Cellulose binding domain assisted immobilization of lipase (GSlip-CBD) onto cellulosic nanogel: characterization and application in organic medium.

A cbd gene was cloned into the C-terminal region of a lip gene from Geobacillus stearothermophilus. The native lipase (43.5 kDa) and CBD-Lip fusion protein (60.2 kDa) were purified to homogeneity by SDS-PAGE. A highly stable cellulosic nanogel was prepared by controlled hydrolysis of microcrystalline cellulose onto which the CBD-lip fusion protein was immobilized through bio-affinity based binding. The nanogel-bound lipase showed optimum activity at 55 °C, and it remains stable and active at pH 10-10.5. Furthermore, the immobilized lipase showed an over two-fold increase of relative activity in the presence of DMSO, isopropanol, isoamyl alcohol and n-butanol, but a mild activity decrease at a low concentration of methanol and ethanol. The immobilized biocatalyst retained ~50% activity after eight repetitive hydrolytic cycles. Enzyme kinetic studies of the immobilized lipase showed a 1.24 fold increase in Vmax and 5.25 fold increase in kcat towards p-NPP hydrolysis. Additionally, the nanogel bound lipase was tested to synthesize a biodiesel ester, ethyl oleate in DMSO. Kinetic analysis showed the km 100.5 ± 4.3 mmol and Vmax 0.19 ± 0.015 mmolmin(-1) at varied oleic acid concentration. Also, the values of km and Vmax at varying concentration of ethanol were observed to be 95.9 ± 13.9 mmol and 0.22 ± 0.013 mmolmin(-1) respectively. The maximum yield of ethyl oleate 111.2 ± 1.24 mM was obtained under optimized reaction conditions in organic medium. These results suggest that this immobilized biocatalyst can be used as an efficient tool for the biotransformation reactions on an industrial scale.

Accumulation of urinary 2-thiothiazolidine-4-carboxylic acid (TTCA) among workers occupationally exposed to carbon disulfide for 1 week.

OBJECTIVES: To investigate if carbon disulfide (CS(2)) accumulates after a 1-week exposure period, and how the work-shift duration and exposure magnitude affects this accumulation for the workers in viscose rayon industry. METHODS: Six 8-h and seven 12-h workers in the spinning department historically known to be exposed to high air CS(2) were recruited as the exposed groups. Seven workers from other non-CS(2)-exposed departments were recruited as non-exposure controls. Exposure monitoring covered a full work shift with personal breathing zone monitoring. Urine was collected pre- and post-shift every day throughout the 5 consecutive days. 2-Thiothiazolidine-4-carboxylic acid levels in the urine (U-TTCA) were determined. RESULTS: No detectable values were found for airborne (<0.6 ppm) and urinary (<35 ng/ml) monitoring for the control groups. The exposure levels for a 12-h shift (11.3+/-1.47) (AM+/-S.D.) were significantly greater than for an 8-h shift (6.3+/-0.64). The linear accumulation trend for daily U-TTCA across the workdays was only significant for the 12-h shift at pre-shift. Statistical significance was found in the regression of the ratios for pre-shift U-TTCA to airborne CS(2) levels on the preceding day to the day of the exposure at pre-shift for a 12-h shift (r=0.98, P=0.02). CONCLUSIONS: The U-TTCA accumulation for occupational exposure to CS(2) was exposure-magnitude-dependent. The linear equations derived in this study indicated that the U-TTCA increment at pre-shift for each additional daily 12-h exposure, after an adjustment for the CS(2) exposure level, was 0.02 mg/g creatinine/ppm of CS(2). The long-term exposure response under such repeated and intermittent conditions should be noteworthy.