An overview on therapeutic potential and various applications of microbial collagenases

Collagen is the most widely distributed class of proteins in the human body. Monomers of collagen are constantly being synthesized and degraded throughout the development of a healthy individual to adulthood. The collagenase subfamily found in human matrix (metalloproteinases), are capable of hydrolyzing native collagen under physiological conditions. Collagenases are produced by specific cells involved in repairs and remodelling processes and plays important role in connective tissue metabolism. Present article focus on the major sources, properties and therapeutic aspects of microbial collagenases in their relation with various diseases and its applications in medical and food industry. Collagenolytic enzymes are highly specific for collagen and have been the focus of much practical interest with respect to cosmetic, medical and food based applications. The most common uses of these enzymes appear to be in medicine as they have been used to treat burns and ulcers, to eliminate scar tissue and play an important role in the successful transplantation of specific organs.

Induction and catabolite repression of alpha-amylase synthesis in Bacilluslicheniformis SKB4

Microbial amylases have an exciting potentiality and are being used extensively in different industries. In this study, regulation of amylase biosynthesis was examined in Bacillus licheniformis SKB4 (wild type) and its mutant strain (8b). The mutant strain was developed by using UV exposure. Expression of the a-amylase gene of Bacillus licheniformis was activated by inducer and subject to catabolite repression. Addition of exogenous glucose or sucrose repressed bio-synthesis of a-amylase which was concentration (0.05-1.0% w/v) dependent. However, mutant strain could enable to overrule the glucose mediated repressive effect. Supplementation of second messenger like cyclic adenosine 3',5'-monophosphate (cAMP, 5 mM) along with glucose could a little bit improve amylase synthesis in wild strain. Antibiotics like rifampicin and tetracycline (ribonucleic acid and protein synthesis inhibitor; 100mg/ml) had stopped the release of enzyme in both wild and mutant strain. Amylase production was also inhibited in presence of respiratory inhibitor 2,4-dinitrophenol (uncoupler) at (5mM) concentration. Thus, the pattern of regulation of a-amylase production in the present strain was in multiple forms; it showed the classical glucose effect without stimulation of second messenger system.

Selective isolation and characterization of rare actinomycetes adopted in glacier soil of Manali ice point and its activity against Mycobacterium spp

An exigent demand of antimicrobial agent against M. tuberculosis was lead to the isolation of novel rare actinomycetes from the unexplored cryophilic environment. Soil samples were collected from glacier ice point of Kullu Manali and processed for further studies. A novel approach was described for the isolation of rare actinomycetes from a heterogeneous population. Isolation was done by conventional and density gradient centrifugation. Sucrose gradient centrifugation showed a maximum of 24 actinomycetes isolates which belong to the genera of Streptomyces sp (12), Micromonospora sp (5) Planomonospora sp(2), Micropolyspora sp (2), Actinopolyspora sp (1) Nocardia sp (1) and Intrasporangium sp (1). Of these 24 actinomycetes, isolate Planomonospora sp (PL-2) showed potent anti-mycobacterial activity against Mycobacterium smegmatis (MTCC300) and M.tuberculosis (MTCC 6). Bioautography reveals that the Rf value of active compound was 0.75 and retains the antimicrobial activity at 75° C. Based on the C13 and H1 NMR the active compound was characterized as 2-(2-ethenylphenyl) heptane-1-ol. Phylogenetic analysis reveals active isolate was closely related to Planomonospora alba and the Genbank accession is JQ280498.

Studies on potential of Withania somnifera root extract against diabetic foot infection pathogens

Withaniasomnifera phytochemical analysis of root extract showed the presence of alkaloids, flavonoids, tannins saponins, carbohydrates, quinines and phenol compounds. Studies on the prevalence of diabetic foot infections showed a total of 88 isolates belongs to seven different genera. The standard antibacterial study reveals that E.coli and S.aureus were only isolated pathogens showed the high degree of resistant pattern against all tested antibiotics. S.aureus showed 100 percent resistant to penicillin, ampicillin and amoxicillin. Out of 40 S.aureus isolates, 23 were found to be methicillin resistant and 12 were vancomycin resistant. Similarly out of 22 E.coli, 18 were resistant to penicillin; ampicillin and nine isolates were resistant to amoxicillin. The antibacterial activity of W.somnifera root extract showed potent antibacterial activity at 5mg/mL against E.coli and S.aureus. Bio assay analysis of extracted compounds reveals that the Rf value of the active fraction is 0.38. Gas Chromatography Mass Spectrum of active fraction shows the presence of 7 different chemical constituents concludes further purification of active compound is necessary. The haemolytic study confirms that the extract is safe to use since there is no haemolysis of human RBCs. Further analysis of purification and characterization of an active fraction is required for structural elucidation.

Solid-state fermentation for production of Pectic enzymes by Aspergillus niger

The production of Pectic enzymes by Aspergillus niger was studied under solid state fermentation (SSF). The effect of fermentation condition such as substrate concentration, inoculum volume, incubation time, moistening agent, inducers and organic and inorganic nitrogen sources was studied for enzyme production. Culture conditions were optimized for maximal yield of enzyme. The solid substrate wheat bran was most suitable for pectic enzyme production under SSF. Enzyme production was found maximum after 10 days of incubation. Lactose was found to be most effective as inducer. Gelatin as organic nitrogen source and ammonium nitrate as inorganic nitrogen source yielded high enzyme titres.

Enzymatic and non-enzymatic antioxidant activity of Pergularia tomentosa against carbon tetra chloride induced hepatic damage in Wistar albino rats

Many oxidative stress related disease are due to accumulation of free radicals in the body which causes cell injury. In this study, the enzymatic antioxidants (Superoxide dismutase, Catalase, Glutathione-s-transferase, Glutathione peroxidase, Ascorbate oxidase and Polyphenoloxidase) and non-enzymatic antioxidants (Total reduced glutathione and Vitamin C) activities were determined using Carbon tetrachloride (CCl4) rat liver as experimental model. The ethanol and chloroform extract showed noticeable increases in enzymatic antioxidant and non- enzymatic antioxidant and thus capability to scavenge the free radicals and protect against oxidative stress causing diseases. Thus, the present study indicates that the plant may be clinically valuable agent in the prevention of hepatic failure caused by CCl4 intoxication.

Bile salt hydrolase and antimicrobial activities of three bile resistant probiotics Lactobacillus plantarum strains isolated from Cameroonian artisanal fermented milk

Probiotics are well known for their efficacy as dietary adjuncts providing benefits to consumers. However, the selection of probiotics before incorporation into diet requires the scrutiny of a well-defined set of criteria. Hence, three Lactobacillus plantarum strains (GGU, GLA51 and GLP56, having KU949009, KU949010 and KU949011 respectively as GenBank accession numbers) previously isolated from Cameroonian traditional fermented milk have been used in this study for the evaluation of their Bile salt hydrolase and antimicrobial activities. Bile tolerance test was carried out by monitoring the bacterial growth at different Oxgall bile concentration (0.3%, 0.5% and 1%). The bile salt hydrolase activity was measured by determining the amount of amino acid liberated from conjugated bile salts by lactobacilli strains. Also, three bshgenes (bsh1 gene specific to Lactobacillus plantarum species, bshA and bshB genes specific to Lactobacillus acidophilus species) involved in the bile salt hydrolase activity were screened. The antimicrobial activity of the strains was evaluated using the agar-spot test. The three Lactobacillus plantarum strains showed survival percentages higher than 90% in the presence of 0.3% of Oxgall bile, and the delays of growth ranged from 0 to 10 min. Their bile salt hydrolase activity ranged from 15.62 ± 3.00 to 23.91 ± 5.82 U/mg towards Oxgall and from 10.47 ± 2.76 to 24.57 ± 6.31 U/mg towards Taurodeoxycholate. Only Lactobacillus plantarum GLA51 was found to harbor thebsh1 gene. Its sequence was deposited in the Genbank under the accession number MF098542. The analysis of thisbsh1 gene sequence from L. plantarum GLA51 indicated that it contained an open reading frame (ORF) of 838 nucleotides encoding a 278 amino acids protein. The three Lactobacilli strains showed inhibitory activity against the pathogenic or spoilage bacteria tested, except for Shigella flexneri. These results suggest that the three Lactobacillus plantarum strain show potential for probiotic applications.Keywords: Bile salt hydrolase activity, antimicrobial activity, Lactobacillus plantarum, bile tolerance

Cellulolytic Bacillus May or May Not Produce β -Glucosidase Due to Their Environmental Origin – A Case Study

Microbial cellulases have been drawing attention worldwide because of their massive capacity to process the most abundant cellulosic biomass into sustainable biofuels and other valuable products. Profitable biomass conversion processes are highly dependent on the use of efficient enzymes for lignocellulose degradation. Among the cellulose degrading enzymes, β-glucosidases are essential for efficient hydrolysis of cellulosic biomass as they relieve the inhibition of the cellobiohydrolases and endoglucanases by reducing cellobiose accumulation. In this study cellulolytic bacteria with potential β-glucosidases activity were isolated and screened from biogas plant effluent and dairy effluent near Jahangirnagar University campus. From initial screening a total of 16 isolates were found to have cellulolytic activity, among them three isolates (B1, B5, D4) were selected based on their superior results. All the three bacterial isolates were identified as B. subtilis (B1), Bacillus amyloliquefaciens (B5) and B. subtilis (D4) respectively based on their morphological, biochemical and molecular characteristics. The β-glucosidases activity of these three potential cellulolytic bacteria was performed by measuring the release of PNP using pNPG as a substrate and interestingly D4 strain was resulted with β-glucosidases negative where B1 strain was found to have efficient for β-glucosidases activity.

Purification, characterization and application of novel alkaline protease from new Bacillus cereus UV-15 mutant

The alkaline protease produced by Bacillus cereus UV-15 mutant was purified by precipitation with ammonium sulphate and gel filtration through sephadex G-100. The enzyme has shown to have a molecular weight of 29kDa by SDS polyacrylamide gel electrophoresis. The extracted protease enzyme was purified by 16.64 fold through ammonium sulphate precipitation and chromatography separation in Sephadex G-100. The purified protease had a specific activity of 2915 (U/mg). The zymogram also revealed a clear hydrolytic zone due to proteolytic activity, which coincided with the band obtained with SDS–PAGE. The enzyme was remained active and stable at pH 8-11, with an optimum at pH 10.0. The protease was stable in the temperature ranging from 40°C to 60°C, but gradually decreased at temperature 70°C. The optimum temperature for protease activity was determined at 60°C. The enzyme showed stability towards non-ionic and anionic surfactants, and oxidizing agents. At 1% concentration of Tween-20 and Tween-80, the enzyme retained 78% and 94% relative activity respectively. Alkaline protease retained 95% activity toward 0.5% concentration of the anionic detergent SDS. The enzyme showed compatibility at 50°C with commercial detergents such as Ariel, Surf excel, Rin, wheel, Tide and Nirma. In the presence of Ariel and Rin the enzyme retained about 72 and 75% of the original activity respectively. The supplementation of the enzyme in detergents could improve the cleansing performance towards the blood stains and suggested to be used as a detergent additive. The enzyme also removed goat hide hairs completely after 15 hr of incubation. These characteristics may make the enzyme suitable for several industrial applications, especially in leather industries.

Production of biopolyester poly(3-hydroxybutyrate) by Bacillus cereus RCL 02, a leaf endophyte of Ricinus communis L.

Endophytic bacteria colonizing the internal tissues of plants have attracted the attention of scientific communities in recent years for production of biodegradable polyesters like polyhydroxyalkanotaes (PHAs). A newly characterized bacterium, Bacillus cereus RCL 02 (GenBank accession no. KX458035), isolated from surface sterilized leaves of Ricinus communis L. has been explored for the production of poly(3-hydroxybutyrate) [P(3HB)], the most common PHA. As revealed by scanning electron microscopy, P(3HB) accumulating cells developed swellings or blebs and released the native granules as a function of autolysis. During growth in glucose containing mineral salts medium under batch fermentation, the isolate produced P(3HB) accounting 68% of its cell dry weight (CDW). Glucose and yeast extract when used in the ratio of 5:1, significantly influenced intracellular biopolyester accumulation (72.2%, CDW and 2.54 g/L). A further increase of polymer production (81%, CDW and 3.17 g/L) was accomplished in presence of 1.5 mM manganese as exogenous metal stress. Moreover, supplementation of the growth medium with non-conventional carbon sources especially refined sugarcane molasses further enhanced the production of both biomass (9.44 g/L) as well as polyester (83.6%, CDW and 7.89 g/L). These finding emphasises exploration of endophytic bacteria of oleaginous plants in general and R. communis L. in particular as potential but hitherto an under exploited bioresource for commercial production of biodegradable polyesters.