Novel phthalate hydrolase EstJ6 as well as coding gene and application thereof

A phthalate ester and hydrolase technology, applied in the fields of hydrolase, application, genetic engineering, etc., can solve the problems of low efficiency of new esterases, non-cultivation, and restrictions on the development and utilization of new enzymes

Active Publication Date: 2020-04-10
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Studies have shown that the efficiency of discovering new esterases through traditional culture techniques is very low, because only a very small number of microorganisms can be cultured under existing experimental conditions, and more than 99% of microorganisms are not cultivable, which greatly limits new types of esterases. Enzyme Development and Utilization

Method used

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  • Novel phthalate hydrolase EstJ6 as well as coding gene and application thereof
  • Novel phthalate hydrolase EstJ6 as well as coding gene and application thereof
  • Novel phthalate hydrolase EstJ6 as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1: the screening of phthalate hydrolase gene in the soil metagenomic library

[0072] 1. Primary screening of positive clones by substrate plate method

[0073] Prepare the primary screening medium. After the LB solid medium is sterilized by high temperature and high pressure, it is cooled to an appropriate temperature (60°C) and the substrate 1mM dibutyl phthalate (dissolved in DMSO) and 100μg / mL ampicillin, shake well and pour plate. Appropriately dilute the cosmid library bacteria liquid on the screening plate, incubate at 37°C for 1-2 days, observe the formation of transparent circles around the colonies, which are positive clones ( figure 1 ).

[0074] 2. Re-screening, HPLC verification of the enzyme activity of its phthalate hydrolase

[0075] Inoculate the monoclonal obtained from primary screening in liquid LB (containing 100 μg / mL ampicillin) medium, culture at 37°C overnight (12h), centrifuge at 12,000×g at room temperature for 8min, discard the...

Embodiment 2

[0081] Embodiment 2: the cloning of phthalate hydrolase

[0082] 1. PCR amplification

[0083] Using primers:

[0084] Upstream primer: 5'-CATG CCATGG GCATGGCAAGTCCGCAACTA-3' (SEQ ID No. 3); and

[0085] Downstream primer: 5'-CCC AAGCTT CGCGGTGATCTGCCGGAC-3' (SEQ ID No.4) amplifies the phthalate hydrolase gene estj6, and the underlines of the upstream and downstream primers indicate the restriction sites of NcoI and HindIII, respectively.

[0086] PCR reaction system (25 μL): 9.5 μL of ultrapure water, 12.5 μL of 2×Taq Master Mix, 1 μL of upstream and downstream primers, and 1 μL of positive subcloning plasmid DNA. PCR reaction conditions: 94°C for 3min; 35 cycles of 94°C for 30s, 63°C for 45s, and 72°C for 1min; 72°C for 10min. The PCR product was electrophoresed and recovered by tapping the gel to obtain a purified PCR product.

[0087] 2. Digestion

[0088] The purified and recovered PCR product was subjected to double enzyme digestion for 3-4 hours. The enzyme dig...

Embodiment 3

[0094] Example 3: Heterologous expression and purification of phthalate hydrolyzing esterase EstJ6

[0095] 1. Conversion

[0096] Take 10uL of the pET28a-esjt6 plasmid obtained in Example 2 and add it to 100uL Escherichia coli BL21 (DE3) competent cells, incubate on ice for 30min, heat shock in a water bath at 42°C for 90s, and add 900uL LB liquid medium after ice bathing for 2min. 180rpm, shaking at 37°C for 1h. The bacteria were resuspended and spread on LB plates containing kanamycin, cultured overnight at 37°C and single colonies were picked.

[0097] 2. Induced expression

[0098] The recombinant bacteria were inoculated into 5 ml of LB liquid medium containing kanamycin, and cultured at 37° C. and 180 rpm for 12 hours. Take 1ml of bacterial liquid and inoculate into 100ml of fresh LB medium, cultivate at 37°C and 200rpm until the OD600nm is about 0.6, add IPTG to the final concentration of 0.5mM, and continue to cultivate at 16°C and 180rpm for 20h.

[0099] 3. Puri...

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Abstract

The invention provides a novel phthalate hydrolase gene derived from a soil metagenome library. The nucleotide sequence and an amino acid sequence of the novel phthalate hydrolase gene are shown as SEQ ID NO.1 and SEQ ID NO.2. After the esterase gene is connected to an expression vector pET28a (+), the esterase gene is transformed into escherichia coli BL21 (DE3) to realize heterologous expression. The molecular weight of the purified recombinase (EstJ6) is 33.31 kDa. The EstJ6 has wide substrate specificity on phthalate, and the EstJ6 not only can hydrolyze phthalate with a simple side chain,but also can hydrolyze diethyl hexyl phthalate and monoethyl hexyl phthalate with complex and longer side chains. In addition, site-specific mutagenesis experiments show that the catalytic triad residue of EstJ6 is S146-E240-H270, and mutation of any amino acid in the three causes the EstJ6 to lose the catalytic ability. The novel phthalate hydrolase disclosed by the invention can be applied to the fields of food industry, agriculture, biotechnology and the like due to the specific activity and enzymatic characteristics of the novel phthalate hydrolase.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a novel phthalate hydrolase obtained by screening from a soil metagenomic library, its coding gene and its application. Background technique [0002] Phthalates (PAEs), a class of toxic organic compounds, are ubiquitous in the environment due to their widespread use as additives or plasticizers in the manufacture of plastics. Six of them (dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), dioctyl phthalate (DNOP), phthalate Diethylhexyl dicarboxylate (DEHP) and butylbenzyl phthalate (BBP) have been listed as priority pollutants by the US Environmental Protection Agency and some of their international counterparts. Among them, DEHP occupies a high proportion (about 31%) in the environment. The massive use of PAEs has resulted in serious environmental problems and a variety of diseases, including respiratory diseases, childhood obesity, and neurop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/70C12N1/21C12R1/19
CPCC12N9/18C12N15/70
Inventor 辛志宏邱佳容张月琦姜俊伟吴盛露李龙祥邵玉庭
Owner NANJING AGRICULTURAL UNIVERSITY
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