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Lactobacillus brevis, aldolase, genes of aldolase and method of preparing statin intermediate

A technology of Lactobacillus brevis and aldolase, which is applied in the field of Lactobacillus brevis, aldolase and its gene, and the preparation of statin intermediates, which can solve the problems of poor substrate tolerance and low enzyme activity

Active Publication Date: 2015-04-29
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide an aldolase with high catalytic activity and strong substrate tolerance for the defects of low enzymatic activity and poor substrate tolerance in the preparation of CTeHP by the current enzymatic synthesis method, expressing the The gene of aldolase and its recombinant expression vector and recombinant expression transformant, the preparation method of said aldolase and the preparation of (3R,5S)-6-chloro-2,4,6-trideoxy Application of -D-erythrohexopyranoside

Method used

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  • Lactobacillus brevis, aldolase, genes of aldolase and method of preparing statin intermediate
  • Lactobacillus brevis, aldolase, genes of aldolase and method of preparing statin intermediate
  • Lactobacillus brevis, aldolase, genes of aldolase and method of preparing statin intermediate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Screening of Lactobacillus brevis CGMCC NO.10135

[0044] The Lactobacillus brevis CGMCC NO.10135 was derived from a soil sample collected by the Shanghai Botanical Garden and named ECU8302. Its separation includes the following steps: more than 100 soil samples from all over the country are screened for microorganisms, and an appropriate amount of soil samples are added to normal saline (0.9% NaCl) to obtain a suspension containing bacteria, and the suspension containing bacteria is diluted and coated separately LB medium plates, isolated single colonies. Inoculate the isolated single colonies into LB medium respectively, culture with shaking at 30°C for 24 hours, centrifuge the culture medium, collect resting cells, add to potassium phosphate buffer (100mM, pH 7.0) containing 100mM acetaldehyde and 50mM chloroacetaldehyde In the method, shake the reaction, detect the concentration of the aldol reaction product, and evaluate the activity of these bacterial strains in ...

Embodiment 2

[0053] Cloning of Aldolase LbDERA Gene

[0054] The strain CGMCC No.10135 was cultured in LB medium, and high-purity, large-fragment total genomic DNA was extracted by the cetyltrimethylammonium bromide (CTAB) method: add CGMCC No.10135 cells to liquid nitrogen Freeze, grind into powder, add 2×CTAB extraction buffer (100mmol / L Tris-HCl, pH 8.0, 20mmol / L EDTA, 1.4mol / L NaCl, 20g / L CTAB, 40mmol / L mercaptoethanol), keep warm at 65°C 10 minutes, shaking intermittently. Then add an equal volume of chloroform / isoamyl alcohol, gently invert the centrifuge tube to mix, centrifuge at 12,000 rpm for 10 min at room temperature, transfer the supernatant to another centrifuge tube, and then add an equal volume of chloroform / isoamyl to the supernatant alcohol, invert the centrifuge tube to mix, and centrifuge at 12,000 rpm for 10 minutes at room temperature. Transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of isopropanol to the upper aqueous phase and mix wel...

Embodiment 3

[0061] Plasmid of recombinant LbDERA and preparation of recombinant bacteria and recombinant enzyme

[0062] With primer pair 1: forward primer 5'-ACGGAATTCATGACATTAACCACA-3' (as shown in SEQ ID NO.3), reverse primer 5'-CCCAAGCTTTTAGTAACCAGCTTT-3' (as shown in SEQ ID NO.4), using a polymerase The chain reaction technology amplifies the nucleotide sequence of LbDERA obtained in Example 1, and the obtained DNA fragment containing the LbDERA gene sequence is double-enzymatically digested with EcoRI and HindIII respectively, and then similarly passed through EcoRI and HindIII double enzymes The cut plasmid pET28a was connected to obtain the plasmid pET28a-LbDERA, and the schematic diagram of its construction can be found in figure 1 .

[0063] The obtained plasmid pET28a-LbDERA was transformed into Escherichia coli E.coli BL21, and the constructed recombinant Escherichia coli was inoculated into LB medium containing 50 μg / ml kanamycin sulfate, cultured with shaking at 37° C. ove...

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Abstract

The invention discloses lactobacillus brevis, aldolase, a gene of the aldolase and a method of preparing a statin intermediate. The lactobacillus brevis is preserved in China General Microbiological Culture Collection Center with a preservation number of CGMCC NO.10135 and is capable of producing the aldolase. Compared with prior art, the aldolase is high in activity, heat stability and substrate tolerance; the method for catalytically preparing (3R, 5S)-6-chloro-2, 4, 6-trideoxy-D-ribofuranose-pyranoside, serving as the statin intermediate, by the aldolase and a mutant of the aldolase has the advantages of mild reaction condition, high substrate concentration, little amount of a catalyst and the like, so that the aldolase has good application prospect in the industrial production.

Description

technical field [0001] The invention belongs to the field of biology, and in particular relates to a lactobacillus brevis, aldolase, a gene encoding the aldolase, a recombinant expression vector containing the gene, a recombinant expression transformant, and a method for preparing aldolase by using the recombinant expression transformant , and a method for preparing statin intermediate (3R,5S)-6-chloro-2,4,6-trideoxy-D-erythrohexopyranoside by using aldolase. Background technique [0002] (3R,5S)-6-Chloro-2,4,6-trideoxy-D-erythrohexopyranoside[(3R,5S)-6-chloro-2,4,6-trideoxy-D-erythro- hexapyranoside, CTeHP] is an important chiral intermediate for the synthesis of statins, and its molecular formula is C 6 h 11 o 3 Cl, CAS No. 714963-27-6. Statins generally refer to hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, through competitive inhibition of endogenous HMG-CoA reductase, blocking intracellular valonate metabolism pathway, making intracellular Choles...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/88C12N15/60C12P17/06C12R1/24
CPCC12N9/88C12P17/06C12N1/205C12R2001/24
Inventor 许建和焦学成潘江陈琦孔旭东
Owner EAST CHINA UNIV OF SCI & TECH
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