Faculty of Applied Sciences
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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 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 Biodiesel synthesis from microalgae using immobilized Aspergillus niger whole cell lipase biocatalyst(Elsevier, 2016) Guldhe, Abhishek; Singh, Poonam; Kumari, Sheena K.; Rawat, Ismail; Permaul, Kugen; Bux, FaizalWhole cell lipase catalysis and microalgal feedstocks make overall biodiesel synthesis greener and sustainable. In this study, a novel approach of whole cell lipase-catalyzed conversion of Scenedesmus obliquus lipids was investigated for biodiesel synthesis. Microalgal biodiesel was characterized for its fuel properties. Optimization of process parameters for immobilized Aspergillus niger whole cell lipase-catalyzed biodiesel synthesis was carried out. Highest biodiesel conversion of 53.76% was achieved from S. obliquus lipids at 35 °C, methanol to oil ratio of 5:1 and 2.5% water content based on oil weight with 6 BSPs (Biomass support particles). Step-wise methanol addition was applied to account for methanol tolerance, which improved biodiesel conversion upto 80.97% and gave 90.82 ± 1.43% yield. Immobilized A. niger lipase can be used for 2 batches without significant loss in conversion efficiency. Most of the fuel properties of biodiesel met the specifications set by international standards.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 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 Advances in synthesis of biodiesel via enzyme catalysis : Novel and sustainable approaches(Elsevier, 2015-01) Singh, Bhaskar; Mutanda, Taurai; Permaul, Kugen; Bux, Faizal; Guldhe, AbhishekBiodiesel, a renewable fuel has a great potential in fulfilling an ever-increasing transport fuel demand. The enzymatic conversion process of feedstock oil to biodiesel is greener when compared to the conventional approach of chemical conversion due to mild reaction conditions and less wastewater generation. Lipases obtained from various microbial sources have been widely applied as catalysts for the conversion of oil to biodiesel. Biodiesel and glycerol obtained by enzymatic conversion have shown a higher purity as compared to that obtained by other conversion techniques. Enzymatic conversion of oil to biodiesel is less energy intensive because of milder reaction conditions and fewer purification steps involved in processing. Lipases, due to their catalytic efficiency and specificity, have emerged as a great tool for converting a wide range of feedstock oils to biodiesel. This manuscript presents an overview of the use of enzymatic conversion for making biodiesel production sustainable and environmentally-friendly. The constraints of enzymatic conversion are the high cost of the enzyme and its inhibition by alcohol and glycerol. The possible solutions to overcome these constraints are discussed. Recent advances to develop an effective process for enzymatic conversion of feedstock oils into biodiesel are critically evaluated. Prospective and challenges in scaling up of this technology are also discussed.Item Biodegradation of glycerol using bacterial isolates from soil under aerobic conditions(Taylor and Francis, 2014) Raghunandan, Kerisha; Mchunu, Siphesihle; Kumar, Ashwani; Kumar, Kuttanpillai Santhosh; Govender, Algasan; Permaul, Kugen; Singh, SurenGlycerol, a non-biodegradable by-product during biodiesel production is a major concern to the emerging biodiesel industry. Many microbes in natural environments have the ability to utilize glycerol as a sole carbon and energy source. The focus of this study was to screen for microorganisms from soil, capable of glycerol utilization and its conversion to value added products such as ethanol and 1,3-propanediol (1,3-PDO). Twelve bacterial isolates were screened for glycerol utilization ability in shake flask fermentations using M9 media supplemented with analytical grade glycerol (30 g/L) at various pH values (6, 7 and 8) and temperatures (30◦C, 35◦Cand 40◦C). Among these, six bacterial isolates (SM1, SM3, SM4, SM5, SM7 and SM8) with high glycerol degradation efficiency (>80%) were selected for further analysis. Highest level of 1,3-PDO production (15 g/L) was observed with isolate SM7 at pH 7 and 30◦C, while superior ethanol production (14 g/L) was achieved by isolate SM9 at pH 8 and 35◦C, at a glycerol concentration of 30 g/L. The selected strains were further evaluated for their bioconversion efficiency at elevated glycerol concentrations (50–110 g/L). Maximum 1,3-PDO production (46 g/L and 35 g/L) was achieved at a glycerol concentration of 70 g/L by isolates SM4 and SM7 respectively, with high glycerol degradation efficiency (>90). Three isolates (SM4, SM5 and SM7) also showed greater glycerol tolerance (up to 110 g/L). The isolates SM4 and SM7 were identified as Klebsiella pneumoniae and SM5 as Enterobacter aerogenes by 16S rDNA analysis. These novel isolates with greater glycerol tolerance could be used for the biodegradation of glycerol waste generated from the biodiesel industry into value-added commercial products.Item Improvement of ethanol production from sugarcane molasses through enhanced nutrient supplementation using Saccharomyces cerevisiae(Academic Journals, 2012-03) Nofemele, Zuko; Shukla, Pratyoosh; Trussler, Arthur; Permaul, Kugen; Singh, SurenSaccharomyces cerevisiae as a yeast cream was utilized for alcoholic fermentation using sugar cane molasses. In the present study, fermentation was optimized for urea and yeast hydrolysate (YH) dosage and the combined effect was evaluated. Total sugars as inverts (TSAI) composition of molasses were -1 determined by HPLC as 39% (m/v). Urea concentrations of 4, 2 and 3 gl showed optimal ethanol -1 production at 30, 35 and 40°C respectively. A YH concentration of 0.5 gl resulted in an ethanol yield of 8.7% (m/v) with a fermentation efficiency of 85.12%. Under optimized conditions (35°C) significant improvements were noticed with ethanol yield of 7.8% (m/v) and efficiency of 76.3%.Item Xylanase superproducer : genome sequence of a compost-loving thermophilic fungus, thermomyces lanuginosus strain SSBP(American Society for Microbiology, 2013-06-20) Mchunu, Nokuthula Peace; Permaul, Kugen; Rahman, Ahmad Yamin; Saito, Jennifer A.; Singh, Suren; Alam, MaqsudulWe report here the draft genome sequence of Thermomyces lanuginosus strain SSBP, which was isolated from soil in South Africa. This fungus produces the largest amount of xylanase ever reported in the literature.