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Laccase gene lblcc9i from Ceruleus bicolor and its application

A technology for decolorization of laccase and dye, applied in the field of laccase LbLCC9I sequence

Active Publication Date: 2019-07-30
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is no large-scale artificial cultivation

Method used

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  • Laccase gene lblcc9i from Ceruleus bicolor and its application
  • Laccase gene lblcc9i from Ceruleus bicolor and its application
  • Laccase gene lblcc9i from Ceruleus bicolor and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 LbLCC9I Gene expression in Pichia pastoris

[0026] 1. Construction of Pichia pastoris expression vector

[0027] The optimized gene sequence is shown as SEQ ID NO 1, and the whole gene synthesis is carried out. The gene fragment was ligated to a T / A cloning vector (Takara). Transform DH5α competent to obtain positive clones. The positive clone plasmid was extracted, and the Bam H1 and Sac 1 After double digestion, insert the correct reading frame into the expression vector pPIC9K of Pichia pastoris, and verify it by sequencing to obtain the recombinant plasmid pPIC9K- LbLCC9I (See Figure 2).

[0028] 2. Transformation of Pichia pastoris

[0029] The GS115 (mut + his - , invitrogen) monoclonal inoculation into 10 mL YPD, cultured overnight at 30°C, 225 rpm, transferred 1 mL to 100 mL fresh YPD and continued to culture for 3-5 h to OD 600 =0.5, 5000 rpm centrifuged to collect the bacterial cells. After washing twice with an equal volume of double...

Embodiment 2

[0039] Example 2 LbLCC9I Gene expression in Arabidopsis

[0040] one, LbLCC9I Construction of Gene Plant Expression Vector

[0041] The optimized gene fragment was ligated with T / A cloning vector (Takara). Transform DH5α competent to obtain positive clones. The positive clone plasmid was extracted, and the Bam H1 and Sac 1 After double digestion, insert the correct reading frame into the plant expression vector pYPX158, and verify it by sequencing to obtain the recombinant plasmid pYPX158- LbLCC9I (See Figure 6).

[0042] 2. Transformation of Arabidopsis

[0043] pYPX158- LbLCC9I Transform Agrobacterium tumefaciens GV3101, or EHA105, or LBA4404 strains (Biovector Co., LTD) by electric shock method to obtain pYPX158- LbLCC9I The recombinant Agrobacterium was spread on a plate containing kanamycin resistance to select transformants. The above transformants were inoculated in YEB liquid medium (containing 100 μg / ml kanamycin and 75 μg / ml rifampicin), and cultured...

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Abstract

The invention discloses a laccase LbLCC9I sequence capable of being expressed in Pichia pastoris and Arabidopsis thaliana after being optimized and application thereof. The nucleotide sequence of the laccase LbLCC9I is disclosed as SEQ ID NO 1, the full-length is 1518bp, and the sequence of the coded protein is disclosed as SEQ ID NO 2 and is composed of 501 amino acids. After being transformed into Pichia pastoris and Arabidopsis thaliana, the sequence can express the active laccase. The laccase can be used for dye decolorization.

Description

technical field [0001] The invention belongs to the field of biological technology, and specifically relates to an optimized laccase LbLCC9I sequence that can be expressed in Pichia pastoris and Arabidopsis thaliana and its application. Background technique [0002] As a biocatalyst, enzymes have the characteristics of high efficiency, high selectivity, mild catalytic reaction conditions and no pollution, and have attracted extensive attention from researchers. Laccase has become a research hotspot in recent years because of its wide application potential in industrial fields such as environmental protection, biological detection, papermaking and food. [0003] Laccase is a copper-containing polyphenol oxidase mainly derived from fungi and plants, and also found in small amounts in bacteria and insects. It has a wide range of substrates, including: phenols, aromatic amines, carboxylic acids and their derivatives, biochromes and steroid hormones, metal organic compounds, etc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/02
Inventor 王波姚泉洪彭日荷朱波许晶田永生李振军付晓燕高建杰韩红娟王丽娟王飞兵张杰
Owner SHANGHAI ACAD OF AGRI SCI
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