Research Publications (Applied Sciences)
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Item Enhanced production of N-acetyl-d-neuraminic acid by whole-cell bio-catalysis of Escherichia coli(Elsevier, 2016) Zhou, Junbo; Chen, Xianzhong; Lu, Liping; Govender, Algasan; Haiquan, Yang; Shen, WieN-acetyl-d-neuraminic acid (Neu5Ac) has been considerably focused due to its promising potential appli-cations in pharmaceuticals and dairy products. A whole-cell biocatalyst process is an important tool for synthesis of pharmaceutical intermediates and fine chemicals. In this study, a whole cell process using engineered Escherichia coli strain was developed and stepwise optimization was employed for Neu5Ac production. N-acetyl-D-glucosamine 2-epimerase and Neu5Ac aldolase were overexpressed in E. coli individually and the activity ratio was optimized by varying recombinant amounts of cell biomass for syn-thesis of Neu5Ac. Moreover, substrate concentrations and ratio of pyruvate and N-acetyl-D-glucosamine (GlcNAc) and detergent concentrations were optimized to increase product synthesis. The resulting process generated 237.4 mM Neu5Ac with a yield of 40.0% mol/mol GlcNAc. Furthermore, transporter pathways involved in Neu5Ac and GlcNAc were engineered and their impact on the Neu5Ac synthesis was evaluated. Using a stepwise optimization, an overall whole-cell biocatalytic process was developed and a maximum titer of 260.0 mM Neu5Ac (80.4 g/L) with a conversion yield of 43.3% from GlcNAc was achieved. The process can be used for industrial large-scale production of Neu5Ac in terms of efficiency and economy.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 Display of phytase on the cell surface of Saccharomyces cerevisiae to degrade phytate phosphorus and improve bioethanol production(Springer Verlag, 2016) Xiao, Yan; Shen, Wei; Govender, Algasan; Zhang, Liang; Xianzhong, ChenCurrently, development of biofuels as an alternative fuel has gained much attention due to resource and environ-mental challenges. Bioethanol is one of most important and dominant biofuels, and production using corn or cassava as raw materials has become a prominent technology. However, phytate contained in the raw material not only decreases the efficiency of ethanol production, but also leads to an increase in the discharge of phosphorus, thus impacting on the environment. In this study, to decrease phytate and its phos-phorus content in an ethanol fermentation process, Saccharomyces cerevisiae was engineered through a surface-displaying system utilizing the C-terminal half of the yeast α-agglutinin protein. The recombinant yeast strain, PHY, was constructed by successfully displaying phytase on the surface of cells, and enzyme activity reached 6.4 U/g wet biomass weight. Ethanol productions using various strains were com-pared, and the results demonstrated that the specific growth rate and average fermentation rate of the PHY strain were higher 20 and 18 %, respectively, compared to the control strain S. cerevisiae CICIMY0086, in a 5-L bioreactor process by simultaneous saccharification and fermentation. More im-portantly, the phytate phosphorus concentration decreased by 89.8 % and free phosphorus concentration increased by 142.9 % in dry vinasse compared to the control in a 5-L bio-reactor. In summary, we constructed a recombinant S. cerevisiae strain displaying phytase on the cell surface, which could improve ethanol production performance and effectively reduce the discharge of phosphorus. The strain reported here represents a useful novel engineering platform for developing an environment-friendly system for bioethanol production from a corn substrate.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 Heterologous expression of the osmotolerant yeast Candida glycerolgenesis glycerol-3-phosphate dehydrogenase gene (CgGPD) in Saccharomyces cerevisiae lacking the HOG pathway(Elsevier, 2013) Chena, Xianzhong; Fang, Huiying; Wang, Zheng-Xiang; Govender, Algasan; Zhuge, Bin; Zhuge, JianThe cytoplasmic glycerol-3-phosphate dehydrogenase of Candida glycerinogenes encoded by CgGPD is a critical enzyme in overproducing glycerol. The function of CgGPD has been characterized, however little information is known about the participation of CgGPD in the high osmolarity glycerol (HOG) pathway. In this study, expression and the function of CgGPD were investigated in Saccharomyces cerevisiae strains lacking a HOG component (hog1, pbs2 and gpd1/gpd2 mutants). Expression of CgGPD in gpd1/gpd2 mutants not only increased viability but also enhanced the salt tolerance and growth capability in a high osmotic medium. Functional comparison of CgGPD, GPD1 and GPD2 showed that expression of either gene significantly improved growth properties of both hog1 and pbs2 mutants under high osmolarity conditions except that the expression of GPD1 had little effect on the growth of hog1 mutants. Expression of CgGPD and GPD1 led to a similar growth pattern in gpd1/gpd2 mutants however, GPD2 had a perceptible impact on its growth properties. Further analysis showed that intracellular glycerol accumulated increasingly in response to high osmolarity conditions in all mutants expressing CgGPD, GPD1 or GPD2. Furthermore, expression of CgGPD was induced by osmo-stress in hog1 and pbs2 mutants however, the transcriptional strength was correspondingly lower when it was expressed in gpd1/gpd2 mutants.