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Pantolactone hydrolase mutant strain and application thereof

A pantolactone and hydrolase technology, applied in the field of pantolactone hydrolase mutant strains

Active Publication Date: 2021-06-29
CHIFENG PHARMA CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the D-pantolactone hydrolase in the prior art is not enough to meet the needs of industrial production, and this area needs to develop new D-pantolactone hydrolase with higher enzyme activity

Method used

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  • Pantolactone hydrolase mutant strain and application thereof
  • Pantolactone hydrolase mutant strain and application thereof
  • Pantolactone hydrolase mutant strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Construction of the genetically engineered strain AR / pHT01-P43-lacton producing D-pantolactone hydrolase 1. Synthetic cloning vector pUC57-lacton:

[0119] Referring to the D-pantolactone hydrolase gene of Fusarium oxysporum (Fusarium oxysporum, GenBank: AB010465) and Fusarium moniliforme (Fusarium moniliforme, GenBank: AY728018), the nucleotide sequences are respectively as SEQ ID NO: 4 and 5. Submit the nucleotide sequence to Suzhou Jinweizhi Biotechnology Co., Ltd. for codon optimization and synthesis of the cloning vector pUC57-lacton. The optimized sequence is shown in sequence SEQ ID NO:6, and the corresponding amino acid sequence is shown in sequence SEQ ID NO:1.

[0120] 2. Construction of expression vector pHT01-P43-lacton

[0121] Amplify the P43 promoter and lacton sequence, obtain P43-lacton by overlapping, digest the vector pHT01 and P43-lacton with BamHI and XbaI respectively, connect them, transform them into E.coli DH10B, and place them on a plate with...

Embodiment 2

[0127] Construction of the genetically engineered strain AR / pHT01-P43-lacton Mu5 producing D-pantolactone hydrolase 1. Construction of the cloning vector pUC57-lacton Mu5:

[0128] Using the plasmid pUC57-lacton as a template, primers lac-mutF1 / lac-mutR1 (see Table 9 for the sequence), PCR amplification was performed for site-directed mutagenesis, and the recovered amplified product was digested with DpnI enzyme at 37°C for 1 hour, and transferred to Ecoli XL1blue competent Cells were cultured overnight at 37° C. on a plate containing 100 mg / L ampicillin antibiotic, and positive clones were obtained by screening. The specific nucleotide sequence is shown in SEQ ID NO: 7, and the corresponding amino acid sequence is shown in SEQ ID NO: 2.

[0129] 2. Construction of expression vector pHT01-P43-lacton Mu5

[0130] Amplify the P43 promoter and lacton sequence, obtain P43-lacton Mu5 by overlapping, digest the vector pHT01 and P43-lacton Mu5 with BamHI and XbaI respectively, connec...

Embodiment 3

[0136] Construction of Genetically Engineered Bacteria AR / pHT01-P43-lacton Mu9 Producing D-Panttolactone Hydrolase

[0137] 1. Construct the cloning vector pUC57-lacton Mu9:

[0138] Using the plasmid pUC57-lacton Mu5 as a template, primers lac-mutF2 / lac-mutR2 (see Table 9 for the sequence), PCR amplification was performed for site-directed mutagenesis, and the recovered amplified product was digested with DpnI enzyme at 37°C for 1 hour, and transferred to Ecoli XL1 blue In the competent cells, cultivate overnight at 37°C on a plate with ampicillin antibiotic 100 mg / L, and screen and identify positive clones. The specific nucleotide sequence is shown in the sequence SEQ ID NO: 8, and the corresponding amino acid sequence is shown in SEQ ID NO: 3 shown.

[0139] 2. Construction of expression vector pHT01-P43-lacton Mu9

[0140] Amplify the P43 promoter and lacton Mu9 sequence, obtain P43-lacton Mu9 by overlapping, digest the vector pHT01 and P43-lacton Mu9 with BamHI and XbaI...

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Abstract

The invention provides a pantolactone hydrolase mutant strain and application thereof. Specifically, the invention provides a mutant pantolactone hydrolase with significantly improved enzyme activity and a host cell for expressing the mutant pantolactone hydrolase. The invention further provides an enzyme preparation containing the mutant pantolactone hydrolase, D-pantolactone in a substrate DL-pantolactone can be completely hydrolyzed into D-pantoic acid, and the mutant pantolactone hydrolase is high in conversion rate, stable in property, capable of being repeatedly used and suitable for industrial production.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a mutant strain of pantolactone hydrolase and application thereof. Background technique [0002] Pantothenic acid is a B vitamin and the main raw material of coenzyme A. It participates in important metabolisms in humans and animals, including fatty acid degradation and synthesis, citric acid cycle, choline acetylation, antibody synthesis, and promotes the absorption and utilization of nutrients. Pantothenic acid is unstable, and its commercial form mainly exists in the form of calcium D-pantothenate (dextrorotary body). Calcium D-pantothenate is widely used in medicine, food, and feed industries, especially as a food and feed additive. The demand is huge. [0003] D-pantolactone is an important chiral intermediate for the manufacture of D-pantothenic acid, and the methods for obtaining D-pantolactone mainly include chemical resolution and microbial enzymatic methods. Among th...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12N11/14C12P7/62
CPCC12N9/18C12N11/14C12P7/62Y02A50/30
Inventor 邵菲左静张莹夏云重朱再玲施明安张国银
Owner CHIFENG PHARMA CO LTD
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