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Serratia lipase mutant, recombinant expression transformant, enzyme preparation and application

A technology of Serratia and lipase, which is applied in the field of bioengineering, can solve the problems of large amount of enzyme, emulsification of reaction solution, difficult product separation, etc., and achieves the effects of less enzyme amount, low cost and high catalytic activity.

Inactive Publication Date: 2016-10-26
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the application of Serratia lipase, due to the low activity of the enzyme, the amount of the enzyme is large, resulting in serious emulsification of the reaction solution and difficulty in product separation

Method used

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  • Serratia lipase mutant, recombinant expression transformant, enzyme preparation and application
  • Serratia lipase mutant, recombinant expression transformant, enzyme preparation and application
  • Serratia lipase mutant, recombinant expression transformant, enzyme preparation and application

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Effect test

Embodiment 1

[0035] Cloning of embodiment 1 Serratia marcescens CGMCC 1219 lipase (SmL) gene

[0036] Wild Serratia sp. (strain preservation number: CGMCC No. 1219) was cultured in LB medium. The genome of Serratia was extracted by the phenol-chloroform method, and the specific steps were as follows:

[0037] The cultured cells were centrifuged, then resuspended with physiological saline (0.85%, w / v), 1.5 mL of the resuspension liquid was drawn, added to a 1.5 mL EP tube, and centrifuged again. Add 500 μL of normal saline to the EP tube, add an appropriate amount of ceramic beads (about 1 mm in diameter, and the total volume of the ceramic beads is 1 / 3 of the solution volume); place the EP tube on a shaker for 5 minutes and place it on ice for 15 minutes , and then oscillated on a shaker for 10 minutes; centrifuged to collect the supernatant; added 500 μL of phenol-chloroform solution to the supernatant, oscillated evenly, and centrifuged at 14,000 rpm for 5 minutes. Then draw 300 μL of ...

Embodiment 2

[0049] The single-point saturation mutation of embodiment 2 lipase SmL

[0050] Using the published crystal structure of Serratia lipase (PDB: 2QUA) as a template, the online modeling tool Swiss-Model was used for homology modeling. Three-dimensional model of Serratia lipase SmL obtained by homology modeling with enzymatic reaction substrate (+)-p-methoxyphenyl glycidic acid methyl ester [(+)-MPGM] as a small ligand molecule Based on the structure, molecular docking was performed with AutoDock software. Centered on the substrate, Within the range of 18 amino acid sites, select 18 amino acid sites, respectively: Y29, T143, D161, G165, F166, H206, L208, A237, S238, P239, V258, A261, L262, L267, S271, W311, L315, Y319, Saturation mutations were performed separately.

[0051] Using the plasmid pET28a as the vector and Escherichia coli BL21 as the host, the NNK saturation mutation library of the above sites was constructed. Taking the site L315 as an example, the primer design...

Embodiment 3

[0062] Example 3 SmL Single Point Mutant Recombinant Expression Transformant Cell and Preparation of Crude Enzyme

[0063] Inoculate the recombinant Escherichia coli containing a single-point mutation recombinant expression plasmid into LB medium containing 50 μg / mL kanamycin sulfate, and culture it with shaking at 37°C until OD 600 reach 1.2, according to the inoculation amount of 1% (v / v), insert into the 500mL Erlenmeyer flask that 100mL LB culture medium is housed, place 37 ℃, 180rpm shaker shaking culture, when the OD of culture solution 600 When it reaches 0.8, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.5mmol / L as an inducer, and induce at 16°C for 18h. The culture solution was centrifuged at 4000 rpm for 20 min, and washed twice with physiological saline to obtain resting cells, which were transformant cells for recombinant expression of the Serratia lipase SmL single point mutant. The obtained cells were suspended in 10 mL of KPB buffer (...

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Abstract

The invention relates to a mutant obtained through molecular modification of serratia marcescens lipase (SmL), a gene of the mutant and an amino acid sequence of the mutant, a recombinant expression plasmid and a recombinant expression transformant containing the gene of the mutant, a preparation method of a recombinant enzyme preparation and application of the enzyme preparation to diltiazem precursor synthesis. Compared with wild serratia marcescens lipase, the activity of the mutant is remarkably improved, excellent stereoselectivity is kept, and the mutant has good application prospects in industrial production of key chiral precursor (-)-(2R,3S)-2,3-epoxy-3-(4methoxyphenyl)propronate of the cardiovascular disease medicine, namely diltiazem.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a mutant of Serratia lipase, a recombinant expression plasmid expressing the mutant, a recombinant expression transformant, a preparation method of the lipase mutant enzyme preparation, and the mutant Application of somatic enzyme preparation in enzymatic resolution and preparation of (-)-methyl p-methoxyphenyl glycidate. Background technique [0002] Diltiazem is a highly selective calcium channel blocker, which can inhibit the movement of calcium ions through vascular smooth muscle and myocardial cell membrane channels, and has the effect of stimulating cardiomyocytes-contraction uncoupling and potently dilating blood vessels. The medicine has good curative effect and little toxic and side effects, and is widely used in the long-term treatment of angina pectoris and hypertension. Synthesis of diltiazem by chemical-enzymatic method can simplify the synthetic r...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12N15/55C12N15/70C12N1/21C12P41/00C12P17/02C12R1/425
CPCC12N9/20C12N15/70C12N2800/101C12P7/6445C12P17/02C12P41/00C12Y301/01003
Inventor 许建和陈科材李春秀陈琦潘江
Owner EAST CHINA UNIV OF SCI & TECH
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