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Lovastatin acylase containing one or several point mutations

An acyltransferase, lovastatin acyl technology, applied in the directions of transferase, enzyme, genetic engineering, etc., can solve the problems of climatic conditions, temperature and humidity sensitivity, easy phage infection, complex processing, etc.

Active Publication Date: 2020-01-17
CSPC ZHONGQI PHARM TECH (SHIJIAZHUANG) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention also provides a Pichia pastoris expression system for lovastatin acyltransferase, in order to solve the problems that the post-treatment of large intestine fermentation is relatively complicated, especially sensitive to climatic conditions, temperature and humidity, and easy to be infected by phages. a new approach

Method used

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  • Lovastatin acylase containing one or several point mutations
  • Lovastatin acylase containing one or several point mutations
  • Lovastatin acylase containing one or several point mutations

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Construction, prokaryotic expression and functional identification of lovastatin acyltransferase mutant coding gene.

[0066] 1. Primer design The wild-type Aspergillus terreus ( A. terreus ) source of lovastatin acyltransferase (LovD) is the gene sequence, the whole gene synthesis of the sequence, and design primers.

[0067] The primer sequence used for cloning in the prokaryotic expression vector pET-21d is: Primer P1: 5'-CATG CCATGGG TTCTATCATTGATGCGGC -3' (the underlined base is the Nco I recognition site); primer 5'-CCC AAGCTT TTAGCCCTGTTGATACTGT -3' (the underlined base is the Hind III recognition site); the LovD gene sequence amplified by the pair of primers is shown in SEQ ID NO.2, and the lovastatin acyltransferase protein encoded by it has SEQ ID NO .1 the amino acid sequence shown.

[0068] Primers for site-directed mutagenesis:

[0069] N45R-P1: 5'-GTAACCTGAGATACACTCGCTGTTTC-3'

[0070] N45R-P2: 5'-GAAACAGCGAGTGTATCTCAGGTTAC-3'

[0071] A...

Embodiment 2

[0122] Example 2: Construction and expression of the recombinant plasmid of Pichia pastoris encoding the lovastatin acylase (LovD) gene.

[0123] Yeast expression vectors for mutants 12, 14, 16, and 18 were respectively constructed and transformed into Pichia pastoris cells. Mutant 16 was used as an illustration below, and the construction methods of other mutants were the same. Mutants 12, 14, 16, and 18 were named LovDm12, LovDm14, LovDm16, and LovDm18, respectively.

[0124] 1. Eukaryotic expression of LovD mutants

[0125] Using P3, P4, P5, and P6 as primers and mutant 16 (synthesized by Nanjing GenScript Biotechnology Co., Ltd.) as a template, amplify LovD mutant 16 (LovDm16 ) gene, and the polymerase used in PCR, corresponding amplification buffer and dNTP solution were all purchased from TaKaRa Company.

[0126] The PCR reaction system is:

[0127] 10×Buffer (Mg 2+ ) 5 μL

[0128] dNTP (2.5mM each) 4μL

[0129] Primer P1 (10 μM) 2 μL

[0130] Primer P2 (10μM) 2μL...

Embodiment 3

[0152] Example 3 Fermenter expression and efficacy experiment of recombinant Pichia pastoris.

[0153] The extracellular expression recombinant pPic9k-LovDm16 strain 3# and pPic9k-LovDm14 strain 1#, which were screened for high enzyme activity, were used as starting strains, inoculated into 150mL YPD liquid medium for overnight cultivation at 28°C to make seed liquid, and then 5% of the inoculum was inserted into 3L of BMGY (PH8.0) liquid medium (5L fermenter), and ammonia and methanol were used as supplements. After 125 hours of cultivation, the enzyme activity of pPic9k-LovDm16 strain 3# reached 266000U / L , pPic9k-LovDm14 strain 1# enzyme activity reached 254000U / L.

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Abstract

The invention relates to a lovastatin acyltransferase mutant. The mutant contains an acyltransferase amino acid sequence represented by SEQIDNO.1 of wild A. terreus and at least 2-6 amino acid mutations selected from N45R, A110P, S145N, Q230M, L293M and L379M. The invention also relates to a nucleic acid nucleic acid for coding the mutant, a relevant expression vector, a host cell, and a use of the mutant in the synthesis of lovastatin.

Description

technical field [0001] The invention relates to the field of medicine production, in particular to the production of simvastatin and an enzyme for production thereof, more specifically to lovastatin acyltransferase and its application in the production of simvastatin. Background technique [0002] Simvastatin is currently one of the best-selling lipid-lowering drugs at home and abroad. It can be used to control blood cholesterol levels and prevent cardiovascular diseases. Its pharmacological effect is to inhibit the rate-limiting enzyme of cholesterol synthesis as a competitive inhibitor: 3-hydroxy- 3-methylglutaryl-CoA reductase activity, thereby reducing cholesterol biosynthesis. As a semi-synthetic derivative of lovastatin, simvastatin can more effectively reduce total cholesterol and low-density lipoprotein cholesterol in serum compared with lovastatin at the same dose. [0003] It is precisely because of the important medical significance of simvastatin that many scien...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/81C12N1/19C12P17/06C12R1/84
CPCC12N9/1029C12P17/06
Inventor 李鹤余允东祝俊吴会广黄学川辛有英任丽梅成志远王翠杜会云
Owner CSPC ZHONGQI PHARM TECH (SHIJIAZHUANG) CO LTD
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