Unveiling the genomic potential of a novel thermostable glycoside hydrolases producing Neobacillus sedimentimangrovi UE25.

Genetic and enzymatic potential of Neobacillus sedimentimangrovi has not been assembled to date. Here, we report a high-quality genome assembly of thermophilic bacterium Neobacillus sedimentimangrovi UE25 using Illumina Hi-seq 2500. The strain was isolated from a crocodile pond Manghopir, Karachi, Pakistan. QUAST quality parameters showed 37.75% GC content and exhibited the genome into 110 contigs, with a total size of 3,230,777 bases. Genome of N. sedimentimangrovi UE25 harbors phage mediated DNA through horizontal gene exchange from the phages, symbiotic and pathogenic bacteria. Most of the phage genome encodes for hypothetical proteins, protease, and phage assembly proteins. Gene clusters encoding the intrinsic resistance to glycopeptides, isoniazid, rifamycin, elfamycin, macrolide, aminoglycosides, tetracycline and fluoroquinolone were identified into the genome. Since, the strain has been reported for the production of many industrially important thermostable enzymes, therefore, the genomic data of thermostable enzymes might be helpful to employ this species in commercial sectors. Probing genes of multiple thermostable glycoside hydrolase enzymes especially xylanases of N. sedimentimangrovi UE25 showed genetic diversity among the genes and confer the industrial importance of this microorganism. Furthermore, the genome of N. sedimentimangrovi will greatly improve our understanding of its genetics and evolution.

Screening for toxigenic Escherichia coli in stool samples of diarrhoeal patients by polymerase chain reaction.

Escherichia coli (E. coli) are normal flora of the intestines of most animals, including humans. Most strains are harmless and beneficial to host by preventing the establishment of pathogenic bacteria within the intestine. However, some E. coli strains can cause a wide variety of intestinal and extra-intestinal diseases, such as diarrhoea, urinary tract infections, septicaemia, neonatal meningitis and renal complications. Several virulence factors including toxins, adhesins, serine proteases, etc. have been reported in these highly adapted clones. The present study was designed to enumerate toxin genotype through PCR assay in local clinical isolates of E. coli. A total of 37 E. coli strains were collected from different clinical laboratories of Karachi and examined for the presence of shiga toxin 1 (stx1) and shiga toxin 2 (stx2) genes of Eenterohemorrhagic E. Coli (EHEC) and heat stable (st) and healt labile (lt) toxin genes of enterotoxigenic E. Coli (ETEC). It was observed that 16 strains out of 37 carried one or more type of toxin genes. The presence of stx1 gene was significantly higher as it was positive in 10 isolates compared to others toxins. Two in above stx1 positive strains were also carrying for stx2 gene. Six out of 37 isolates were positive for lt gene, and none of the strains are carrying st gene. Although, the study was carried out with fewer isolates, yet it demonstrated the trend of dispersion of toxin genes and findings can be used to correlate the gastro-intestinal infections and their complications in Pakistan.

Purification and characterization of novel α-amylase from Bacillus subtilis KIBGE HAS.

Purification of extracellular α-amylase from Bacillus subtilis KIBGE HAS was carried out by ultrafiltration, ammonium sulfate precipitation and gel filtration chromatography. The enzyme was purified to homogeneity with 96.3-fold purification with specific activity of 13011 U/mg. The molecular weight of purified α-amylase was found to be 56,000 Da by SDS-PAGE. Characteristics of extracellular α-amylase showed that the enzyme had a Km and V (max) value of 2.68 mg/ml and 1773 U/ml, respectively. The optimum activity was observed at pH 7.5 in 0.1 M phosphate buffer at 50 °C. The amino acid composition of the enzyme showed that the enzyme is rich in neutral/non polar amino acids and less in acidic/polar and basic amino acids. The N-terminal protein sequence of 10 residues was found to be as Ser-Ser-Asn-Lys-Leu-Thr-Thr-Ser-Trp-Gly (S-S-N-K-L-T-T-S-W-G). Furthermore, the protein was not N-terminally blocked. The sequence of α-amylase from B. subtilis KIBGE HAS was a novel sequence and showed no homology to other reported α-amylases from Bacillus strain.

Wild Halophytic Phragmites karka Biomass Saccharification by Bacterial Enzyme Cocktail.

Biofuel derived from halophytic biomass is getting attention owing to the concerns of energy versus food crisis. The disadvantages associated with edible bioenergy resources necessitate the need to explore new feedstocks for sustainable biofuel production. In this study, biomass from locally available abundant halophytes (Panicum antidotale, Phragmites karka, Halopyrum mucronatum, and Desmostachya bipinnata) was screened for saccharification by an enzyme cocktail composed of cellulase, xylanase, and pectinase from Brevibacillus borstelensis UE10 and UE27, Bacillus aestuarii UE25, Aneurinibacillus thermoaerophilus UE1, and Bacillus vallismortis MH 1. Two types of pretreatment, i.e., with dilute acid and freeze-thaw, were independently applied to the halophytic biomass. Saccharification of acid-pretreated P. karka biomass yielded maximum reducing sugars (9 mg g-1) as compared to other plants. Thus, the factors (temperature, pH, substrate concentration, and enzyme units) affecting its saccharification were optimized using central composite design. This statistical model predicted 49.8 mg g-1 of reducing sugars that was comparable to the experimental value (40 mg g-1). Scanning electron microscopy and Fourier-transform infrared spectroscopy showed significant structural changes after pretreatment and saccharification. Therefore, halophytes growing in saline, arid, and semi-arid regions can be promising alternative sources for bioenergy production.

Banana Peels: A Promising Substrate for the Coproduction of Pectinase and Xylanase from Aspergillus fumigatus MS16.

Banana peels (BP), an under-utilized waste material, was studied for the production of xylanase and pectinase by Aspergillus fumigates MS16. The factors affecting the co-production of both the enzymes were separately studied for their influence under submerged (Smf) and solid-state fermentation (SSF) of BP. The strain was cultivated in the presence of mineral salt (MS) solution containing BP powder as a sole source of carbon and physical and nutritional factors varied to observe the change in the enzyme titers. The data revealed that the MS-based medium was appropriate for the production of both the enzymes; therefore, in subsequent experiments, the same medium was used. A temperature of 30-35°C was found better for the production of the two enzymes under Smf; however, the titers of pectinase dropped significantly at 40°C. Contrarily, xylanase production was inhibited at 40°C under SSF but not under Smf. Whereas, supplementation of xylan or pectin to BP induced the production of xylanase and pectinase, respectively. Lowering the pH value favored the production of both the enzymes under Smf; however, the production of pectinase improved significantly when a higher concentration of BP (1%) was used compared to the concentration (0.25%) required for the production of xylanase. Interestingly, the enzyme preparation obtained under SSF exhibited optimal activities of both the enzymes at higher temperatures when compared to those obtained under Smf. The data indicated that the physiology of the fungus differed greatly when the cultivation pattern varied from Smf to SSF and, hence, the enzymes produced were characteristically distinct.Banana peels (BP), an under-utilized waste material, was studied for the production of xylanase and pectinase by Aspergillus fumigates MS16. The factors affecting the co-production of both the enzymes were separately studied for their influence under submerged (Smf) and solid-state fermentation (SSF) of BP. The strain was cultivated in the presence of mineral salt (MS) solution containing BP powder as a sole source of carbon and physical and nutritional factors varied to observe the change in the enzyme titers. The data revealed that the MS-based medium was appropriate for the production of both the enzymes; therefore, in subsequent experiments, the same medium was used. A temperature of 30­35°C was found better for the production of the two enzymes under Smf; however, the titers of pectinase dropped significantly at 40°C. Contrarily, xylanase production was inhibited at 40°C under SSF but not under Smf. Whereas, supplementation of xylan or pectin to BP induced the production of xylanase and pectinase, respectively. Lowering the pH value favored the production of both the enzymes under Smf; however, the production of pectinase improved significantly when a higher concentration of BP (1%) was used compared to the concentration (0.25%) required for the production of xylanase. Interestingly, the enzyme preparation obtained under SSF exhibited optimal activities of both the enzymes at higher temperatures when compared to those obtained under Smf. The data indicated that the physiology of the fungus differed greatly when the cultivation pattern varied from Smf to SSF and, hence, the enzymes produced were characteristically distinct.