15/70C12N15/81C12N1/21C12N1/19C12P41/00C12P7/42C12R1/19C12R1/645
CPCC12N9/18C12N15/70C12N15/81C12P41/005C12P7/42
Inventor 万南微陈永正张秋华周中平
Owner 江西兄弟医药有限公司
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