Ocean cold-adapted esterase as well as coding gene E40 and application thereof

A technology of cold-adapted esterase and cold-adapted ester, which is applied in marine cold-adapted esterase and its coding gene E40 and its application in the field of biology to achieve the effect of ensuring safety

Active Publication Date: 2015-04-29
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reported bacterial HSL esterases are mainly thermostable mesophilic enzymes and thermophilic enzymes, while there are few reports on low-temperature esterases and cold-adapted esterases

Method used

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  • Ocean cold-adapted esterase as well as coding gene E40 and application thereof
  • Ocean cold-adapted esterase as well as coding gene E40 and application thereof
  • Ocean cold-adapted esterase as well as coding gene E40 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] A marine cold-adapted esterase gene E40, the nucleotide sequence of which is shown in SEQ ID NO.1. The above gene encodes a marine cold-adapted esterase E40, the amino acid sequence of which is shown in SEQ ID NO.2.

[0032] The gene E40 is 894bp in total, which contains an open reading frame of 894bp, which encodes cryogenic esterase E40, the start codon is located at 1bp, and the stop codon is located at 892bp, encoding a total of 297 amino acids.

Embodiment 2

[0033] Example 2: Determination of the gene sequence encoding cold-adapted esterase E40

[0034] The source of the strain: Escherichia coli EPI300 clone E40-6B in the E505 metagenomic library of the South China Sea seabed sediment sample.

[0035] Specific steps are as follows:

[0036] 1.1 For the extraction of the large fragment plasmid fosmid in E. coli EPI300 clone E40-6B, refer to the instructions of the BAC / PAC DNA extraction kit from OMEGA Company

[0037] (1) Take 1.5-5ml clone E40-6B bacteria solution, centrifuge at 13,000rpm for 3min, discard the supernatant, collect the bacteria, and suck up the supernatant as much as possible;

[0038] (2) Add 200 μl of RNase A-added buffer T1 to the cells obtained in step (1) to resuspend the cells, shake and mix to obtain a resuspension;

[0039] (3) Add 200 μl of buffer T2 to the resuspension prepared in step (2), mix by gently inverting 5-10 times to obtain a clear lysate, and place at room temperature for 5 minutes;

[0040...

Embodiment 3

[0053] Example 3: Cloning, heterologous expression and separation and purification of esterase E40

[0054] 2.1 Amplification of E40 gene sequence by PCR

[0055] (1) Design two specific primers according to the E40 gene sequence:

[0056] 40F: CGG CATATG GCCAAAAGCCCAGAGTT (SEQ ID NO.3), underlined is the NdeI restriction site;

[0057] 40R: GCC AAGCTT TCAGCCGATCTGCTTCCGC (SEQ ID NO.4), underlined is the HindIII restriction site;

[0058] Primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0059] (2) Using 40F and 40R as primers and using the fosmid where E40 is located as a template, FastPfu DNA polymerase (purchased from Transgen) was used to amplify the target gene fragment.

[0060] The PCR reaction conditions were: pre-denaturation at 95°C for 2 min; then denaturation at 95°C for 20 sec; annealing at 55°C for 20 sec; extension at 72°C for 20 sec, after 30 cycles; extension at 72°C for 10 min.

[0061] (3) PCR amplification product is carried out ...

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Abstract

The invention relates to ocean cold-adapted esterase as well as a coding gene E40 and an application thereof. The nucleotide sequence of the gene E40 of ocean cold-adapted esterase is shown by SEQ ID No.1. The esterase E40 provided by the invention has relatively high catalytic efficiency at 0-20 DEG C and can hydrolyze butterfat at a low temperature or normal temperature to generate short-chain fatty acid, and the substances can enhance the flavor of the dairy product and avoid the influence of long-time high temperature on the food quality and the taste and nutritional ingredients; and meanwhile, when the cold-adapted esterase E40 is used for treating the ester-containing waste and wastewater from the catering, the application is safe and efficient and is of very positive significance to environmental protection.

Description

technical field [0001] The invention relates to a marine cold-adapted esterase, its encoding gene E40 and its application, and belongs to the technical field of biotechnology. Background technique [0002] Lipolytic enzymes include esterases and lipases, which represent a large class of hydrolytic enzymes that can catalyze the hydrolysis and synthesis of ester bonds. Esterase usually acts on simple esters or short-chain glycerides with less than 10 carbon atoms, while lipase usually acts on long-chain glycerides (≥10 carbon atoms) that are poorly soluble in water . Ester hydrolases widely exist in animals, plants and microorganisms. Microbial resources are abundant, and the use of microbial fermentation to produce enzymes has the advantages of convenient industrial production and easy purification. Therefore, ester hydrolases derived from microorganisms have been widely used in agriculture, food brewing, medicinal chemistry, sewage treatment, and bioremediation. The appli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/18C12N15/70C12N1/21A23L1/30C02F3/34B09B3/00A23L33/10
Inventor 张玉忠李平一陈秀兰解彬彬张熙颖宋晓妍苏海楠秦启龙石梅周百成
Owner SHANDONG UNIV
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