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Alkaline pectinase poly lactic acid (PLA) and gene and application thereof

A pectinase and alkaline technology, applied in the field of genetic engineering, can solve the problems of low expression of alkaline pectinase and not being widely used

Active Publication Date: 2012-07-11
WUHAN SUNHY BIOLOGICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression level of alkaline pectinase is still very low, and it has not been widely used so far due to cost issues.

Method used

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  • Alkaline pectinase poly lactic acid (PLA) and gene and application thereof
  • Alkaline pectinase poly lactic acid (PLA) and gene and application thereof
  • Alkaline pectinase poly lactic acid (PLA) and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] The cloning of embodiment 1 Klebsiella pectinase coding gene plA

[0095] Klebsiella sp. is derived from the soil samples of Guangxi fruit juice factory, and the genomic DNA of Klebsiella sp. is extracted: centrifuge the bacterial liquid cultured in LB medium at 30°C for 2 days at 10,000rpm for 10min , Weigh 50 mg of bacteria sludge and wash with 500 μL of sterile water, and centrifuge to get the precipitated bacteria. Resuspend the precipitated bacteria in 500 μL of lysozyme mixture, incubate at 37°C for 1 hour, add 100 μL of enzyme solution and continue to incubate at 45°C for 30 minutes until the bacteria solution is transparent, add 10% SDS to a final concentration of 2%, and stir for about 5 minutes Until the viscosity of the bacterial liquid drops significantly, centrifuge at 15,000 rpm for 10 minutes to remove debris. The supernatant was extracted with equal volumes of phenol, phenol:chloroform, and chloroform in sequence. Take the upper layer solution and add ...

Embodiment 2

[0102] Enzyme activity analysis of embodiment 2 pectinase

[0103] The enzyme activity was determined by the DNS method. Under the conditions of pH 9.0 and 50°C, 1 mL of the reaction system includes 100 μL of appropriate diluted enzyme solution, 900 μL of substrate, reacted for 10 min, added 1.5 mL of DNS to terminate the reaction, and boiled for 5 min. After cooling, the OD value was measured at 540 nm. One enzyme activity unit (U) is defined as the amount of enzyme that releases 1 μmol of reducing sugar per minute under given conditions.

Embodiment 3

[0104] Example 3 Preparation of recombinant pectinase.

[0105] Design primers based on sequencing results:

[0106] PLAF (5'-CTG CCATGG CAGGAGAACGAGCGCCTGAGCAGCGTAAAGC-3', the underline is the Nco I site) and PLAR (5'-CTT GCGGCCGC TTATTTTTTGCCGTTCGGTTTCAGCTGCTCAC-3'), the underline is the Not I site) for PCR amplification. Plasmid pET22b(+) and gene fragments were digested and ligated to form vector pET22b(+)-plA to prepare BL21(DE3) competent cells, and the recombinant plasmid pET22b-plA was electroporated to transform the host bacteria. Positive recombinants were identified and induced to detect enzyme activity. Take the BL21(DE3) strain containing the recombinant plasmid and the BL21(DE3) strain containing the pET22b(+) empty plasmid (as a control), and inoculate them in 3 mL LB (adding 100 μg / mL ampicillin) culture solution, 37 Cultivate overnight with rapid shaking. Add 100 μL of overnight culture solution to 10 mL of LB culture solution (1% inoculum size) contain...

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Abstract

The invention relates to the field of gene engineering, in particular to alkaline pectinase poly lactic acid (PLA) and a gene and application thereof. According to the alkaline pectinase PLA, the amino acid sequence of the alkaline pectinase PLA is show as the sequence identity number 1 or 2 (SEQ ID NO. 1 or 2). The optimum potential of hydrogen (pH) for alkaline pectinase is 9.0, and more than 80% of enzymatic activity exists between pH 7.5 to pH 10.0. The alkaline pectinase PLA has good pH stability and is stabilized between the pH5.0 to pH10.5. Specific activity of the alkaline pectinase PLA is 797U / mg, and the alkaline pectinase PLA is good in alkaline protease resistance and easy in industrial fermenting production. Serving as a novel enzyme preparation, the alkaline pectinase PLA has application values in the industries of textile, papermaking, detergent, plant fiber processing, tea and coffee fermentation, oil extraction, treatment of industrial waste water containing pectin, biological technology and the like.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to an alkaline pectinase PLA and its gene and application. Background technique [0002] Pectin is a group of polysaccharides widely present in the primary wall and intercellular space of higher plants, and plays a role of "glue" in the cell tissue of plants. The basic unit of pectin is galacturonic acid with different degrees of esterification. D-galacturonic acid is connected by α-1,4 glycosidic bonds as the main chain, and rhamnose, arabinose, galactose, Xylose and other sugars are attached to its main chain as branches (Thakur BR, et al. Crit Rev Food Sci Nutr. 37:47-73, 1997). [0003] Pectinase (pectinases) is a general term for various enzymes that decompose pectin. Due to the complex chemical structure of pectin, there are many kinds of pectinases that can catalyze its decomposition. Pectinases can be divided into pectin hydrolases, pectin lyases ...

Claims

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Application Information

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IPC IPC(8): C12N9/26C12N15/56C12N15/63C12N1/15C12N1/19C12N1/21
Inventor 詹志春张菁苑鹏
Owner WUHAN SUNHY BIOLOGICAL
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