Bioprospecting potential of microbial communities in solid waste landfills for novel enzymes through metagenomic approach.

Landfills are repository for complex microbial diversity responsible for bio-degradation of solid waste. To elucidate this complexity, samples from three different landfill sites of North India (sample V: Bhalswa near Karnal byepass road, New Delhi, India; sample T: Chandigarh, India and sample S3: Una, H.P., India) were analyzed using metagenomic approach. Selected landfill sites had different geographical location, varied in waste composition, size of landfill and climate zone. For comparison, one sample from high altitude (sample J) having less human interference was taken in this study. The aim of this study was to explore microbial diversity of communities responsible for degradation of landfill. Samples were characterized by 16S rRNA gene sequencing. Data from three landfill sites showed abundance of phylum Proteobacteria while less contaminated sample from high altitude showed abundance of phylum Cholroflexi followed by phylum Proteobacteria. The most abundant genus was unknown suggesting that these landfills could be repository for various novel bacterial communities. Sample T was relatively more active in terms of microbial activity. It was relatively abundant in enzymes responsible for dioxin degradation, styrene degradation, steroid degradation, streptomycin biosynthesis, carbapenem biosynthesis, monobactam biosynthesis, furfural degradation pathways while sample J was predicted to be enriched in plant cell wall degrading enzymes. Co-occurrence analysis revealed presence of complex interaction networks between microbial assemblages responsible for bio-degradation of hydrocarbons. The data provides insights about synergetic interactions and functional interplay between bacterial communities in different landfill sites which could be further exploited to develop an effective bioremediation process.

A novel approach to produce glucose from the supernatant obtained upon the dilute acid pre-treatment of rice straw and synergistic action of hydrolytic enzymes producing microbes.

The present work refers to a process involving the use of dilute nitric acid pretreatment and enzymatic hydrolysis for the transformation of rice straw into simple sugars. Acid pre-treated rice straw was separated into the pulp and supernatant through centrifugation and filtration. The two fractions are then converted into simple sugars by combined action of microbes producing cellulase and laccase enzymes. These microbes were isolated from soil samples which were collected from different locations with varying altitudes, expected to harbour microbes with high-hydrolysing activity. The nitric acid pretreatment was carried out at 30 °C, 200 rpm for 72 h. After 72 h, the culture supernatants were analysed for the presence of glucose with the help of HPLC. The supernatant fraction separated after the acid pre-treated rice straw produced highest amount of glucose (205 mg/g of rice straw) upon subsequent hydrolysis with synergistic action of cellulase and laccase-producing microbes.

Processing-Independent Extracellular Production of High Purity C-Phycocyanin from Spirulina platensis.

C-Phycocyanin (C-PC) is a natural blue colored protein of Spirulina platensis. Several methods based on chemical, physical, and enzymatic processes have been reported for the extraction of C-PC. However, most of the processes are either costly and/or time-consuming and/or produce C-PC with less purity. In view of this, a very simple and effective method was developed to extract C-PC with high purity and without involving any energy-consuming process from S. platensis. Wet biomass of S. platensis was harvested and incubated in an optimized 2-(N-morpholino)ethanesulfonic acid buffer with no supplement of any other compounds under dark, anaerobic, and still conditions. These conditions facilitated the leaching out of C-PC from the cells. The purity ratio of the blue color C-PC supernatant obtained was enhanced by precipitation and dialysis. The purity ratio of C-PC produced was 0.644 and increased to 1.345 upon simple dialysis of the crude C-PC. The purity of C-PC was 6.17 upon purifying through column chromatography. Hence, a simple process was developed for producing C-PC of much higher purity than therapeutic grade (>4). Furthermore, the molecular regulation of the extracellular release of C-PC from S. platensis cells was monitored through relative expression levels of α and ß subunits of C-PC genes during specific buffer vis-à-vis water as a control. Expression levels of genes encoding α and ß subunits of C-PC were found to be upregulated in the case of the 2-(N-morpholino)ethanesulfonic acid buffer treated algal cells compared to water treated algal cells. The higher level expression of these genes documented the higher and specific production of C-PC by the S. platensis upon 2-(N-morpholino)ethanesulfonic acid buffer treatment.

mbtJ: an iron stress-induced acetyl hydrolase/esterase of Mycobacterium tuberculosis helps bacteria to survive during iron stress.

AIM: mbtJ from Mycobacterium tuberculosis H37Rv is a member of mbt A-J operon required for mycobactin biogenesis. MATERIALS & METHODS: The esterase/acetyl-hydrolase activity of mbtJ was determined by pNP-esters/native-PAGE and expression under iron stress by quantitative-PCR. Effect of gene on growth/survival of Mycobacterium was studied using antisense. Its effect on morphology, growth/infection was studied in Mycobacterium smegmatis. RESULTS: It showed acetyl hydrolase/esterase activity at pH 8.0 and 50°C with pNP-butyrate. Its expression was upregulated under iron stress. The antisense inhibited the survival of bacterium during iron stress. Expression of mbtJ changed colony morphology and enhanced the growth/infection in M. smegmatis. CONCLUSION: mbtJ, an acetyl-hydrolase/esterase, enhanced the survival of M. tuberculosis under iron stress, affected the growth/infection efficiency in M. smegmatis, suggesting its pivotal role in the intracellular survival of bacterium.

Xylanase from Acinetobacter pittii MASK 25 and developed magnetic cross-linked xylanase aggregate produce predominantly xylopentose and xylohexose from agro biomass.

Most of the chemical and biochemical processes used for the de-polymerization of structural polymers of lignocellulosic biomass are environment unfriendly and costly. Here an efficient process based on xylanase, produced by Acinetobacter pittii MASK25 (MTCC 25132), hydrolysis of only physically treated rice straw and corn cob has been developed for the production of xylooligosaccharides. Bacterial strain isolated from soil was found to produce maximum xylanase at 30°C and pH 7. While the optimum temperature and pH of xylanase were characterized as 40°C and 5. Process was further improved by developing magnetic-xylanase CLEA. Crude xylanase and magnetic-xylanase CLEA could convert respectively more than 45% and 60% xylan of the powdered rice straw and corn cob into xylooligosaccharides. Interestingly, hydrolysis by both types of enzymatic forms was found to produce predominantly xylopentose and xylohexose. Hence, the process is environment friendly and the predominant production of xylopentose and xylohexose could find unique prebiotic applications.

mesT, a unique epoxide hydrolase, is essential for optimal growth of Mycobacterium tuberculosis in the presence of styrene oxide.

AIM: mesT of Mycobacterium tuberculosis, a hypothetical/putative epoxide hydrolase, is predicted to convert toxic epoxides to the more water-soluble and less toxic diols. Detailed characterization of the protein was carried out. RESULTS: mesT demonstrated esterase as well as epoxide hydrolase activity. It was membrane bound and was upregulated under hypoxic conditions. The enzyme was able to degrade styrene oxide. The presence of antisense against this gene resulted in the inhibition of in vitro bacterial growth/survival in the presence of styrene oxide. Conclusion & future perspective: We demonstrated that mesT possessed epoxide hydrolase activity and styrene oxide might be its physiological substrate. Inhibition of mesT reduced the growth of the bacteria in presence of styrene oxide and its expression under hypoxic condition suggested its role in intracellular survival of bacteria.