ABSTRACT
An enzyme hyaluronidase (hyase) producing halotolerant bacterium was isolated from dental caries and identified as Brevibacterium halotolerans DC1. Higher growth and hyase production were observed in nutrient broth fortified with hyaluronic acid at pH 7.0, temperature 37 °C, 120 rpm upon 48 h of incubation. Hyase was purified using salt precipitation, DEAE cellulose ion exchange, and Sephadex G-100 gel filtration chromatography. The enzyme was purified to 13-fold with 67.19% recovery of activity and 26.37 U/mg of specific activity. SDS-PAGE and zymography revealed it to be near to homogeneity showing a relative molecular weight of about 43 kDa that was confirmed by MALDI-TOF MS. This hyase was very active and stable at pH 7.0 and temperature 40 °C. The presence of metal ions Ca2+ and Mg2+ increased its activity while Zn2+ and Cu2+ severely inhibited it. Being stable at 2 M NaCl, hyase exhibited its halotolerant nature. This enzyme showed wide substrate specificity where hyaluronic acid (HA) was the best substrate. The kinetic studies revealed that Km and Vmax were 91.3 µg/mL and 306.2 µg/mL/min respectively. This is the first report of hyaluronidase from a halotolerant Brevibacterium spp. which can find applications under high salinity.
Subject(s)
Bacillus/enzymology , Bacterial Proteins/chemistry , Hyaluronoglucosaminidase/chemistry , Salt Tolerance , Bacillus/isolation & purification , Bacillus/pathogenicity , Bacterial Proteins/metabolism , Copper/chemistry , Dental Caries/microbiology , Enzyme Stability , Humans , Hyaluronoglucosaminidase/metabolism , Sodium Chloride/chemistry , Zinc/chemistryABSTRACT
The finding described in this study is the first report of leaf spot disease of cotton caused by Curvularia verruculosa surveyed in the state of Maharashtra (India). The isolated phytopathogenic fungal strain was identified using morphological characteristics and molecular identification of ITS gene sequence (MF784436) and D1D2 region of LSU gene (KY978073). The ability of fungal strain to secrete hydrolytic enzymes viz., pectinase, xylanase, protease, cellulase and lipase was detected. The secretion profile of hydrolytic enzymes by C. verruculosa was also examined in planta and in vitro. The secretion of cellulase, xylanase and protease was found to be inducible on cotton-stalk powder containing media; while secretion of pectinase and lipase was constitutive in glucose containing medium. The hydrolytic enzymes secretion during etiological progression of disease was detected on cotton leaves at regular interval of 24 h up to 10 days. A significant correlation (P < 0.05) was observed between hydrolytic enzymes secretion and disease severity index. The increased level of hydrolytic enzymes in infected plant sample indicates their role in disease progression. The newly documented fungal phytopathogen Curvularia verruculosa was deposited at National Fungal Culture Collection of India, Pune with accession number of NFCCI-4119.
