Tertiary alcohol ester hydrolase, encoding gene, carrier, engineering bacteria and application thereof

A technology of hydrolase and tertiary alcohol ester, applied in the direction of genetic engineering, hydrolase, application, etc., to achieve the effect of high resistance to organic solvents and stability

Active Publication Date: 2013-11-27
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Tertiary alcohol substrates are a class of very valuable chiral platform-based compounds, which can be widely used in the synthesis of pharmaceutical intermediates, but chiral tertiary alcohol substrates have strong steric hindrance, so their chemical synthesis process is constructed It is very challenging. At present, there is still a very urgent need in the development of a large number of synthetic high-purity chiral tertiary alcohols, especially the green and environmentally friendly technology.

Method used

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  • Tertiary alcohol ester hydrolase, encoding gene, carrier, engineering bacteria and application thereof
  • Tertiary alcohol ester hydrolase, encoding gene, carrier, engineering bacteria and application thereof
  • Tertiary alcohol ester hydrolase, encoding gene, carrier, engineering bacteria and application thereof

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Experimental program
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Effect test

Embodiment 1

[0067] Embodiment 1: Acquisition of full-length esterase Est076 gene

[0068] Soil samples at 30 cm below the surface were collected in the Gaofeng Forest in West Hubei, Hangzhou, and the total metagenomic DNA was extracted using the Mo Bio Power Soil DNA isolation kit (M) (Carlsbad, CA) kit, and the Copy Control Fosmid Library production kit (Epicentre, USA) library construction kit to construct environmental metagenomic library. The metagenomic DNA of the library was screened by esterase using the plate substrate transparent circle method, and the surrounding hydrolysis was obtained by screening with Luria-Bertani (LB) solid medium containing 1% tributyrin substrate (polyvinyl alcohol emulsion). Positive clones with transparent circles. High-copy induction of the Fosmid recombinant plasmid in the positive clones (see the Copy Control Fosmid Library production kit manual for specific methods), and then extract the Fosmid recombinant plasmid. Use endonuclease Sau3A I to inco...

Embodiment 2

[0069] Embodiment 2: Construction of the expression vector system containing esterase Est076 target gene

[0070] According to the full-length gene sequence of esterase Est076, the upstream primer Est076F (5′-CCG) was designed to amplify the complete coding reading frame GAATTC ATGAGTCAGCTTCCAGCAG-3′) and the downstream primer Est076R (5′-GCG GCCGCC TTCAAATACTTGTCC-3'), respectively introduce restriction endonuclease sites on the upstream and downstream primers (upstream introduced enzyme cutting site is EcoR I, downstream is Not I). Taking the plasmid containing the esterase gene sequence described in the present invention as a template, after PCR amplification, use the esterase Est076 nucleotide fragment obtained by restriction endonuclease EcoR I and Not I double enzyme digestion amplification, ligation To the expression vector pET21a with corresponding cuts (digested by EcoR I and Not I), transformed into E.coil DH5α, the recombinant expression vector was identified und...

Embodiment 3

[0071] Embodiment 3: Expression and purification preparation of esterase Est076

[0072] The esterase expression system E.coilBL21(DE3) / pET21a / Est076 obtained in Example 2 was inoculated into Luria-Bertani (LB) liquid medium containing 100 μg / ml ampicillin (Sigma, USA), and cultured at 37°C to logarithmic growth phase (OD 600After adding IPTG (genview, USA) with a final concentration of 0.15mM, induce culture at 15°C for about 20 hours, collect the bacteria by centrifugation, resuspend in 50mM Tris-HCl (pH8.0) buffer, and ultrasonically break The bacterial suspension was centrifuged to remove the precipitate, and the supernatant was obtained as a crude enzyme solution containing esterase protein. The crude enzyme solution was filtered through a 0.45 μm cellulose acetate filter, followed by nickel ion affinity chromatography to obtain the pure enzyme protein (SEQ ID No.1) of esterase Est076 described in the present invention.

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Abstract

The invention provides tertiary alcohol ester hydrolase, a gene for encoding the same, a carrier containing the encoding gene, engineering bacteria and application thereof. The amino acid sequence of the tertiary alcohol ester hydrolase is shown as SEQ ID No.2, and the amino acid sequence of the encoding gene is shown as SEQ ID No.1. The tertiary alcohol ester hydrolase is high in short-carbon chain ester hydrolytic activity; and the enzymatic activity for catalyzing hydrolysis of p-nitrophenol butyrate in a Tri-HCl buffer solution with pH of 8.5 at the temperature of 50 DEG C can reach 3,010 U / mg. The esterase is high in strong organic solvent resistance stability and activity improving characteristic; and after the esterase is incubated in strong organic solvents such as normal hexane, toluene and dimethylbenzene, of which the partition coefficient Log P is greater than 3, the enzymatic activity still can be improved by 51 percent, 44 percent and 49 percent respectively. Moreover, the esterase has tertiary alcohol ester hydrolytic activity, so that the esterase can be widely used for hydrolyzing different tertiary alcohol ester substrates. The esterase, the encoding gene, the carrier, and the recombinant bacteria can be widely applied to ester catalysis of synthesis and resolution of chiral compounds related to tertiary alcohol substrates in medicine industry, food industry, chemical industry and the like.

Description

(1) Technical field [0001] The invention relates to a tertiary alcohol ester hydrolase, a gene encoding the enzyme, a carrier containing the encoding gene, engineering bacteria and application thereof. (2) Background technology [0002] Esterase is an enzyme that can hydrolyze fatty acid esters to produce free fatty acids, hydrolyze oils into fatty acids and glycerol, and also catalyze ester synthesis and transesterification. As an important class of biocatalysts, esterases are widely used in light industry, chemical industry, medicine, food and other industries because of their high selectivity, mild catalytic reaction conditions, and no pollution. In recent years, with the deepening of non-aqueous enzymology and interfacial enzymology, the application of esterase has also been continuously expanded, and it is widely used in ester synthesis, resolution of chiral compounds, selective group protection of chemical synthesis intermediates, Polymer synthesis, peptide synthesis,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/63C12N1/21C12P7/04C12P41/00C12P7/62
CPCY02P20/582
Inventor 王秋岩谢恬金鹏杜鹏飞熊小龙吴慧丽
Owner HANGZHOU NORMAL UNIVERSITY
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