Cold-active enzymes have received little research attention although they are very useful in industries. Since the structure bases of cold adaptation of enzymes are still unclear, it is also very difficult to obtain cold adapted enzymes for industrial applications using routine protein engineering methods. In this study, we employed directed evolution method to randomly mutate a mesophilic cellulase, endoglucanase Ⅲ (EG Ⅲ) from Trichoderma reesei, and obtained a cold adapted mutant, designated as w-3. DNA sequence analyses indicate that w-3 is a truncated form of native EG III with a deletion of 25 consecutive amino acids at its C-terminus. Further examination of enzymatic kinetics and thermal stability shows that mutant w-3 has a higher Kcat value and becomes more thermolabile than its parent. In addition, activation energy of w-3 and wild type EGIII calculated from Arrhenius equation are 13.3kJ.mol-1 and 26.2kJ.mol-1, respectively. Therefore, the increased specific activity of w-3 at lower temperatures could be resulted from increased Kcat value and decreased activation energy.

A bacterial strain BZS21 with enantioselective epoxide hydrolase activity to trans ethyl 3-phenylglycidate was screened. The strain was identified as Pseudomonas sp. The enantiomeric excess of the remaining epoxide, (2R, 3S) ethyl 3-phenylglycidate, was significantly influenced by the conditions of cultivation and kinetic resolution. With 1% sucrose as carbon source, 0.2% beef extract, 0.2% peptone and 1.5% urea as mixed nitrogen source, the strain produced epoxide hydrolase with the highest enantioselectivity. And (2R, 3S) ethyl 3-phenylglycidate was obtained with ee of 94.6% and yield of 26.2%.

The DNA fragment encoding the cellulose binding domain of endoglucanase Ⅲ (CBDEG Ⅲ) from Trichoderma reesei was subcloned and expressed in E. coli. The CBDEG Ⅲ had a high affinity for cellulose. The morphological and structural changes of cellulose after treatment with CBDEG Ⅲ indicated a 17.0% decrease in number of hydrogen bonds and a 16.5% decrease in crystalline index. X-ray diffraction and IR spectra analyses indicated that the destabilization and breakage of the hydrogen bonds in crystalline cellulose accounted for the non-hydrolytic disruption of the structure of cellulose.

main product was separated and purified by HPLC and characterized as sophorolipid by nuclear magnetic resonance (NMR) and mass spectroscopy (MS). This is the first report on sophorolipids produced from W. domercqiae. Subsequently, the cytotoxic effects of the sophorolipid on cancer cells of H7402, A549, HL60 and K562 were investigated by MTT assay. The results showed a dose dependent inhibition ratio on cell viability according to the drug concentration ≤62.5μg/ml. These findings suggested that the sophorolipid produced by W. domercqiae have anticancer activity.

A Pseudomonas sp. was isolated with enantioselective epoxide hydrolase activity to ethyl 3-phenylglycidate. Cells grown on sucrose and suspended in 10% DMF (v/v) as cosolvent produced (2R,3S) ethyl 3-phenylglycidate with 95% ee and 26% yield in 12h from 0.2% (w/v) of the racemate.