Faculty of Applied Sciences
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Item Microbial production of phytases for combating environmental phosphate pollution and other diverse applications(Taylor and Fancis Online, 2016) Kumar, Ashwani; Chanderman, Ashira; Makolomakwa, Melvin; Perumal, Kugen; Singh, SurenConcerns of phosphorus pollution and its impact on environments have driven the biotechnological development of phytases. Phosphoric acid, inositol phosphate, or inositols are produced after hydrolysis of phosphate from phytate, initiated by phytase. Research over the last two decades on microbial phytases has deepened our understanding of their production, optimization, and characterization. Despite the wide availability of phytase producing microorganisms, only a few have been commercially exploited. The current high cost of phytases, inability to withstand high temperatures (>85 C), a limited pH range, and poor storage stability are a major bottleneck in the commercialization of phytases. The development of novel phytases with optimal properties for various applications is a major research challenge. In this paper, recent advances in microbial phytase production, application of tools to optimize higher enzyme production, and characterization of phytases along with potential biotechnological applications are reviewed. Additionally the development of phytase assay methods and functions of phytate and phytate degradation products are discussed.Item Purification and characterization of an Endoinulinase from Xanthomonas campestris pv. phaseoli KM 24 Mutant(University of Zagreb, 2015) Naidoo, Kameshnee; Kumar, Ajit; Sharma, Vikas; Permaul, Kugen; Singh, SurenAn extracellular endoinulinase from Xanthomonas campestris pv. phaseoli KM 24 mutant was purifi ed to homogeneity by gel fi ltration chromatography and showed a specifi c activ-ity of 119 U/mg. The optimum pH and temperature of the purifi ed enzyme were found to be 6.0 and 50 °C, respectively. The enzyme was stable up to 60 °C, retaining 60 % of residu-al activity for 30 min, but inactivated rapidly above 60 °C. The enzyme was found to be stable at pH=6–9 when it retained 100 % of its residual activity. The Lineweaver-Burk plot showed that the apparent Km and vmax values of the inulinase when using inulin as a sub-strate were 1.15 mg/mL and 0.15 μM/min, respectively, whereas the kcat value was found to be 0.145 min–1. The calculated catalytic effi ciency of the enzyme was found to be 0.126 (mg·min)/mL. The purifi ed inulinase can be used in the production of high fructose syr-ups.Item Molecular dynamics simulation of chitinase I from Thermomyces lanuginosus SSBP to ensure optimal activity(Taylor and Fancis Online, 2016-09-22) Khan, Faez Iqbal; Bisetty, Krishna; Gu, Ke-Ren; Singh, Suren; Permaul, Kugen; Hassan, Md. Imtaiyaz; Wei, Dong-QingThe fungal chitinase I obtained from Thermomyces lanuginosus SSBP, a thermophilic deuteromycete, has an optimum growth temperature and pH of 323.15 K and 6.5, respectively. This enzyme plays an important task in the defence mechanism of organisms against chitin-containing parasites by hydrolysing β-1, 4-linkages in chitin. It acts as both anti-fungal and biofouling agents, with some being thermostable and suitable for the industrial applications. Three-dimensional model of chitinase I enzyme was predicted and analysed using various bioinformatics tools. The structure of chitinase I exhibited a well-defined TIM barrel topology with an eight-stranded α/β domain. Structural analysis and folding studies at temperatures ranging from 300 to 375 K using 10 ns molecular dynamics simulations clearly showed the stability of the protein was evenly distributed even at higher temperatures, in accordance with the experimental results. We also carried out a number of 20 ns constant pH molecular dynamics simulations of chitinase I at a pH range 2–6 in a solvent. This work was aimed at establishing the optimum activity and stability profiles of chitinase I. We observed a strong conformational pH dependence of chitinase I and the enzyme retained their characteristic TIM barrel topology at low pH.Item Biocontrol of Listeria monocytogenes ATCC 7644 on fresh-cut tomato (Lycopersicon esculentum) using nisin combined with organic acids(The Korean Society of Food Science and Technology, 2015) Oladunjoye, Adebola Olubukola; Singh, Suren; Ijabadeniyi, Oluwatosin AdemolaThe biocontrol of Listeria monocytogenes on fresh-cut tomato using nisin and organic acids was investigated. Fresh-cut samples inoculated with 108 CFU/mL of L. monocytogenes, treated with nisin (5,000 IU/mL), a combination of nisin and organic acids (acetic and citric acids at 3 and 5% each), with chlorine at 200 ppm as a control, and stored for six days at 4, 10, and 25oC were used to evaluate certain physicochemical qualities (pH, titratable acidity, soluble solid content, vitamin C content, and color). Nisin treatment significantly (p<0.05) reduced bacterial population by 1.91-3.07 log CFU/mL. Nisin-citric acid combination provided 2.65-3.29 log CFU/mL reduction, while nisin-acetic acid combination provided 2.93-4.15 log CFU/mL reduction. The control treatment provided <1-2 fold log reductions. Slight variations in physicochemical properties of fresh-cut tomato were observed. Nisin and organic acids can be used to improve the microbial safety of fresh-cut tomato.Item Chitinase from Thermomyces lanuginosus SSBP and its biotechnological applications(Springerlink, 2015) Khan, Faez Iqbal; Bisetty, Krishna; Singh, Suren; Permaul, Kugen; Hassan, Md. ImtaiyazChitinases are ubiquitous class of extracellu-lar enzymes, which have gained attention in the past few years due to their wide biotechnological applications. The effectiveness of conventional insecticides is increasingly compromised by the occurrence of resistance; thus, chi-tinase offers a potential alternative to the use of chemical fungicides. The thermostable enzymes from thermophilic microorganisms have numerous industrial, medical, envi-ronmental and biotechnological applications due to their high stability for temperature and pH. Thermomyces lanug-inosus produced a large number of chitinases, of which chi-tinase I and II are successfully cloned and purified recently. Molecular dynamic simulations revealed that the stability of these enzymes are maintained even at higher tempera-ture. In this review article we have focused on chitinases from different sources, mainly fungal chitinase of T. lanug-inosus and its industrial application.Item Thermostable chitinase II from Thermomyces lanuginosus SSBP: Cloning, structure prediction and molecular dynamics simulations(Elsevier, 2015) Khan, Faez Iqbal; Govender, Algasan; Permaul, Kugen; Singh, Suren; Bisetty, KrishnaThermomyces lanuginosus is a thermophilic fungus that produces large number of industrially-significant enzymes owing to their inherent stability at high temperatures and wide range of pH optima, including thermostable chitinases that have not been fully characterized. Here, we report cloning, characterization and structure prediction of a gene encoding thermostable chitinase II. Sequence analysis revealed that chitinase II gene encodes a 343 amino acid protein of molecular weight 36.65 kDa. Our study reports thatchitinase II exhibits a well-defined TIM-barrel topology with an eight-stranded α/β domain. Structural analysis and molecular docking studies suggested that Glu176 is essential for enzyme activity. Folding studies of chitinase II using molecular dynamics simulations clearly demonstrated that the stability of the protein was evenly distributed at 350 K.Item Cloning, expression, and molecular dynamics simulations of a xylosidase obtained from Thermomyces lanuginosus(Taylor and Francis Online, 2015-10-19) Gramany, Vashni; Khan, Faez Iqbal; Govender, Algasan; Bisetty, Krishna; Singh, Suren; Permaul, KugenthirenThe aim of this study was to clone, express, and characterize a β-xylosidase (Tlxyn1) from the thermophilic fungus Thermomyces lanuginosus SSBP in Pichia pastoris GS115 as well as analyze optimal activity and stability using computational and experimental methods. The enzyme was constitutively expressed using the GAP promoter and secreted into the medium due to the alpha-mating factor secretion signal present on the expression vector pBGPI. The 1276 bp gene consists of an open reading frame that does not contain introns. A 12% SDS–PAGE gel revealed a major protein band at an estimated molecular mass of 50 kDa which corresponded to zymogram analysis. The three-dimensional structure of β-xylosidase was predicted, and molecular dynamics simulations at different ranges of temperature and pH were performed in order to predict optimal activity and folding energy. The results suggested a strong conformational temperature and pH dependence. The recombinant enzyme exhibited optimal activity at pH 7 and 50°C and retained 80% activity at 50°C, pH 7 for about 45 min. This is the first report of the cloning, functional expression, and simulations study of a β-xylosidase from Thermomyces species in a fungal host.Item The multi-chitinolytic enzyme system of the compost-dwelling thermophilic fungus Thermomyces lanuginosus(Elsevier, 2014-12-03) Zhang, Meng; Puri, Adarsh Kumar; Govender, Algasan; Wang, Zheng-Xiang; Singh, Suren; Perumal, KugenthirenThe recent sequencing of the Thermomyces lanuginosus SSBP genome by our group has revealed four putative family 18 chitinases. In this study, three novel chitinase genes (chit2, chit3 and chit4) and the previously-reported chit1 gene were cloned from T. lanuginosus SSBP. chit1, encoding a 44.1 kDa protein, and chit2, encoding a 36.6 kDa protein, were successfully expressed in Pichia pastoris. The recombinant Chit1 and Chit2 enzymes exhibited optimum activity at pH 5.0 and pH 4.0, respectively. Chit1 had optimal activity at 50 ◦C and retained 56% of its activity at 60 ◦C after 30 min, while Chit2 was optimally active at 40 ◦C and retained 71% of its activity at 50 ◦C after 60 min. Both enzymes produced chitobiose as the major product using different substrates. Chit2 displayed antifungal activity against Penicillium verrucosum and Aspergillus niger. These activities could be useful in the environmental degradation of chitinous wastes as well as for biotechnological applications.Item Thermostable chitinase II from Thermomyces lanuginosus SSBP : Cloning, structure prediction and molecular dynamics simulations(Elsevier, 2015-04-08) Khan, Faez Iqbal; Govender, Algasan; Permaul, Kugen; Singh, Suren; Bisetty, KrishnaThermomyces lanuginosus is a thermophilic fungus that produces large number of industrially-significant enzymes owing to their inherent stability at high temperatures and wide range of pH optima, including thermostable chitinases that have not been fully characterized. Here, we report cloning, characterization and structure prediction of a gene encoding thermostable chitinase II. Sequence analysis revealed that chitinase II gene encodes a 343 amino acid protein of molecular weight 36.65 kDa. Our study reports that chitinase II exhibits a well-defined TIM-barrel topology with an eight-stranded α/β domain. Structural analysis and molecular docking studies suggested that Glu176 is essential for enzyme activity. Folding studies of chitinase II using molecular dynamics simulations clearly demonstrated that the stability of the protein was evenly distributed at 350 K.Item Influence of the alkyl group on thermophysical properties of carboxylic acids in 1-butyl-3-methylimidazolium thiocyanate ionic liquid at various temperatures(Elsevier, 2015) Redhi, Gan G.; Ebenso, Eno E.; Singh, Suren; Bahadur, Indra; Ramjugernath, DereshIn the present study, influence of the alkyl group and temperature on the interactions between the carboxylic acid and ionic liquid (IL) mixtures were discussed in term of density and sound velocity measurements. The IL used in this study was 1-butyl-3-methylimidazolium thiocyanate ([BMIM]+[SCN]−). The density (ρ), and sound velocity (u), of the IL, acetic acid, propionic acid, and their corresponding binary systems {[BMIM]+[SCN]− (x1) + acetic or propionic acid (x2)} have been measured at T = (293.15, 298.15, 303.15, 308.15 and 313.15) K and at p = 0.1 MPa. The excess molar volumes, View the MathML sourceVmE, isentropic compressibility, κs, and deviation in isentropic compressibility, Δκs, were calculated using experimental density and sound velocity data, respectively. The Redlich–Kister polynomial equation was used to fit the excess/deviation properties. These results are useful for describing the intermolecular interactions that exist between the IL and carboxylic acid mixtures.