Preparation method and application for biphenyl dioxygenase mutant of polychlorinated biphenyl degrading bacteria

A biphenyl dioxygenase and polychlorinated biphenyl technology, which is applied in the fields of genetic engineering and enzyme engineering, can solve problems such as the inability to meet the needs of actual treatment, and achieve a wide range of substrate degradation, substrate spectrum and degradation rate improvement. , the effect of a wide range of applications

Active Publication Date: 2020-04-10
YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI +1
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Problems solved by technology

Although many attempts have been made in the mutation of biphenyl dioxygenase, its degradation rate and degradation types, especially the degradation of high-chlorinated biphenyls, still cannot meet the needs of actual treatment.

Method used

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  • Preparation method and application for biphenyl dioxygenase mutant of polychlorinated biphenyl degrading bacteria
  • Preparation method and application for biphenyl dioxygenase mutant of polychlorinated biphenyl degrading bacteria
  • Preparation method and application for biphenyl dioxygenase mutant of polychlorinated biphenyl degrading bacteria

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Embodiment

[0037] This example relates to a method for preparing a biphenyl dioxygenase mutant of a polychlorinated biphenyl degrading bacterium, comprising the following steps: mutating the serine at position 283 of the engineering bacterium BphAEp4 into methionine to obtain a biphenyl dioxygenase mutant Oxygenase mutant BphAEp4-S283M.

[0038] The present invention mutates the serine at position 283 of BphAEp4 to methionine through site-directed mutation, and obtains a biphenyl that has a wider range of degradation substrates than BphAEp4, significantly degrades highly chlorinated polychlorinated biphenyls, and has higher enzymatic activity. Dioxygenase mutant BphAEp4-S283M, see figure 2 , image 3 shown.

[0039] The principle is that the amino acid at position 283 is located at the entrance of the catalytic cavity of biphenyl dioxygenase, and the size and structure of the amino acid residues affect the binding of the enzyme to substrates such as polychlorinated biphenyls. By muta...

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Abstract

The invention provides a preparation method and an application for a biphenyl dioxygenase mutant of polychlorinated biphenyl degrading bacteria. The preparation method comprises the following step: allowing serine at the 283rd position of engineering bacteria BphAEp4 to be mutated into methionine so as to obtain a biphenyl dioxygenase mutant BphAEp4-S283M. According to the invention, through changing of a transcription regulation factor, the expression quantity of related enzymes is increased, so the degradation effect is improved. Compared with parent biphenyl dioxygenase, the biphenyl dioxygenase mutant provided by the invention has the following advantages: the substrate spectrum and the degradation rate are significantly improved; and the degradation rate is improved to three times ormore of an original degradation rate, such as degradation of hexachlorobiphenyl at 2, 2', 3, 4, 5 and 5' positions. The mutant BphAEp4-S283M obtained by using the preparation method provided by the invention has significantly improved enzyme activity compared with a parent, and has a kcat / Km value improved by two times or more of the kcat / Km value of the parent.

Description

technical field [0001] The invention relates to the technical field of genetic engineering and enzyme engineering; in particular, it relates to a preparation method and application of a biphenyl dioxygenase mutant of polychlorinated biphenyl degrading bacteria. Background technique [0002] Polychlorinated biphenyls (PCBs) are a synthetic organic pollutant, theoretically composed of 209 congeners, with the characteristics of high toxicity, environmental persistence, bioaccumulation, semi-volatility, and long-distance migration ability. In 2001, it became one of the first 12 types of persistent organic pollutants announced by the Stockholm Convention. Due to the electrically insulating and nonflammable properties of PCBs, many of their counterparts are used in industrial and commercial applications such as transformer oil, plastics, and other applications. From 1929 to 1978, approximately 1.5 million tons of PCBs were manufactured worldwide. PCBs are gradually released in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/70
CPCC12N9/0071C12N15/70C12Y114/12018
Inventor 胡晓珂孙成成李俊德王媛
Owner YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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