Recombinant ECB (echinocandin B) deacylase mutant and application

A technology of deacylase and echinocandin, applied in the direction of recombinant DNA technology, bacteria, hydrolase, etc., can solve the problem of low enzyme activity and achieve the effect of improving catalytic performance

Active Publication Date: 2019-06-18
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the constructed ECB deacylase secreted and expressed by Streptomyces lividans has a defect of low enzyme activi

Method used

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  • Recombinant ECB (echinocandin B) deacylase mutant and application
  • Recombinant ECB (echinocandin B) deacylase mutant and application
  • Recombinant ECB (echinocandin B) deacylase mutant and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Homology Modeling and Molecular Docking

[0040] In the SWISS-MODEL database (https: / / www.swissmodel.expasy.org / ), the amino acid sequence of the deacylase AuEBDA (shown in SEQ ID No.2, the nucleotide sequence of the coding gene auadea is SEQ ID No. 1) for comparison. The model protein with the highest degree of homology is the antibiotic acyltransferase MacQ (PDB ID: 5C9i_1) derived from Acidovoraxsp.MR-S7, whose amino acid sequence has a homology of 40.77% with the starting deacylase AuEBDA.

[0041] Modeler 9.14 software was used for program coding, and the MacQ protein sequence was used as a template to construct a three-dimensional space model of the AuEBDA protein sequence, and the best model was obtained. Subsequently, the ChemDraw software was used to draw and convert the three-dimensional model of the substrate ECB, and the AutoDock 4.2 software was used for molecular docking. Using Pymol1.6.0.0 to analyze the three-dimensional structure relationsh...

Embodiment 2

[0044] Embodiment 2: Construction of Escherichia coli cloning vector by PCR method

[0045] 1. pSET152-auebda plasmid

[0046] Actinoplanes utahensis ZJB-08196 (Wang Y J, Liu L L, Feng Z H, et al. Optimization of media composition and culture conditions foracarbose production by Actinoplanes utahensis ZJB-08196[J].World Journal of Microbiology&Biotechnology, 2011, 27 (12): 2759-2766.) Carry out streak activation on the synthetic No. V solid medium without resistance, pick single bacterium colony in the seed medium without resistance, 28 degrees Celsius, 200 revs per minute and cultivate 3 On the next day, the cells of Actinomyces uthaensis ZJB-08196 were obtained, and the genome was extracted. Using the extracted genome as an amplification template, EcoR I and Xba I restriction sites were added to both ends of the gene sequence, and auebda-F and auebda-R were used as primers for PCR amplification (the amplification system and conditions are shown in Table 1 and Table 2), the...

Embodiment 3

[0072] Embodiment 3: Streptomyces lividans expression vector construction

[0073] 1. The plasmids in Example 2 are transformed into E.coli ET12567 / pUZ8002 respectively, and single colonies of positive clones are picked in 10 milliliters of LB medium (containing 50 micrograms per milliliter of kanamycin, 25 micrograms per milliliter of chloramphenicol Incubate overnight at 37°C and 200 rpm in a test tube with 25 micrograms per milliliter of prunamycin. Overnight bacterial solution was transferred to 50 milliliters of LB liquid medium containing corresponding antibiotics with an inoculum of 1% in volume concentration, cultivated at 37 degrees Celsius and 200 rpm for 6 to 8 hours, and the OD 600 Reach 0.4 to 0.6. Wash twice with an equal volume of sterile LB medium, then suspend with 0.5 ml of fresh sterile LB medium, which is the E.coli suspension, and measure the concentration of E.coli with a hemocytometer.

[0074] 2. Prepare the spore suspension of Streptomyces lividans. ...

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Abstract

The invention discloses a recombinant ECB (echinocandin B) deacylase mutant and an application in synthesis of ECB parent nucleus from the ECB by biocatalysis. The deacylase mutant is obtained throughsingle mutation or double mutation of the 287th and 527th sites of an amino acid sequence shown in SEQ ID No.2. AuEBDA primary structure and enzyme conformation are changed through mutation of an auebda gene sequence, and the catalytic performance of AuEBDA is improved. Influences of G287Q and R527V substitution on activity of ECB deacylase are systemically analyzed. The enzyme activity of constructed double mutant AuEBDA-G287Q/R527V is improved by 2.5 times than that of parent wild AuEBDA, and the catalytic efficiency kcat/Km is increased by 2.9 times.

Description

technical field [0001] The invention relates to a deacylase AuEBDA of actinomycetes uthaensis capable of efficiently catalyzing the hydrolysis of the amide bond of echinocandin B and its coding gene auebda. Semi-rational design and molecular modification of AuEBDA were carried out through bioinformatics, homology modeling and molecular docking methods, and the robust AuEBDA mutant enzyme using molecular biology techniques was realized for the first time. Wild-type AuEBDA increased by 2.5 times, catalytic efficiency k cat / K m Increased by 2.9 times. technical background [0002] In recent years, fungal infections have seriously threatened human life and health, especially deep fungal infections. According to statistics, 70% of the death cases of fungal infection in Europe are related to Aspergillus and Candida infection. The mortality rate of patients infected by invasive candida is as high as 30%, and the fatality rate is close to 100% without timely and effective treat...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N1/21C12N15/76C12P21/04C12R1/465
Inventor 王亚军程英男邹树平郑裕国
Owner ZHEJIANG UNIV OF TECH
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