Screening and morphological observations of endophytic mold origin of pennywort (Centella asiatica (L.) Urban) as extracellular enzyme producer

Pennywort is a biological plant that is included in the medicinal plant species. The analysis was carried out to obtain information and to find out that endophytic molds from pennywort (Centella asiatica (L.) Urban) can produce extracellular enzymes (amylase, cellulase, pectinase, protease, glucanase, and laccase). Based on the analysis that has been carried out, several conclusions are obtained, endophytic fungi from pennywort with isolate codes MB 20, MM 1, MM 6, MM 8, and MM 16 capable of producing extracellular amylase enzymes, endophytic fungi from pennywort with isolate codes MM 1, MM 12, MM 16, MM 18, MM 19, MM 20 and MM 21 were able to produce extracellular cellulase enzymes, endophytic fungi from pennywort with isolate codes MB 1, MB 3, MB 4, MB 6, MM 1, MM 9, MM 11, MM 13, MM 14, MM16, MM 19, MM 20 and MM 21 were able to produce extracellular glucanase enzymes, endophytic mold isolates from pennywort were proven to be unable to produce extracellular enzymes laccase, pectinase, and protease, and endophytic molds from pennywort with isolate codes MB 1, MB 20, MM 14 and the MM 16 is capable of producing siderophores.Keywords: Centella asiatica (L.) Urban, extracellular enzymes, endophytic typite, isolatesABSTRAKSkrining pengamatan morfologi kapang endofit asal tanaman pegagan (Centella asiatica (L.) Urban) sebagai penghasil enzim ekstraselulerTanaman pegagan merupakan tanaman hayati yang termasuk dalam jenis tanaman obat. Analisis dilakukan untuk mendapatkan informasi dan mengetahui bahwa kapang endofit asal tanaman pegagan (Centella asiatica (L.) Urban) dapat menghasilkan enzim ekstraseluler (Amilase, selulase, pektinase, protease, glukanase, dan lakase). Berdasarkan analisis yang telah dilakukan di peroleh beberapa kesimpulan, diantanya. yaitu kapang endofit asal tanaman pegagan dengan kode isolat MB 20, MM 1, MM 6, MM 8 dan MM 16 mampu menghasilkan enzim amilase ekstraseluler, kapang endofit asal tanaman pegagan dengan kode isolat MM 1, MM 12, MM 16, MM 18, MM 19, MM 20 dan MM 21 mampu menghasilkan enzim selulase ekstraseluler, kapang endofit asal tanaman pegagan dengan kode isolat MB 1, MB 3, MB 4, MB 6, MM 1, MM 9, MM 11, MM 13, MM 14, MM16, MM 19, MM 20 dan MM 21 mampu menghasilkan enzim glukanase ekstraseluler, isolat kapang endofit asal tanaman pegagan terbukti tidak mampu menghasilkan enzim ekstraseluler lakase, pektinase dan protease, dan kapang endofit asal tanaman pegagan dengan kode isolat MB 1, MB 20, MM 14 dan MM 16 mampu menghasilkan siderofor.Kata Kunci: Centella asiatica (L.) Urban, enzim ekstraseluler, kapang endofit, isolat

Extracellular amylase is required for full virulence and regulated by the global post-transcriptional regulator RsmA in Xanthomonas campestris pathovar campestris

As with many phytopathogenic bacteria, the virulence of Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot disease in cruciferous plants, relies on secretion of a suite of extracellular enzymes that includes cellulase (endoglucanase), pectinase, protease and amylase. Although the role in virulence of a number of these enzymes has been assessed, the contribution of amylase to Xcc virulence has yet to be established. In this work, we investigated both the role of extracellular amylase in Xcc virulence and the control of its expression. Deletion of XC3487 (here renamed amyAXcc), a putative amylase-encoding gene from the genome of Xcc strain 8004, resulted in a complete loss of extracellular amylase activity and significant reduction in virulence. The extracellular amylase activity and virulence of the amyAXcc mutant could be restored to the wild-type level by expressing amyAXcc in trans. These results demonstrated that amyAXcc is responsible for the extracellular amylase activity of Xcc, and indicated that extracellular amylase plays an important role in Xcc virulence. We further found that the expression of amyAXcc is strongly induced by starch and requires activation by the global post-transcriptional regulator RsmA. RsmA binds specifically to the 5’-untranslated region (5’UTR) of amyAXcc transcripts, suggesting that RsmA regulates amyAXcc directly at the post-transcriptional level. Unexpectedly, in addition to post-transcriptional regulation, the use of a transcriptional reporter demonstrated that RsmA also regulates amyAXcc expression at the transcriptional level, possibly by an indirect mechanism.

Production and Purification of Amylase from Bacillus subtilis Isolated from Soil

In spite of progress in biotechnology and enzymology, the enzymes have been industrialized in recent years for the mounting up the product development in various arena. The ultimate goal of this study comprises the production and purification the amylase enzyme from the bacterial strain. A powerful amylase producer, Bacillus subtilis ISOLATE-4 was isolated, screened and identified from the soil sample. In order to produce extracellular amylase, various physico-chemical parameters were optimized. During optimization, the maximal production of amylase by the isolate at 48 hrs of incubation in 100 rpm was found to be 6.93U/ml, 5.94U/ml, 6.0U/ml at 45ºC, pH 6 with 1% substrate concentration respectively. Ammonium sulphate fractionation was done for rapid precipitation of the amylase at a concentration of 60% and exposed to dialysis showed the 25% purification fold of an enzyme. The dialyzed product was further subjected to DEAE-Cellulose column chromatography resulted in an increase up to 75% purification fold than crude enzyme. The amylase enzyme might be suitable for the liquefaction of starch, detergent, textile and several additional industrial applications.