The aim of present study was the production of protease from local Bacillus subtilis through solid state fermentation. Response Surface Methodology (RSM) was used for the optimization of all the culture conditions. Casein (1% w/v) was used as a substrate in nutrient agar medium for the screening of enzyme production potential and showed maximum zone of clearance (4.6 cm). It was identified as Bacillus subtilis K-5 by genetic identification based on 16S rRNA and blast technology of NCBI. Among culture conditions, incubation temperature, incubation time, pH of the medium and moisture level of the substrate were optimized. Maximum protease production was observed at 37^oC, pH 9.0 with incubation time of 36 h and moisture to substrate ratio of 1: 0.75. Maximum protease production of 70.21 U/mL was recorded when wheat bran was used as an agro-industrial substrate. The activity of crude protease was observed 99.63 % at 60^oC and pH 10.0 with protein concentration 0.63 mg/mL and specific activity of 111.56 U/mg. Protein contents of 0.57 mg/mL (specific activity of 124.72 U/mg) and protein contents of 0.44 mg/mL (specific activity of 143.65 U/mg) were observed by 70% saturation with ammonium sulphate and gel chromatography, respectively. Line Weaver Burk plot was used to find its Vmax and Km, which were 344. 83 mg/mL/min and 100.04 mg/mL, respectively. The study concluded that Bacillus subtilis K-5 is thermophilic and alkaliphilic strain which produces active protease and can be used as potential microorganism for industries.

Aspergillus flavus was used to produce alkaline protease. Solid state fermentation (SSF) strategy was adopted to explore the most favorable physical and nutritional conditions for enzyme production. Maximum production was achieved at pH 6.0 and a temperature of 30 °C after 84 h of growth period. For the optimization of the chemical parameters, different carbon and nitrogen sources were used including glucose, fructose, sucrose, ammonium sulphate, and urea. Maximum production was observed with 0.3% concentration of all the compounds. Ammonium sulphate salting out and gel chromatography was used to purify the enzyme. The enzyme was completely precipitated out at 80% saturation. The value of Vmax was 3.9 U/mL, while the value of Km was 1.9 mg/mL. The enzyme was tested for its compatibility with a few famous detergents available on the market. With the alkaline protease under study, the enzyme displayed a maximum retention of its activity i.e. 80.8% in the presence of commercial detergent Surf excel. The activity dropped down to 61.5% when the enzyme was allowed to work in the presence of another locally used detergent, i.e., Bonus. Protease production from A. flavus was carried out on rice bran and wheat bran and the wheat bran gave better results.

Many agents, including those from herbal sources, have been sought as preventives or cures for hepatotoxicity. The pollen of Pinus brutia Ten., known as red pine (Pinaceae), is used against liver diseases in Anatolian folk medicine. In the current study, pollen ethanol extract of P. brutia was investigated for its possible hepatoprotective activity using a mouse model of CCl4-induced hepatotoxicity. Swiss albino mice were divided into five groups, and extract-treated groups were compared with a silymarin-treated group as the reference. The extract was tested at 100, 200, and 300 mg/kg (b.w.). Phenolic acids were analyzed using high-performance column chromatography (HPLC) in the extracts as pollens are usually known to be rich in phenolics. Our data revealed that the extract displayed the best hepatoprotection at a dose of 100 mg/kg when compared with silymarin (Legalon®), the reference drug. HPLC analysis indicated presence of protocatechuic acid (0.176 mg/g extract), p-hydroxybenzoic acid (0.001 mg/g extract), vanillic acid (VA) (0.537 mg/g extract), syringic acid (0.050 mg/g extract), and tr-cinnamic acid (0.310 mg/g extract), while the major phenolic acid was VA. The outcomes of this study allow us to conclude that red pine pollen extract can serve as a promising hepatoprotective agent. Among the phenolic acids analyzed in the pollen extract, vanillic acid as the major one besides some other phenolic acids detected seems to be responsible for its remarkable hepatoprotective effect.

The world population is genetically predisposed to metabolic syndrome (MetS) and its components, also known as cardiometabolic risk phenotypes, which can cause severe health complications including coronary heart disease (CHD). Genetic variants in the 9p21 locus have been associated with CHD in a number of populations including Pakistan. However, the role of the 9p21 locus in MetS and cardiometabolic risk phenotypes (such as obesity, hypertension, hyperglycemia, and dyslipidemia) in populations with CHD or no established CHD has not been explored. Therefore, the present study was designed to explore the association of the minor/risk allele (C) of 9p21 locus SNP rs1333049 with MetS or its risk phenotypes regardless of an established CHD, in Pakistani subjects. Genotyping of rs1333049 (G/C) was performed on subjects under a case-control study design; healthy controls and cases, MetS with CHD (MetS-CHD⁺) and MetS with no CHD (MetS-CHD⁻), respectively. Genotype and allele frequencies were calculated in all study groups. Anthropometric and clinical variables (Means ± SD) were compared among study groups (i.e., controls, MetS + CHD and MetS-CHD) and minor/risk C allele carriers (GC + CC) vs. non-carriers (Normal GG genotype). Associations of the risk allele of rs1333049 SNP with disease and individual metabolic risk components were explored using adjusted multivariate logistic regression models (OR at 95% CI) with a threshold p-value of ≤0.05. Our results have shown that the minor allele frequency (MAF) was significantly high in the MAF cases (combined = 0.63, MetS-CHD⁺ = 0.57 and MetS-CHD⁻ = 0.57) compared with controls (MAF = 0.39). The rs1333049 SNP significantly increased the risk of MetS, irrespective of CHD (MetS-CHD⁺ OR = 2.36, p < 0.05 and MetS-CHD⁻ OR = 4.04, p < 0.05), and cardiometabolic risk phenotypes; general obesity, central obesity, hypertension, and dyslipidemia (OR = 1.56–3.25, p < 0.05) except hyperglycemia, which lacked any significant association (OR = 0.19, p = 0.29) in the present study group. The 9p21 genetic locus/rs1333049 SNP is strongly associated with, and can be a genetic predictor of, MetS and cardiometabolic risks, irrespective of cardiovascular diseases in the Pakistani population.

The current study was performed to optimize three different industrial textile effluent biodegradation potentials of a brown rot fungus, Piptoporus betulinus IEBL-3, to reduce environmental pollution. The Response Surface Methodology under the Box Bhenken Design was used for the optimization steps. Three ligninolytic enzymes named lignin peroxidase, manganese peroxidase and laccase were also studied during the biodegradation process. The biodegradation rate of the 3 industrial effluents varied between 67 and 76% at the initially optimized conditions. There was a 10%, 7% and 9% increase in the biodegradation of Mujahid textile (MT), Five Star textile (FST) and Sitara textile (ST) effluent, respectively, after the addition of various additional carbon and nitrogen sources in different ratios. The biological treatment decreases the Biological Oxygen Demand and Chemical Oxygen Demand values of the effluents well below the WHO-recommended values for the industrial effluents. The HPLC monitoring of the effluent’s biodegradation showed the appearance of new peaks, some of which may correspond to secondary amines. Study of ligninolytic enzymes during the biodegradation process confirmed their role in the biodegradation process, with lignin peroxidase having highest activity among the others. These findings suggest that P. betulinus is a potential fungus for the biodegradation of the dyes and effluents and can be a suitable candidate for this process.