DNA molecule for expressing collagenase in Escherichia coli and method and application of recombinant collagenase

A technology of DNA molecules and Escherichia coli, applied to the DNA molecules of collagenase, the application field of collagenase in degrading collagen, can solve the problems of complex extraction process and high production cost of high-purity collagenase, and achieve significant application prospects and strategies Significant, large yield, significant effect

Inactive Publication Date: 2015-10-21
TIANJIN UNIV OF COMMERCE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the production and preparation process of high-purity microbial collagenase has become a research hotspot at home and abroad. The application of microbial collagenase in my country is still in its infancy. D

Method used

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  • DNA molecule for expressing collagenase in Escherichia coli and method and application of recombinant collagenase
  • DNA molecule for expressing collagenase in Escherichia coli and method and application of recombinant collagenase
  • DNA molecule for expressing collagenase in Escherichia coli and method and application of recombinant collagenase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The construction of embodiment 1 engineering bacterium ColB-RZ1

[0041] (1) Construction of pET28a-Collagenase recombinant plasmid

[0042] Using the genomic DNA of Bacillus cereus R75E with the preservation number CGMCC No.8614 in the General Microbiology Center of the China Microbiological Culture Collection Management Committee as a template, the genomic DNA was extracted according to the instructions of the bacterial genomic DNA extraction kit. The upstream primer P1 shown in SEQ ID NO:3 and the downstream primer P2 shown in SEQ ID NO:4 were designed according to the collagenase gene sequence shown in SEQ ID NO:2. The PCR reaction was carried out with the reaction system and conditions shown in Table 1 below.

[0043] Table 1. PCR reaction system for amplifying Bacillus cereus collagenase gene

[0044] Loading order name Volume (μL) 1 10×PCR buffer 5 2 Upstream primer P1 (10mM) 1 3 Downstream primer P2 (10mM) 1 4 MgSO 4 (...

Embodiment 2

[0060] Induction, expression and purification of embodiment 2 recombinant collagenase

[0061] (1) Induced expression and detection of recombinant collagenase

[0062] After culturing the successfully constructed strain ColB-RZ1 in Example 1, a single clone was picked and placed in 5 ml of LB liquid medium containing 34 μg / ml kanamycin, and cultured at 37°C and 150 r / min for 12 hours. The resulting culture was inoculated into 1 L of LB liquid medium containing 34 μg / ml kanamycin, and cultured at 37°C for 2.5-3 hours until the OD600 value of the strain ColB-RZ1 was 0.6-0.8. Add IPTG to the culture solution to The final concentration is 0.1mmol / L. Shake induction culture at 22°C and 150r / min for 16h.

[0063] Centrifuge 1L of the culture medium of ColB-RZ1 strain induced by IPTG at 12000rpm / min at 4°C for 30min to collect the bacterial cells, discard the supernatant, and then resuspend the bacterial pellet with 50ml of buffer 1. Among them, the composition of buffer 1 is: 20m...

Embodiment 3

[0066] Example 3 Activity Analysis of Recombinant Collagenase

[0067] (1) Determination of specific activity of recombinant collagenase

[0068] Determination of recombinant collagen by the general Mandl collagenase activity assay method (Mandl, I., Maclennan, J., D., Howes, E., L., J. Clin. Invest., 12 (1953) 1323-1329.) Enzyme specific activity. In this method, 1 unit of activity (U) is defined as: every milliliter of collagenase reacts with bovine Achilles tendon-derived type I collagen under the reaction conditions of 37°C and pH value of 7.4, and each unit of activity produced within 5 hours 1 μmoL free amino acid corresponds to 1 activity unit (1U). The specific activity of collagenase is represented by the ratio (U / mg) of the total number of enzyme activity units (U) per milliliter of collagenase to the amount of collagenase protein per milliliter (mg). The specific steps of viability determination are:

[0069] a, L-glycine standard curve drawing

[0070] ①Use bu...

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Abstract

The invention discloses Escherichia coli for expressing a collagenase and a construction method and application thereof. An amino acid sequence (shown in SEQ ID NO:1) and a nucleotide sequence (shown in SEQ ID NO:2) of the collagenase excreted by Bacillus cereus R75E are provided. The invention provides an expression bacterium comprising the collagenase genes in the Escherichia coli. The recombinant Escherichia coli containing the expressed DNA molecule is an Escherichia coli strain obtained by guiding an Escherichia coli recombinant expression vector into host bacteria. The invention provides a method for purifying the recombinant collagenase and an application for degrading type I collagen in fish scales. The recombinant collagenase can efficiently degrade natural type-I collagen fibers, is remarkable in effect, and can be applied to development and utilization of collagen in agricultural and sideline products, processing and utilizing of collagen of aquatic products, and production in medical and food industries.

Description

Technical field [0001] The present invention involves the field of biotechnology, especially DNA molecules involving a collagense, and its application of the expression, purifying and restructuring collapse in E. coli, and the application of collagen enzymes in derivative collagen. Background technique [0002] The chemical name of collagen enzymes is collagenase (Collagenase), which can hydrolyze the three -dimensional spiral structure of natural collagen under physiological pH and temperature without damage other protein and tissue.According to the source of collaonenase, it can be divided into animal collains and microbial collagense. [0003] Among them, the advantages of microbial collagen enzymes are widely used in the advantages of wide range of substrates, large enzyme cutting points, and low production costs.The microorganisms that have been discovered are currently distributed in the genus of Bacillus. The typical types are: Clastridiumhistolycum, BacillusSubtilis, and ...

Claims

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Application Information

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IPC IPC(8): C12N9/48C12N15/57C12N15/70C12N1/21C12P21/06C12R1/19
Inventor 阮海华张西轩李晔杜康龙张真
Owner TIANJIN UNIV OF COMMERCE
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