Current study was undertaken to carry out the genome-wide analysis of a multipotent isolate from desert soil which was previously identified as
Bacillus tequilensis
based on 16S rDNA analysis. This ...study also aims to characterize the serine protease and its biocatalytic potentials implying a combination of empirical and in-silico approaches. Next generation sequencing and short read de novo assembly generated the 4,235,084 bp draft genome of
Bacillus
sp. ZMS-2. Genome sequence analysis by digital DNA:DNA hybridization (dDDH) and average nucleotide identity classified the isolate as
Bacillus subtilis ZMS-2
(Bioproject ID: PRJNA691551)
.
Genome annotation revealed 10 antibiotic resistance genes, 8 antibiotic/antifungal gene clusters and 25 genes encoding proteases including subtilisin E, an extracellular alkaline protease. This extracellular protease (ZMS-2 protease) was produced using a statistically optimized medium, purified partially and characterized as alkaline serine protease. The partially purified ZMS-2 protease (780 U/mL) showed a 21 mm zone of casein hydrolysis and dehaired goat skin by pulling out hair with roots. These catalytic potentials of ZMS-2 protease were further confirmed using scanning electron microscopy of casein beads and dehaired skin. The study concludes
B
.
subtilis
ZMS-2 as a potent producer of a protease with promising potentials of commercial importance.
Proteolytic enzymes are the most versatile and commercially viable group of enzymes comprising over 65% share in the global enzyme market amongst which alkaline proteases have extensive applications ...in detergent and leather industry. Current study was designed to assess the potential of an alkaline serine protease from Bacillus subtilis ZMS-2 as a bating agent in leather processing. Initially, the production parameters were investigated through Response Surface Methodology (RSM) using Plackett-Burman Design, which identified substrate, agitation speed and incubation temperature as the most significant factors. The optimal levels of these factors were determined through the Box-Behnken experimental analysis as 0.436% substrate concentration, 36.5 °C incubation temperature and 56 rpm agitation speed. The statistical optimization experiments increased the volumetric production of enzyme by 3.94 times (2246 U mL−1) than the initial titer (571 U mL−1). The enzyme was partially purified and characterized as metal ions and detergent compatible serine protease having optimum activity at pH 8 and 60 °C. During the pilot-scale application as a bating agent, the enzyme (340 U mL−1) successfully removed the hair roots and other unwanted proteins from goat skins as observed during scudding and confirmed through Scanning Electron Microscopy. The processed skins displayed enhanced porosity, thumb impression, smoothness and pliability. These findings provide a strong basis for the use of this protease as an efficient and eco-friendly alternative for bating of animal skins in leather tanneries.
Microbial populations within the rhizosphere have been considered as prosperous repositories with respect to bioremediation aptitude. Among various environmental contaminants, effluent from textile ...industries holds a huge amount of noxious colored materials having high chemical oxygen demand concentrations causing ecological disturbances. The study was aimed to explore the promising mycobiome of rhizospheric soil for the degradation of azo dyes to develop an efficient system for the exclusion of toxic recalcitrants. An effluent sample from the textile industry and soil samples from the rhizospheric region of Musa acuminata and Azadirachta indica were screened for indigenous fungi to decolorize Congo red, a carcinogenic diazo dye, particularly known for its health hazards to the community. To develop a bio-treatment process, Aspergillus terreus QMS-1 was immobilized on pieces of Luffa cylindrica and exploited in stirred tank bioreactor under aerobic and optimized environment. Quantitative estimation of Congo red decolorization was carried out using UV-Visible spectrophotometer. The effects of fungal immobilization and biosorption on the native structure of Luffa cylindrica were evaluated using a scanning electron microscope. A. terreus QMS-1 can remove (92%) of the dye at 100 ppm within 24 h in the presence of 1% glucose and 1% ammonium sulphate at pH 5.0. The operation of the bioreactor in a continuous flow for 12 h with 100 ppm of Congo red dye in simulated textile effluent resulted in 97% decolorization. The stirred tank bioreactor was found to be a dynamic, well maintained, no sludge producing approach for the treatment of textile effluents by A. terreus QMS-1 of the significant potential for decolorization of Congo red.
Kohl (Surma/Kajal) is a widely used eye cosmetic having traditional and religious importance in subcontinent and other parts of the world since ancient times. However, the use of Kohl has become ...hazardous for human health in recent times due to its changing compositions. In present study, we studied the antimicrobial potential of Kohl, presence of microbial pathogens and chemical contaminants in twenty Kohl samples collected from Karachi city, Pakistan. The samples included 85% branded and 15% unbranded Kohl samples. Our results showed that 75% of the samples possess higher antibacterial activity, while 30% of the samples showed antifungal potential. Moreover, pathogenic Bacillus and Aspergillus species were isolated from these Kohl samples as major microbial contaminants. Higher levels of arsenic, lead and cadmium were detected in 80%, 35% and 30% of the Kohl samples respectively, when compared against the standard limits. Current findings showed that presence of toxic metals and microbial pathogens in Kohl samples is hazardous for humans. Furthermore, Kohl shall not be used until the safe limits of carcinogenic metals and other contaminants in the Kohl are certified by the manufacturers.
Due to industrialization and over population, surface water resources are out of reach from many people so consumption of ground water is the only choice to overcome the water scarcity. Naturally, ...ground water is one of the significant and potable water resource but some geographical conditions and anthropogenic activities deteriorate the water quality and make it objectionable for drinking. This study was conducted to evaluate the ground water quality of Karachi, Pakistan. For this, 42 ground water samples were collected from different districts of Karachi and analyzed their physicochemical and microbiological characteristics and compared with both international (WHO) and national (SEQS) drinking water standards. Observations of the study declared that overall contamination (physicochemical and microbial) in the ground water samples of different districts of Karachi was as follow West (21%), South (20%), Central (17%), Malir (16%), Korangi (14%) and East (12%). Physical assessment of the study area declared that pH and turbidity of the ground water samples varies in the range of (6.54-7.9) and (0-1.01 NTU) which exist in the standard prescribed limit. Whereas, detection of chemical contaminants particularly TDS (457-12090 mg/L), hardness (118.8-3645 mg/L) and chloride (190-4918 mg/L) content in most of the samples were also exceed from the prescribed limit. Additionally, arsenic was abundantly present ranging from 3.52-13.63 mg/L in all collected samples of Karachi city while the concentration of cadmium (range: 0.0005-0.5012 mg/L) and lead (range: 0.201-1.817 mg/L) were also high in few samples, from the permissible limit of drinking water. Microbial contamination was also detected in which coliforms were present in the range of 0-150 CFU/100mL, which also unfit the water quality. This deteriorated ground water quality of Karachi can be improved by maintenance of proper sanitary conditions of the communities and implementation of water treatments, otherwise consumption of such water may develop serious health related consequences in the consumers.
Serine proteases are important enzymes widely used in commercial products and industry. Recently, we identified a new serine protease from the desert bacterium Bacillus subtilis ZMS-2 that showed ...enhanced activity in the presence of Zn2+, Ag+, or H2O2. However, the molecular basis underlying this interesting property is unknown. Here, we report comparative studies between the ZMS-2 protease and its homolog, subtilisin E (SubE), from B. subtilis ATCC 6051. In the absence of Zn2+, Ag+, or H2O2, both enzymes showed the same level of proteolytic activity, but in the presence of Zn2+, Ag+, or H2O2, ZMS-2 displayed increased activity by 22%, 8%, and 14%, whereas SubE showed decreased activity by 16%, 12%, and 9%, respectively. In silico studies showed that both proteins have almost identical amino acid sequences and folding structures, except for two amino acids located in the protruding loops of the proteins. ZMS-2 contains Ser236 and Ser268, whereas SubE contains Thr236 and Thr268. Replacing Ser236 or Ser268 in ZMS-2 with threonine resulted in variants whose activities were not enhanced by Zn2+ or Ag+. However, this single mutation did not affect the enhancement by H2O2. This finding may be used as a basis for engineering better proteases for industrial uses.
Zn2+, Ag+, or H2O2 increased the activity of ZMS-2 protease but decreased the activity of subtilisin E (SubE). They are different in only two amino acid residues located in the protruding loops of the proteins. Site-directed mutagenesis confirmed the role of Ser236 and Ser268 in the increased activity of ZMS-2. Display omitted
•ZMS-2 and subtilisin E showed opposite responses toward Zn2+, Ag+, or H2O2.•There are only two amino acids different between the two proteins.•Ser236 and Ser268 in ZMS-2 are replaced by Thr236 and Thr268 in subtilisin E.•They are located in the protruding loops of the proteins.•Site-directed mutagenesis confirmed their roles in the observed opposite responses.
Microbial alkaline proteases are dominating the global enzyme market with a share of over 65% due to their multifarious catalytic potentials. Hence, production of proteases with novel properties of ...commercial significance is highly desirable to meet the global enzyme demand. Here, we report the purification, characterization, and pilot-scale application of a serine protease from the desert soil bacterium Bacillus subtilis ZMS-2 with novel properties as dehairing agent in leather processing. The enzyme was purified 16.5-fold with a specific activity of 1543.5 U mg-1 and recovery percentage of 33.6% using ammonium sulfate precipitation, ion exchange, and gel filtration chromatography. The purified enzyme was characterized as a metal ion-, surfactant-, and denaturant-compatible alkaline serine protease having a molecular weight of 36.1 kDa with an optimum activity at pH 8.5 and 60 °C. The catalytic activity of the enzyme was enhanced by Zn+2 (204%), Ag+ (110%), H2O2 (123%), Triton X-100 (110%), iso-octane (109%), chloroform (110%), ethanol (105%), ethyl acetate (110%), and acetonitrile (128%). During pilot-scale applications, the optimum condition was found to be a combination of enzyme (1.5%, 460 U mL-1), sodium sulfide (2%), and calcium hydroxide (lime) (3%). Under this condition, the time required for complete dehairing was 90 min. Chemoenzymatically processed skins exhibited better physical properties than chemically processed skin, including tensile strength (16.35 ± 6.68 N/mm), ball burst (452.88 ± 6.06 N/mm), percent elongation (38.85 ± 1.06 N), tear strength (50.16 ± 4.42 N/mm), and softness (6.5 mm). Electron microscopy analysis of the treated skin showed complete removal of hairs with roots, confirming the keratin specificity of the enzyme. Moreover, the enzyme-assisted dehairing process reduced chemical oxygen demand (COD), biochemical oxygen demand (BOD), total dissolved solids (TDS), and total suspended solids (TSS) by 68, 77, 34, and 39%, respectively. Thus, the alkaline serine protease from B. subtilis ZMS-2 is a potential dehairing agent for the eco-friendly processing of animal skins on industrial scales.
Candidiasis is a significant fungal infection with high mortality and morbidity rates worldwide. Candida albicans is the most dominant species responsible for causing different manifestations of ...candidiasis. Certain virulence traits as well as its resistance to antifungal drugs contribute to the pathogenesis of this yeast. This study was designed to determine the production of some virulence factors, such as biofilm formation and extracellular hydrolytic enzymes (esterase, coagulase, gelatinase, and catalase) by this fungus, as well as its antifungal resistance profile. A total of 304 clinical C. albicans isolates obtained from different clinical specimens were identified by a conventional diagnostic protocol. The antifungal susceptibility of C. albicans strains was determined by disk diffusion technique against commercially available antifungal disks, such as nystatin 50 μg, amphotericin B 100 unit, fluconazole 25 μg, itraconazole 10 μg, ketoconazole 10 μg, and voriconazole 1 μg. The assessment of biofilm formation was determined by the tube staining assay and spectrophotometry. Gelatinase, coagulase, catalase, and esterase enzyme production was also detected using standard techniques. A total of 66.1% (201/304) and 28.9% (88/304) of C. albicans strains were susceptible-dose dependent (SDD) to nystatin and itraconazole, respectively. Among the antifungal drugs, C. albicans strains showed high resistance to ketoconazole 24.7% (75/304); however, no statistically significant relationship between the clinical origin of C. albicans isolates and antifungal drug resistance pattern was detected. For virulence factors, the majority of the C. albicans strains actively produced biofilm and all hydrolytic enzymes. Biofilm formation was demonstrated by 88% (267/304) of the strains with a quantitative mean value 0.1762 (SD ± 0.08293). However, 100% (304/304) of isolates produced catalase enzyme, 69% (211/304) produced coagulase, 66% (197/304) produced gelatinase, and 52% (157/304) produced esterase enzyme. A significant relationship between the source of specimens and biofilm formation by C. albicans was observed; nevertheless, there was no significant relationship between different sources of C. albicans strains and the production of different enzymatic virulence factors. The study found that C. albicans strains have excellent potential to produce virulence markers and resistance to antifungals, which necessitates surveillance of these opportunistic pathogens to minimize the chances of severe invasive infections.
•C.albicans of clinical origin shows increasing resistance against antifungals, particularly ketoconazole.•The isolates from urine and high vaginal swabs are more resistant to antifungals.•Biofilm formation and catalase production are the predominant virulence traits among these isolates.
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