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Pectate lyase coding gene cloned from Streptomyces griseorubens, as well as expression in vitro and application thereof

A technology of Streptomyces grisea and pectin lyase, which is applied in the field of genetic engineering to achieve the effects of convenient purification, guaranteed biological activity, and guaranteed activity

Inactive Publication Date: 2017-05-10
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Pectin lyase has a wide range of sources, bacteria, fungi and actinomycetes have produced pectin lyase, but there are few examples of pectin lyase produced and successfully applied to industrial production

Method used

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  • Pectate lyase coding gene cloned from Streptomyces griseorubens, as well as expression in vitro and application thereof
  • Pectate lyase coding gene cloned from Streptomyces griseorubens, as well as expression in vitro and application thereof
  • Pectate lyase coding gene cloned from Streptomyces griseorubens, as well as expression in vitro and application thereof

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Experimental program
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Effect test

Embodiment

[0038] Step 1 Extraction and sequencing of the genome of Streptomyces griseus

[0039] The spore suspension of Streptomyces griseus was inoculated into LB liquid medium and cultured at 37° C. and 150 rpm for 3 days, then 2.0 mL of bacterial liquid was collected and centrifuged at 12000 rpm for 2 min. Discard the supernatant, collect the bacterial pellet, add 180μL lysozyme (20mg / mL) and 20μl EDTA solution (0.5M, pH8.0), treat at 37°C for 45min, add 4μL RNase A (100mg / mL), shake and mix for 15s , Placed at room temperature for 5 minutes, and then follow the instructions of the bacterial DNA extraction kit to complete the remaining operations to obtain high-purity genomic DNA. The quality of genomic DNA was determined by 0.8% agarose gel electrophoresis. The test results showed no obvious RNA bands, and the genomic bands were clear, complete, free of degradation and pollution.

[0040] Use the second-generation sequencing technology to construct libraries of different insert lengths...

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Abstract

The invention discloses a novel pectate lyase coding gene cloned from Streptomyces griseorubens, an expression in vitro method and an enzymological characteristic. According to the invention, a pectate lyase coding gene sequence is connected to an expression vector, and a transgenic recombinant escherichia coli for inducing the synthesis of pectate lyase is obtained. The invention overcomes defects that pectate lyase is relatively low in expression quantity in vivo and activity and causes seriously limited application range and effect, and high-efficiency expression of pectate lyase in vitro is realized. In addition, the activity and the expression quantity of the recombinant pectate lyase are improved and the following purification of an expression product is facilitated by adding a soluble poly-L-histidine tag; determination results show that the pectate lyase is an alkaline high-temperature-resistant lyase with relatively strong resistant ability to various heavy metal ions and enzyme activity inhibitors. The invention lays a strong foundation for development and preparation of novel pectate lyase preparations.

Description

Technical field [0001] The present invention relates to the technical field of genetic engineering, in particular to a pectin lyase encoding gene sequence cloned from Streptomyces griseus, an expression vector containing the gene sequence, a transgene expression strain and the special enzymatic activity of a recombinant pectin lyase . Background technique [0002] Pectinase is a general term for a type of compound enzyme composed of many single enzymes that is widely present in fruits and a variety of microorganisms can act on pectin. Pectinase has main application significance in the fields of ramie degumming, wood preservation and alcohol fermentation, and has been widely used in the food industry. [0003] Pectin lyase is an important part of pectinase. It can catalyze the breaking of polygalacturonic acid α-1,4 bond. It is a depolymerase that can degrade plant cell walls and cause plant tissues to soften or even die. Pectin lyase acts on the 5th β-carbon atom of galacturonic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12R1/19
CPCC12N9/88C12Y402/0201
Inventor 周培支月娥冯海玮孙玉静毛亮聂文翰
Owner SHANGHAI JIAO TONG UNIV
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