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Gene of paper-making cholesterase, expression vector, strain and applications thereof

A sterol lipase and expression vector technology, applied in the field of bioengineering, can solve the problems of paper breakage, complex composition, increase of paper holes, dust spots, etc., and achieve the effect of stable operation efficiency

Active Publication Date: 2017-05-10
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, due to its complex composition, waste paper contains a large amount of fine components and anionic impurities. Among these impurities, especially stickies, it is difficult to remove. On the one hand, the existence of stickies will increase the holes and dust spots on the paper, reducing the quality of the paper. On the other hand, its deposition in the forming wire, press section and drying section can easily cause paper breakage, resulting in poor drainage performance and reduced paper machine speed. Therefore, the treatment of stickies is also a major issue in waste paper pulping

Method used

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  • Gene of paper-making cholesterase, expression vector, strain and applications thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036]Embodiment 1 synthetic sterol esterase gene (CHE)

[0037] The sterol esterase (CHE) gene sequence and protein structure information of S.lavendulae H646-SY2 was obtained from the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov / ) and the Protein Database (PDB, http: / / www.rcsb.org / pdb / search / advSearch.do).

[0038] According to the CHE gene sequence published on NCBI, the sequence was optimized, and all the codons preferred by Pichia pastoris were replaced, and then the optimized gene sequence was used with DNAWORKS software ( http: / / mcl1.ncifcrf.gov / lukowski.html ), design primers for whole gene synthesis, and design 2 primers, wherein 2 primers introduce EcoRI and NotI enzyme cutting sites and enzyme cutting protection bases respectively, and the primer sequences are:

[0039] CHE-F: 5′-CCG GAATTC ATGTCCTCCCAAGTT-3′; (the horizontal line is the EcoRI restriction site)

[0040] CHE-R: 5′-ATAAGAAT GCGGCCGC TTAATGG-3'; (the horiz...

Embodiment 2

[0042] Embodiment 2 prepares the plasmid containing CHE gene

[0043] The PCR product obtained in Example 1 was electrophoresed, the gel was cut to recover the target band at about 700bp, the recovered product was digested with EcoRI and NotI, and the expression vector pPICZαA was also digested with EcoRI and NotI and recovered from the gel with T4DNA Ligase ligation. The 10 μL volume reaction system is as follows: pPICZαA 1 μL (50ng), add 6 μL of fully synthetic gene product, 1 μL of 10× Buffer containing ATP, T4 DNA ligase 1 μL, add ddH 2 O to make up to 10 μL. Slightly centrifuged, connected in a water bath at 16°C overnight, and transformed into E.coli Top10 (invitrogen), and positive transformants were screened on LB plates containing Zeocin (bleomycin, 25 μg / mL). Randomly pick a certain amount of transformants, prepare a small amount of plasmids, and perform electrophoresis analysis after double digestion with restriction endonucleases EcoRI and NotI. It is estimated ...

Embodiment 3

[0044] Example 3 High expression of sterol lipase CHE in Pichia pastoris

[0045] The pPICZαA-CHE of Example 2 that had been completely linearized by Sac I was transformed into Pichiapastoris X33 (invitrogen) by the LiCl method. The transformants were spread on MD plates and cultured at 30°C for 2 days. Transformants on the MD plate were inoculated on YPD plates containing Zeocin 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, and 500 μg / mL, and cultured at 30°C for 3 days. Single clones were picked from the transformants appearing on the Zeocin-YPD plate with a higher concentration. Extract yeast genomic DNA as a template according to the Invitrogen operating guide, and use the PCR primers of the target gene sequence for yeast genome PCR identification. The total reaction volume is 20 μL, and the amount of Taq enzyme is 2U. Take 2 μL of PCR products for 0.8% agarose gel electrophoresis identification.

[0046] The PCR amplification product was detected by 1% agarose gel electro...

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Abstract

The invention discloses a gene of paper-making cholesterase, an expression vector, a strain and applications thereof, and belongs to the field of bioengineering. A nucleotide coding sequence of the gene is shown as SEQ ID NO:1. The cholesterase gene provided by the invention, on the basis of such technologies as codon optimization, escherichia coli expression, pichia pastoris expression and the like, achieves high expression in pichia pastoris; and the enzymatic activity of the obtained cholesterase is about 0.4U / mL. The expressed cholesterase has the properties of resisting high temperature and resisting weak base; the cholesterase provided by the invention can be applied to waste paper de-inking, namely ink particles can be removed by hydrolyzing adhesives on the surface of waste paper and between paper pieces; and the stable quality of recycled paper and the operating efficiency of a paper-making machine can be guaranteed.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a gene capable of efficiently expressing sterol lipase for papermaking, an expression vector, a bacterial strain and an application thereof. Background technique [0002] The paper industry is one of the important industries supporting my country's national economy, but problems such as lack of resources, energy shortages and serious pollution are restricting the development of the paper industry, and expanding the use of waste paper is an effective way to solve these problems. However, due to its complex composition, waste paper contains a large amount of fine components and anionic impurities. Among these impurities, especially stickies, it is difficult to remove. On the one hand, the existence of stickies will increase the holes and dust spots on the paper, reducing the quality of the paper. On the other hand, its deposition in the forming wire, press section and dryin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/18C12N15/81C12N1/19D21C5/00D21C5/02C12R1/84
CPCC12N9/18C12N15/815C12N2800/102C12N2800/22C12Y301/01013D21C5/005D21C5/025Y02W30/64
Inventor 韩双艳高士鹏李玲林影郑穗平
Owner SOUTH CHINA UNIV OF TECH
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