Bhaumik R. Dave

Optimization of Process Parameters for Cellulase Production by Bacillus licheniformis MTCC 429 using RSM and Molecular Characterization of Cellulase Gene

World is threaten to energy crisis which has advances research in bioenergy and specifically development of biofuels to replace petroleum products have increased the use of microbial enzyme like cellulases and xylanases as well as amylases for generation of reducing sugars for their conversion into bioethanol. Extensive research has been carried out in this view but alkaline cellulase production and molecular characterization is not studied in detail so far, this study will aid to achieve it. Optimization of fermentation parameters for production of cellulase was evaluated with the help of Response Surface Methodology (RSM) a statistical design, initial pH (9), moisture ratio (1:1) and incubation time (72 h) (run no.4) were found to be ideal parameters for optimum production of cellulase, substrate Jatropha seed cake without any pre-treatment was found to be an ideal source for cellulase production by Bacillus licheniformis under solid state fermentation. Cellulase gene of size 786 bp was isolated later using PCR techniques, confirmed with sequence analysis and ligated to pRSET A vector for the transformation to E. coli DH5α. Positive clones were identified and sequenced to justify the cloning. Sequence of Bacillus licheniformis endo-β-1,4-glucanase (Cel12A) gene showed 100% similarity with endoglucanase gene sequence from Bacillus licheniformis ATCC 14580 genome, shows successful cloning of Cel12A gene into pRSET A vector.

Purification and properties of an endoglucanase from Thermoascus aurantiacus

An Endo-cellulase was purified to homogeneity using ammonium sulfate precipitation, ion exchange and size exclusion chromatography from newly isolated strain of Thermoascus aurantiacus RBB-1. The recovery and purification fold were 13.3% and 6.6, respectively, after size exclusion chromatography. The purified cellulase has a molecular mass (M) of 35 kDa. Optimum temperature for the enzyme was found to be 70 °C and stability was upto 80 °C for 1 h. Along with higher stability at 80 °C, enzyme showed half lives of 192 h and 144 h at 50 and 70 °C respectively. The purified cellulase was optimally active at pH 4.0 and was stable over a broad pH range of 3.0–7.0. The enzyme purified showed apparent Km and Vmax values of 37 mg/ml and 82.6 U/min/mg protein respectively with higher salt tolerance of 10% for 1 h.

Enhanced catalysis of l-asparaginase from Bacillus licheniformis by a rational redesign

L-Asparaginase (3.5.1.1) being antineoplastic in nature are used in the treatment of acute lymphoblastic leukemia (ALL). However glutaminase activity is the cause of various side effects when used as a drug against acute lymphoblastic leukemia (ALL). Therefore, there is a need of a novel L-asparaginase (L-ASNase) with low or no glutaminase activity. Such a property has been observed with L-ASNase from B. licheniformis (BliA). The enzyme being glutaminase free in nature paved the way for its improvement to achieve properties similar to or near to the commercially available L-ASNases. Rational enzyme engineering approach resulted in four mutants: G238N, E232A, D103V and Q112H. Among these the mutant enzyme, D103V, had a specific activity of 597.7IU/mg, which is higher than native (rBliA) (407.65IU/mg). Moreover, when the optimum temperature and in vitro half life were studied and compared with native BliA, D103V mutant BliA was better, showing tolerance to higher temperatures and a 3 fold higher half life. Kinetic studies revealed that the mutant D103V L-ASNase has increased substrate affinity, with Km value of 0.42mM and Vmax of 2778.9μmolmin(-1).

Nickel accumulation by Colocassia esculentum and its impact on plant growth and physiology

The remediation of heavy metal-contaminated sites using plants presents a promising alternative to current methodologies. In this study, the potential of Colocassia esculentum for Nickel (Ni) accumulation was determined. C. esculentum plants exposed to Ni, demonstrated capability to accumulate on average, more in shoots as compared to roots, suggesting better translocation of Ni from root to shoot. High metal content in soil caused reduction in growth parameters and an increase in oxidative stress. Under heavy metal stress, an increase in catalase, peroxidase, ascorbic acid and proline were observed in the roots along with some anatomical changes. Lipid peroxidation showed a slight increase by Ni treatment along with some anatomical changes in the root. This work demonstrates that metal induced oxidative stress occurs by the presence of heavy metals at higher concentrations. It also suggests that superior antioxidative defenses, particularly catalase activity, may play an important role in C. esculentum. The outcome of this study corroborate that C. esculentum is a suitable candidate for the phytoremediation of Ni contaminated soil and could be considered as a potential Ni hyperaccumulator plant species.