Novel esterase as well as coding gene and application of esterase

An esterase and encoding technology, which is applied in the fields of application, genetic engineering, and plant gene improvement, can solve the problems of unreported biocatalytic methods, and achieve the effects of no pollution in product quality, wide application prospects, and low energy consumption in production

Inactive Publication Date: 2014-11-12
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology

[0004] The current reports on the synthesis of ethyl isobutyrate mainly involve the ...

Method used

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  • Novel esterase as well as coding gene and application of esterase
  • Novel esterase as well as coding gene and application of esterase
  • Novel esterase as well as coding gene and application of esterase

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Embodiment 1

[0026] 1. Isolation and identification of Bacillus

[0027] Bacillus sp. SCSIO15121 was isolated from the Indian Ocean (89°29.22′E, 10°00.12′N)-3400m deep sea mud, and the cultured bacteria were used for the extraction of total DNA.

[0028] 2. Cloning and expression of esterase EST04211 gene and preparation of enzyme powder

[0029] According to the analysis of the EST04211 gene sequence in the whole genome, corresponding primers were designed to amplify the gene and connected to the expression vector pET-28a(+), and then introduced into Escherichia coli BL21(DE3) for high-level expression.

[0030] Design the primers as follows: the upstream primer is 5′-TGCTAGC CATATG TATGAAACGACTGTCCAAACGTG-3′; downstream primer is 5′-C GAATTC CTAAACCTGCAGGTTTGAGGCTG-3', NdeI and EcoRI restriction sites (underlined parts) were designed at the 5' ends of the upstream and downstream primers respectively, and the genomic DNA of Bacillus sp. SCSIO15121 was used as a template for PCR amplif...

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Abstract

The invention discloses a novel esterase as well as a coding gene and an application of esterase. The esterase EST04211 has an amino acid sequence shown in SEQ ID NO. 2. The amino acid sequence of the gene of the esterase EST04211 is shown in SEQ ID NO. 1. The novel esterase disclosed by the invention is obtained by cloning Bacillus SCSIO15121 derived from deep sea of the Indian Ocean. The esterase has the largest characteristic that the esterification rate is 97% in catalytic synthesis of ethyl isobutyrate, and under similar conditions, the esterase can also catalyze propionic acid, butyric acid, valeric acid, caproic acid and ethanol, propanol, butanol, pentanol and hexanol to synthesize corresponding short-chain aromatic ester flavors, such as ethyl propionate, propyl propionate and butyl propionate, the esterification rate mostly reaches 85%-95%. The esterase can be applied in the industrial bio-manufacturing of the short-chain aromatic flavors, such as ethyl isobutyrate and the ester flavors are high in quality, belongs to natural products and can be applied in production industries, such as foods, cigarettes and daily chemical products.

Description

Technical field: [0001] The invention belongs to the technical field of genetic engineering and biocatalysis, and in particular relates to an esterase gene from the deep-sea bacillus in the Indian Ocean, the esterase expressed by the gene, and the esterase can be used for biological production of ethyl isobutyrate. Background technique: [0002] Ethyl isobutyrate, as an edible flavor allowed in the "Usage Standards of Food Additives", is mainly used to prepare cream and all fruit flavors such as strawberries and cherries, and can also be used as flavoring materials for cigarettes, daily chemical products or other products. It is also an excellent organic solvent. The method of industrially producing ethyl isobutyrate is mainly to directly esterify isobutyric acid and ethanol, and strong acids (such as concentrated sulfuric acid, p-toluenesulfonic acid, etc.) have been used as catalysts. Although strong acid has ideal catalytic activity and low price as an esterification syn...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12P7/62C12R1/07
CPCC12N9/18C12P7/62
Inventor 胡云峰梁甲元孙爱君张云吴正超邓盾李洁田新朋
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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