Enzyme composition for high-efficiency decomposition of cellulose

A technology of cellulase and enzyme composition, applied in the direction of drilling composition, detergent composition, non-surface active detergent composition, etc., to achieve the effects of increased production, enhanced enzyme activity, and obvious effects

Inactive Publication Date: 2010-01-06
郑荣
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current research and development of cellulase can not meet the needs of converting plant fiber into simple sugar as a renewable energy source, so it is of great significance to develop cellulase with high-efficiency hydrolysis of cellulose

Method used

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  • Enzyme composition for high-efficiency decomposition of cellulose
  • Enzyme composition for high-efficiency decomposition of cellulose
  • Enzyme composition for high-efficiency decomposition of cellulose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Acquisition of cellulase gene

[0069] The gene fragment in the present invention is obtained from a thermophilic archaea Pyrococcus horikoshii OT3 (JCM9974), which is purchased from Riken (the Institute of Physical and Chemical Research), JAPAN. The entire genomic DNA of this thermophilic archaea has been sequenced.

[0070] The thermophilic bacterium pyrococcus horikoshii OT3 was cultured statically at 95°C for 16 hours (see the catalog of the Japan Collection of Microorganisms for specific culture methods). Genomic DNA was extracted by QIAamp DNA mini Kit (Qiagen.com, Valencia, CA, USA. Cat#51304). The inventors designed the following primers based on the genomic DNA sequence of the thermophile pyrococcus horikoshii OT3, and obtained two cellulase genes of different lengths and two subunit genes of Prefoldin (PFD) by polymerase chain reaction.

[0071] 1.1 Endo-1,4-glucanase PH1171

[0072] The optimized gene constructs with full-length gene and partial ...

Embodiment 2

[0103] Example 2 Positive clone identification of cellulase gene

[0104] 2.1 Determination of biological activity of endo-1,4-glucanase:

[0105] The transformants (pET / PH1171-458 full-length gene or its optimized construct) containing the insert fragment were respectively copied to a medium containing 0.5% carboxymethyl cellulose (CMC) and 1 mM IPTG by lithographic method. On the LB agar medium plate (SIGMA company, C-5678), copy the same transformant to another LB agar medium plate containing 100 μg / ml ampicillin at the same time, and place the two plates in a 37-degree incubator for cultivation After 20 hours, put the ampicillin LB agar medium plate with colonies in a 4-degree incubator for preservation. Another LB agar medium plate containing CMC was fumigated with chloroform vapor to lyse the cells, then stained with 0.5% Congo red solution for 20 minutes, decolorized with 1M NaCI solution for 20 minutes, and then observed for hydrolysis circles. Extract the plasmid th...

Embodiment 3

[0114] Example 3 Cellulase optimization construct activity research

[0115] The steps of producing heat-resistant recombinant protein with various engineering bacterial strains in Example 1 are similar. The original strains stored at -70°C were streak cultured on LB plates containing 50 μg / ml ampicillin, and cultured overnight at 37°C. A single colony was selected, inoculated in LB liquid medium containing 100 μg / ml ampicillin, and cultured with shaking at 37°C. When the OD600 of the bacterial solution concentration was between 0.4 and 0.7, isopropylthio-β-D-galactoside (IPTG) was added to a final concentration of 1 mM. After continuing to cultivate for 6 hours, the cells were collected by centrifugation, and the culture solution was discarded. The cells were uniformly dispersed in an appropriate amount of 100 mM citrate solution with pH 5.0 by shaking. The suspended bacteria are lysed by ultrasonic waves (reference parameters are: ultrasonic instrument Sonifier 250 intens...

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Abstract

The invention relates to an enzyme composition for the high-efficiency decomposition of cellulose, which is a recombinant biologic enzyme capable of effectively decomposing the cellulose under the conditions of high temperature and strong acid. The enzyme composition comprises two enzymes from pyrococcus horikoshii and one auxiliary protein, namely, 1, internally cutting 1,4 glucanase, 2, externally cutting beta-glycuronide and 3, forward folding element protein; the proportion of the three proteins in the enzyme composition is preferably 1:1:0.1-1. The enzyme gene of an amino acid residue sequence of the former two enzymes after being analyzed by deletion and optimized and the other two alpha and beta subgroup genes containing the auxiliary protein (the forward folding element protein) are respectively regulated and controlled by a heat promoter of a temperature sensitive type and are expressed with high efficiency in bacteria; the auxiliary protein has the function of greatly enhancing the activity of the two enzymes; therefore, the cellulose enzyme composition can decompose the cellulose with high efficiency into monosaccharide under the conditions of high temperature and strong acid; and the potential application of the cellulose enzyme composition is the decomposition of cellulose class wheat straws for providing a raw material for the production of biologic petroleum.

Description

technical field [0001] The present invention relates to the field of biology, in particular to a recombinant biological enzyme preparation capable of effectively decomposing cellulose under high temperature and strong acid conditions. and pharmaceutical applications. Background technique [0002] The cellulose produced by photosynthesis in the world reaches 100 billion tons a year, which is the most abundant renewable resource. Using cellulase to decompose plant fibers as industrial raw materials will be a major contribution to human beings, which can make people get rid of their absolute dependence on grains and alleviate the shortage of world resources. [0003] Cellulose is the most abundant renewable biomass resource on earth synthesized by plants from carbon dioxide and water through photosynthesis. According to Wikipedia (http: / / zh.wikipedia.org), cellulose is the most widely distributed and most abundant polysaccharide in nature. Regardless of annual or perennial p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/21C12N1/19C12N1/15C12C7/04C11D7/42D06M16/00B09B3/00C02F3/00A21D8/04A23P1/02A23P1/00C10G3/00C09K8/58C12R1/19C12R1/07C12R1/645
CPCY02P30/20
Inventor 郑荣郑春阳
Owner 郑荣
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