Isolation and characterization of potent antifungal strains of the Streptomyces violaceusniger clade active against Candida albicans

Streptomyces strains were isolated from a sagebrush rhizosphere soil sample on humic acid vitamin (HV) agar and water yeast extract (WYE) agar supplemented with 1.5% (w/w) phenol as a selective medium. Acidic, neutral and alkaline pH conditions were also used in the isolation procedures. The phenol treatment reduced the numbers of both actinomycetes and non-actinomycetes on plates under all three pH conditions. From phenol-amended HV and WYE agar, 16 strains were isolated in pure culture; 14 from the HV agar and two from the WYE agar. All the isolates were tested for their antifungal activities against Pythium ultimum P8 and five yeast strains, including two antifungal drug-resistant Candida albicans strains. HV isolates that showed broad-spectrum antifungal antibiotic activities were all found to be members of the Streptomyces violaceusniger clade, while those that did not were non-clade members. The phenol treatment was not selective for S. violaceusniger clade members. Therefore, we tested the spores of both S. violaceusniger clade and non-clade members using two biocides, phenol and hydrogen peroxide, as selection agents. Spores of non-clade members, such as S. coelicolor M145 and S. lividans TK 21, survived these two biocides just as well as S. violaceusniger clade members. Thus, in our hands, biocide resistance was not S. violaceusniger clade specific as previously reported. However, isolates showing broad-spectrum antifungal and antiyeast activity were all members of the clade. We conclude that screening of isolates for broad-spectrum antifungal/antiyeast activity is the preferred method of isolating S. violaceusniger clade strains rather than biocide-based selection. Phylogenetic analysis of the phenol-resistant isolates revealed that the HV isolates that exhibited broad-spectrum antifungal antibiotic activity were all clustered and closely related to the S. violaceusniger clade, while the isolates that did not exhibit antifungal antibiotic activity were all non-clade members

Purification and characterization of chitinase from <i>Streptomyces </i><i style="mso-bidi-font-style:normal">violascens</i> NRRL B2700

64-71<span style="font-size:11.0pt;font-family: " times="" new="" roman","serif";mso-fareast-font-family:timesnewroman;mso-bidi-font-family:="" mangal;mso-ansi-language:en-gb;mso-fareast-language:en-us;mso-bidi-language:="" hi"="" lang="EN-GB">Chitinase is one of the important enzymes as it is directly linked to Chitin that has wide<span style="font-size:11.0pt;font-family: " times="" new="" roman","serif";mso-fareast-font-family:"times="" roman";mso-bidi-font-family:="" mangal;mso-ansi-language:en-gb;mso-fareast-language:en-us;mso-bidi-language:="" hi"="" lang="EN-GB"> applications in industrial, medical and commercial fields for its biocompatibility and biodegradability. Here, we report extracellular chitinase production by Streptomyces violascens NRRL B2700 under submerged fermentation condition. Chitinase production started after 10 h of incubation and reached to maximum level at 72 h of cultivation. Studies on the influence of additional carbon and nitrogen sources on chitinase production revealed that maltose, xylose, fructose, lactose, soybean meal and ammonium nitrate served as good carbon and nitrogen sources to enhance chitinase yield by 1.6 to 6 fold. Medium supplemented with 1% colloidal chitin produced high chitinase concentration (0.1714 U/mg). The enzyme chitinase was purified from the culture broth by 75% ammonium sulphate precipitation, DEAE-cellulose ion-exchange and sephadex G-100 gel filtration. The molecular mass of the purified chitinase was 65 kDa as estimated by SDS-PAGE. The apparent Michaelis constant (<span style="font-size:11.0pt; font-family:" times="" new="" roman","serif";mso-fareast-font-family:"times="" roman";="" mso-bidi-font-family:mangal;mso-ansi-language:en-gb;mso-fareast-language:en-us;="" mso-bidi-language:hi;mso-bidi-font-weight:bold"="" lang="EN-GB">Km) and the maximum rate <span style="font-size:11.0pt; font-family:" times="" new="" roman","serif";mso-fareast-font-family:"times="" roman";="" mso-bidi-font-family:mangal;mso-ansi-language:en-gb;mso-fareast-language:en-us;="" mso-bidi-language:hi;mso-bidi-font-weight:bold"="" lang="EN-GB">(Vmax) of the enzyme for colloidal chitin were 1.556 mg/mL and 2.680 µM/min/mg, respectively suggested high affinity towards chitin. Possibly, it is the first report on production of chitinase from S<span style="mso-bidi-font-style: italic">. violascens NRRL B2700. The findings were encouraging, especially for cost effective production, and further warrants media and purification optimization studies for enhanced yield.</span

Screening of biologically active microbial strains having therapeutic applications

244-251Natural sources, particularly microbes yield active molecules that have wide application in food and pharmaceutical industries, degradation of hazardous bacterial biofilms, etc. Safety and acceptability of such drugs attract researchers’ attention for new drug discovery. Here, we explored biologically active microbial strains having therapeutic applications isolated from five different geographical areas of India. On screening, we found 10 strains capable of producing chitinase (Chi), seven cholesterol oxidase (COD), five glutaminase (Gln) and two heparinase (Hep) producing strains. Most of the isolated strains were found to be actinomycetes. Morphological and biochemical characterization of the strains suggest that the selected 13 isolates belong to the genus Streptomyces. Out of which, four were characterized through 16S ribosomal RNA gene analysis as Streptomyces xanthochromogenes MTCC 11937 (S1), Streptomyces violascens (N1), Streptomyces xanthopheus MTCC 11938 (H1) and Streptomyces rimosus MTCC 10792 (Ay). Results suggest that the soil isolated Streptomyces strains continue to act as a fascinating source of clinical and commercially importance enzymes. Partially purified enzymes were found to possess a broad range of pH and temperature stability indicating their capability to be used in clinical and pharmaceutical fields

Screening of biologically active microbial strains having therapeutic applications

244-251Natural sources, particularly microbes yield active molecules that have wide application in food and pharmaceutical industries, degradation of hazardous bacterial biofilms, etc. Safety and acceptability of such drugs attract researchers’ attention for new drug discovery. Here, we explored biologically active microbial strains having therapeutic applications isolated from five different geographical areas of India. On screening, we found 10 strains capable of producing chitinase (Chi), seven cholesterol oxidase (COD), five glutaminase (Gln) and two heparinase (Hep) producing strains. Most of the isolated strains were found to be actinomycetes. Morphological and biochemical characterization of the strains suggest that the selected 13 isolates belong to the genus Streptomyces. Out of which, four were characterized through 16S ribosomal RNA gene analysis as Streptomyces xanthochromogenes MTCC 11937 (S1), Streptomyces violascens (N1), Streptomyces xanthopheus MTCC 11938 (H1) and Streptomyces rimosus MTCC 10792 (Ay). Results suggest that the soil isolated Streptomyces strains continue to act as a fascinating source of clinical and commercially importance enzymes. Partially purified enzymes were found to possess a broad range of pH and temperature stability indicating their capability to be used in clinical and pharmaceutical fields

Isolation, screening and identification of novel isolates of Actinomycetes from India for antimicrobial applications

The search for novel bioactive compounds from the natural environment has been rapidly increased with the increase in multi-drug resistant (MDR) pathogens. In the present study, the antimicrobial potential of novel actinomycetes has been evaluated by initial screening of six soil samples. Primary and secondary screening was performed against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Candida albicans, Candida tropicalis, Trichophyton rubrum, and other MDR bacterial and fungal test strains, and at the end thirteen active isolates were selected for further study. Microbial strains were identified on the basis of growth conditions and other biochemical characters. Five most active microbial strains were identified using 16S rRNA sequence homology and designated as Streptomyces xanthophaeus MTCC 11938, Streptomyces variabilis MTCC 12266, Streptomyces xanthochromogenes MTCC 11937, Streptomyces levis EU 124569 and Streptomyces sp. NCIM 5500. Four antibacterial and three antifungal compounds isolated from the above five isolates were purified and partially characterized using UV absorption and IR spectra. Two antibacterial metabolites, belong to chromone and peptide antibiotic, respectively. The antifungal compounds were found to be of non-polyene nature. In conclusion, we study the isolation of novel bacterial strains of actinomycetes for producing novel compounds having antibacterial and antifungal activities from the unexplored agro-ecological niches of India. Also, this study paves the way for further characterization of these isolates of Streptomyces sp. for their optimum utilization for antimicrobial purposes