Archive \ Volume.10 2019 Issue 5

New Tannase-Producing Lactobacillus Sp. Nrc10: Gene Cloning, Enzyme Purification, And Characterization‎‎‎

Sanaa Tork , Magda M. Aly , Mona O. Albureikan
Abstract

Tannins are natural polyphenolic compounds that considered nutritionally undesirable because they form water-insoluble complexes with protein, starch, and digestive enzymes and cause a reduction in the nutritional value of food and inactivate the enzymes. This study aimed to characterize an efficient tannin-degrading microorganism, able to grow under acidic conditions. Samples were collected from different places in Jeddah city. The samples were plated onto tannic acid plates containing tannic acid as the sole carbon source. The efficient tannase-producing microorganism was selected for further work. Out of 10 tannase-producing microbial isolates, the best tannase-producing, acid-tolerant bacterium was selected and identified based on morphological, physiological, and biochemical characteristics. The best tannase producer was selected and identified as a species belonging to the genus Lactobacillus and identified as Lactobacillus sp. NRC10. The results of identification were confirmed by 16S rRNA studies. Improving tannase production was carried out. The tannase –coding gene was amplified using the PCR technique. The cloning of tannase gene (Tanlp1) was carried out and its complete sequence was determined. The cloning and efficient expression of tannase gene (Tanlp1) were carried out. Precipitation and purification of the tannase enzyme in addition to factors affecting enzyme production were studied. The enzyme molecular weight was determined to be 50 KDa using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The best temperature and pH were determined to be 30°C and 6, respectively‎‎‎‎.


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