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High-throughput expression method of alkaline bacterial laccase in yeast

An expression method and high-throughput technology, applied in the field of bioengineering, can solve the problems of low enzyme activity, easy formation of inclusion bodies and yield, etc., and achieve the effects of stable and high-efficiency expression, excellent thermal stability and easy operation.

Active Publication Date: 2014-05-28
HUBEI UNIV
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Problems solved by technology

[0003] In order to overcome the deficiencies in the prior art, the present invention provides a high-throughput expression method of alkaline bacterial laccase in yeast, which helps the modeler to complete the model more efficiently and reasonably through semantic-based model discovery Combined modeling, thus solving the problem of easy formation of inclusion body and low yield and enzyme activity of alkaline bacterial laccase in prokaryotic expression system

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Embodiment Construction

[0028]The technical solution of the present invention will be further described below in conjunction with the examples.

[0029] The study on the pH stability of a Pseudomonas 511 laccase expressed by the present invention shows that the 511 laccase has the best stability at a pH of 6.5, and still has 90% activity after 20 hours. pH7.5 and pH8.5 have great stability, and the relative enzyme activity still exceeds 80% after 16 hours of treatment. It also has certain stability in the pH environment of weak acid and weak alkali. In the environment of pH 3.0 and pH 9.5, it still has 75% activity after 12 hours of treatment, and has about 60% activity after 16 hours of treatment. Among them, about 40% of the relative enzyme activity was left after 20 hours of pH9.5 treatment.

[0030] The above is only the best implementation mode of the present invention, any simple changes or equivalent replacements of the technical solutions that can be clearly obtained by any person skilled in...

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Abstract

The invention discloses a high-throughput expression method of alkaline bacterial laccase in yeast. The high-throughput expression method comprises the following steps: (A) a pichia pastoris expression vector carrying with alkaline bacterial laccase genes is constructed; (B) positive transformants are screened; (C) the yeast expression vector is linearized; (D) the yeast galvanic competence is prepared; (E) recombinants are expressed and identified; (F) fusion proteins are purified; (G) enzyme kinetic parameters are determined. The high-throughput expression method helps modelers to finish the compositional modeling of models more efficiently and reasonably through the model discovery based on the semantics, so that the problems of easy formation of inclusion bodies by the alkaline bacterial laccase in the prokaryotic expression system, low yield and low enzymatic activity are solved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a high-throughput expression method of alkaline bacterial laccase in yeast. Background technique [0002] Laccase (Laccase EC1.10.3.2) is a copper-containing polyphenol oxidase that can catalyze the oxidation of phenols and aromatic compounds to generate corresponding quinones, and at the same time reduce molecular oxygen with the transfer of electrons into water. Because laccase can oxidize aromatic compounds and some other non-aromatic organic substances, it has a wide range of substrate specificity, so it is used in pulp bleaching, textile dye decolorization, removal of toxic waste, bioremediation, medical diagnosis or preparation of pseudo-anticancer drugs Catalysts and biosensors have great application potential. Laccase exists not only in plants, but also in fungi. The latest research shows that some insects and even bacteria can also produce substances with laccase...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/81C12R1/84C12R1/38
CPCC12N9/0061C12N15/815C12Y110/03002
Inventor 李洋王行国彭梦王慧敏
Owner HUBEI UNIV
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