Effects of Structural Modification on the Thermostability of F1 Protease from Bacillus Stearothermophilus F1

A thermophilic Bacillus stearothermophilus F1 was found to produce an extremely thermostable serine protease. The F1 protease sequence was modeled onto the crystal structure of thermitase with 61% sequence identity. The F1 protease contains a catalytic triad comprising of Asp39, His72 and Ser226. The predicted structure of F1 protease comprised 10 α-helices and 10 β-sheets arranged in a single domain. Comparison of the predicted 3D structure of F1 protease with the crystal structure of serine proteases from mesophilic bacteria and archaea, led to the identification of features related to protein stabilization. Higher themostability was found to be correlated with an increased number of residues involved in ion pairs or networks of ion pair. In order to investigate F1 protease stability, two mutated (W200R and D58S) F1 protease were designed. The analysis of molecular dynamics simulations of W200R mutant revealed that an additional three new ion pairs between Arg200 and Asp202, however there was no ion pair interaction at position Ser58. To confirm the role of ion pair, site-directed mutagenesis was carried out. Both mutated F1 proteases were designed, cloned into pGEX-4T1 and expressed in E. coli BL21 (DE3) pLysS. The optimum expression level for the wild type F1 protease, W200R and D58S mutants were 94 U/mL, 112 U/mL and 68 U/mL, respectively. The wild type F1 protease, W200R mutant and D58S mutant were purified by affinity chromatography and heat-treatment. A single band was visible at SDS-PAGE at approximately 33.5 kDa. The purified wild type, W200R mutant and D58S mutant showed 95%, 115% and 64% recovery with purification fold of 21.7, 33.8, and 17.2, respectively. In the presence of 2 mM CaCl2, the wild type had half-lives of 60 min and 7 min at 85 ºC and 90 ºC. Meanwhile, the W200R mutant had half-lives of 75 min and 12 min at 85 °C and 90 ºC, which was more stable than wild type. The stability of W200R mutant was 1.25 times higher than that of the F1 protease. The enhanced thermostability can be correlated to the increase in the number of residues involving ion pairs and ion pairs networks. In contrast, the D58S mutant showed half-life of 45 min at 85 ºC but there was no enzymatic activity at 90 ºC. Thus, the D58S mutant was less thermostable than that wild type which could be due to the removal of ion pairs on this mutated F1 protease (computational work). Far-UV CD at 221 nm was used to detect the denatured proteins. As the temperature was increased from 50 °C to 90 °C, the change in ellipticity at 221 nm revealed a sigmoidal monophasic transition curve of mutant’s proteins which indicated unfolding of a protein.The melting point at pH 8.0 of the wild type F1 protease, W200R mutant and D58S mutant were 70 °C, 72 °C and 63 °C, respectively. Far-UV CD measurements studies indicated the overall features of the secondary structure of protein. The wild type contains 25.5% of α-helix, 17.7% of β-sheet, 22.5% of turn and 34.0% of random coil. There was some loss of β-sheet in W200R but a 3.8% and 1.7% increase in an α-helix and a random coil, respectively, indicated that Arg200 stabilized the α-helix content. The β-sheet was reduced to less than 4% and random coil rose up to more than 8%. A small decrease in the α-helix was observed in D58S mutant. This could be due to the substitution of Asp58 to Ser causing disruption of four ion pairs between Asp58 and Arg103, located at β-sheet. The results obtained confirmed the important role of intermolecular ion pairs in the stability of the whole structure of F1 protease. These results also showed that simulation procedures and molecular biology techniques can be used together to direct protein engineering and/ or site-directed mutagenesis

Effect of 1-butyl-3-methyl-imidazodium chloride (BMIMCl) pretreatment on structural and glucose yield of the rice husk

Ionic liquid (IL) are of great interest as solvents for production of fuels from lignocellulosic biomass. The aim of this research is to determine the effect of ionic liquid, 1-butyl-3-methylimidazolium (BMIMCl) pretreatment on rice husk (Oryza sativa) based on it structural changes and glucose yield production. The pretreatment was conducted by heating 5% (w/w) rice husk in BMIMCl solution at 80 °C for 48 hours. The structural changes of regenerated rice husk were observed and characterized using X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). It was found that the regenerated rice husk was less crystalline and higher amorphous upon BMIMCl treatment. The total sugar yield before and after fermentation by saccharomyces cerevisiae was analysed using dinitrosalicyclic acid (DNS) method. The regenerated rice husk produces higher total sugar yield compared with untreated rice husk

Wax removal in textile manufacturing wastewater using surface modification of rice straw

In this study, the effect of modification surface on lignocellulosic adsorbent (rice straws) was developed and examined. The surface of lignocellulosic adsorbent was treated with Cetylpyridinium chloride (CPC) solution, sodium hydroxide (NaOH) solution and mixture of NaOH and CPC solution at 90 ± 5°C with different chemical treatments method. The surface morphologies of treated and untreated rice straws were observed by Scanning Electron Microscope (SEM). The treated rice straws adsorb more wax compared to untreated rice straws

Thermostable proteases

New proteases are constantly isolated. Proteases, mainly the alkaline proteases are widely used in the detergent formulation. Thermostable alkaline proteases are of great interest in the detergent industry because these enzymes are stable and able to retain its activities over broad range of pH and temperatures. Most thermostable alkaline proteases reported are from Bacillus spp. In this chapter, we introduce a new thermostable alkaline protease produced from Bacillus stearothermophilus strain F1. The protease production requirements by strain F1, the properties and characterization of this enzyme are discussed. The protease F1 gene was successfully cloned and expressed into E. coli XL1-Blue. The bacteriocin release protein(BRP)system was utilized; to release the recombinant F1 protease into the culture medium. The purified native and recombinant F1 protease showed a pH optimum of 9.0 and thermostabilities ranging from a half-life of 25 min at 90◦C to a half-life of 4 h at 85◦C. The optimumtemperatures were 85◦C and 80◦C respectively

Structural conformation of Bacillus stearothermophilus F1 protease and effect of modification on its thermostability

The extracellular F1 serine protease, produced by a thermophilic Bacillus stearothermophilus F1, has been isolated and characterized as one of a serine protease. F1 protease was stable in the pH range of 8.0 to 10.0, with an optimum activity at pH 9.0. The enzyme was stable for 24h at 70°C (Rahman et al., 1994)

Knowledge and Attitudes of Maintaining Bone Health among Post-Menopausal Women in Malaysia

Introduction: Public awareness of osteoporosis is low among women in the developing countries. Health education was shown to be effective in improving knowledge and awareness on maintaining bone health. This study aims to identify the level of knowledge and attitudes among post-menopausal women in Malaysia on achieving bone health throughout the menopausal transition period.Methods: A total of 116 post-menopausal female patients of orthopedic menopause clinic were recruited using a purposive sampling approach. Data on osteoporosis awareness and knowledge were collected using validated structured questionnaires Osteoporosis Prevention and Awareness Tool and Osteoporosis Attitude Knowledge Test. The chi-square test was used to determine the association between post-menopausal women’s socio-demographic characteristics and their knowledge and attitude towards maintaining bone health.Results: Participants’ age ranged between 49 and 82 years (61.84, SD=7.87). The knowledge of osteoporosis varied significantly by age (p=0.014) and education (p=0.001) among the studied population. No significant diffrences were found for participants’ attitude towards bone health.Conclusion: This study showed that the age and education levels have significantly different knowledge of bone health

Fracture risk prediction in post-menopausal women with osteopenia and osteoporosis: preliminary findings

Objective: The study aims to identify the risk of obtaining a fracture among post-menopausal women with osteopenia and osteoporosis. Method: This work was a cross-sectional study involving a purposive sample of 87 post-menopausal women who attended the orthopedic and menopause clinics of Hospital Tengku Ampuan Afzan, Kuantan. The data were entered into the WHO fracture risk assessment tool (FRAX®) to predict major fracture and risk for hip fracture in 10 years’ time. Results: The mean age of the respondents was 61.6 years (SD = 7.9). Among the respondents, 50.6% had osteopenia and nearly half (48.3%) had osteoporosis. The mean number of menopausal years of the respondents was 11.9 (SD = 8.5), ranging between 1 and 44 years. The FRAX findings indicated 9.7% major osteoporotic fracture probability and 3.5% hip fracture probability, which were denoted as high risk. A Pearson correlation coefficient was computed to assess the relationship between menopausal years and the FRAX major osteoporotic fracture probability. A significant positive correlation was found between the two, but the correlation was weak (r = 0.581, n = 87, p < 0.001). Conclusions: The present findings indicate that menopausal years have a positive correlation with the risk of obtaining a fracture

Bone health status among postmenopausal women in Malaysia

Objectives: The study has evaluated bone health status among post-menopausal women in a public hospital of Malaysia. Materials and Methods: A total of 116 post-menopausal women, who met the criteria, participated in this study. The purposive sampling method was used to achieve the criteria of participants. Results: This cross-sectional study revealed that more than half of the respondents (52.6%, n=61) had osteopenia; whereas, 47.4% (n=55) of the respondents had osteoporosis. There was a significant correlation between the age and menopausal years with the bone health status (i.e. P=0.004 and P=0.028, respectively) in postmenopausal women. Conclusions: Menopausal women experienced deterioration of bone mineral density (BMD) with advancing age and menopausa

Zero-valent iron nanoparticles for environmental Hg (II) removal: a review

Mercury is a natural, long-lasting, and bio-accumulative contaminant found in both soil and water. Mercury is toxic and its organic derivative, methylmercury (MeHg), could be lethal. The increasing level of mercury in the environment is a threat, as it can easily enter the food chain upon exposure. Zero-valent iron nanoparticle (nZVI), an environmentally friendly nanomaterial, is envisaged as an ideal candidate for the remediation of metal pollutions in soil and water bodies. Due to low toxicity and decent activity, nZVI and its corrosion products have shown huge potential for the removal of heavy metals from soil and water. It has been widely applied for the removal of heavy metals including mercury and other organic and inorganic contaminants. In this review, the current preparation methodology, characterization techniques, reductive mechanism for heavy metal removal with focus on mercury is reviewed. This review discusses the use of nZVI for the removal of mercury and demonstrates that nZVI possesses high reactivities for mercury removal and have great application prospects in environmental remediation. Some recommendations are proposed and conclusions drawn for future research