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Novel lipase gene, lipase production strain and application

A kind of lipase gene and lipase technology, applied in Pichia pastoris strains, the application field of waste newsprint deinking, can solve the problems of limited development, low expression of original strains, not seen, etc.

Active Publication Date: 2013-03-13
SOUTH CHINA UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there have been no reports about the application of this enzyme in production since then. The reason may be that the expression level in the original strain is low, which limits its further development as an industrial enzyme preparation.

Method used

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  • Novel lipase gene, lipase production strain and application
  • Novel lipase gene, lipase production strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The synthesis of embodiment 1 novel lipase gene

[0023] Based on the Achetobacter radioresistens CMC-1lipase (GenBank: AF073953.1) gene sequence published on the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov / ), the sequence was optimized , replaced with the codon preferred by Pichia pastoris to obtain a new lipase gene, its sequence is shown in SEQ ID NO.1, and then the new lipase gene was cloned through the whole gene synthesis of a biotechnology company (GenScript Biotechnology Co., Ltd.) To the pUC57 plasmid to obtain the plasmid pUC57-ARL.

Embodiment 2

[0024] Example 2 Construction of plasmid pPICZαA-ARL containing novel lipase gene and strain Escherichia coli TOP10 / pPICZαA-ARL carrying novel lipase gene

[0025] (1) Design primers for amplifying the gene according to the sequence of the novel lipase gene:

[0026] Upstream primer F1: 5'-CG GAATTC TGTAATGACGACCACGACGA-3', containing EcoRI restriction site (shown underlined) and protective base;

[0027] Downstream primer R1: 5'-ATTAAATA GCGGCCGC CTGAATTGGCATAAGACT-3', containing the NotI restriction site (shown underlined) and the protection base.

[0028] (2) Perform PCR amplification using the plasmid pUC57-ARL in Example 1 as a template and F1 and R1 as primers; PCR amplification procedure: pre-denaturation at 94°C for 2 minutes; denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 68°C 90s, a total of 30 cycles; the 30th cycle was extended at 68°C for 7 minutes.

[0029] (3) The PCR product and plasmid pPICZαA (purchased from Invitrogen) were digest...

Embodiment 3

[0032] Example 3 Construction of Lipase Production Strain Pichia Pastoris X33 / pPICZαA-ARL

[0033]The linearized pPICZαA-ARL (digested with SacⅠ enzyme) plasmid in Example 2 was transformed into Pichia pastoris X33 (purchased from Invitrogen) by lithium chloride (LiCl) method, and Zeocin (bleomycin) at 0.1-2 mg / mL ) resistance YPD plate for screening. The genomic DNA of the screened transformants was used as a template, and the upstream primer F1 and downstream primer R1 were used as primers for PCR identification. The correct transformant identified by PCR was the lipase producing strain Pichia Pastoris X33 / pPICZαA-ARL.

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Abstract

Belonging to the field of enzymatic deinking, the invention discloses a novel lipase gene, a lipase production strain and application. The novel lipase gene is obtained by codon optimization, and can realize high expression in Pichia Pastoris. By connecting the gene to a Pichia Pastoris expression plasmid pPICZ alpha, a plasmid pPICZ alpha A-ARL can be obtained. Then the plasmid pPICZ alpha A-ARL is employed to convert a Pichia Pastoris host bacterium Pichia Pastoris X33, thus obtaining the lipase production strain Pichia Pastoris X33 / pPICZ alpha A-ARL. The strain can achieve high-efficiency lipase expression. The expressed lipase has an activity of 2500U / mL, and has medium and high temperature resistance as well as alkali resistance, thus being applicable to old newspaper deinking effectively.

Description

technical field [0001] The invention relates to the field of enzymatic deinking, in particular to a novel lipase gene, a Pichia strain producing the enzyme, and applications of the gene and the Pichia strain in deinking waste newsprint. Background technique [0002] The pulp and paper industry is a pillar industry of the national economy. In recent years, in order to alleviate the pressure on resources, energy and environmental protection, they have turned their attention to the reuse of secondary fibers. With more participation of final consumers in the paper cycle, More old newspapers (ONP) are provided. The key to ONP recycling and reuse is deinking. Due to the use of a large amount of strong alkaline chemicals in traditional chemical deinking, the COD value of deinking wastewater is high and the pollution load is large; in addition, the disadvantages of chemical deinking efficiency are low, fiber strength loss is large, and deinking pulp quality is poor. The substantia...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N15/63C12N1/21C12N1/19D21C5/02C12R1/19C12R1/84
CPCY02W30/64
Inventor 林影韩双艳赵小兰郑穗平龚艳
Owner SOUTH CHINA UNIV OF TECH
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