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.

Error-prone PCR was used to randomly mutate the endoglucanase (EG Ⅲ) gene of Trichoderma reesei followed by screening for clear-hole-forming colonies on carboxymethyl cellulose (CMC) plates at high pH. A mutant was selected with a shift of pH optimum from 4.8-5.0 to 5.4, and its specific enzyme activity decreased in low pH and increased in high pH. The sequencing of the mutant gene showed that the asparagine residue at 321 position was substituted by threonine. Two site-directed mutations, N321D and N321H, were designed to study the role of EG Ⅲ-321. It was discovered that the pH activity profiles of these two mutants had obvious change compared to the wild-type enzyme. It suggested that the residue at position 321 is a key amino acid residue in determining the pH-activity profile of the EG Ⅲ from T. reesei. The rule accorded with the alkali-tolerant mechanism of alkaliphilic cellulase K.

Asparagine (Asn)-71 of the xylanase (XYN) from Bacillus pumilus A-30 was found highly conserved in alkaline xylanases of family G/11. The mutated gene fragments containing different substitutions of Asn-71 was obtained by site-directed mutagenesis to study its role in the alkali-tolerant mechanism of xylanase. The xylanase activity was completely lost if Asn-71 residue was replaced by alkaline arginine (Arg) or lysine (Lys) residues, but obviously depressed with a shift in the pH optimum of the enzyme from 6.7 to 6.3 if substituted by serine (Ser) or aspartate (Asp) residues. No mutant with a shift of the pH optimum to a more basic value was found. Furthermore, N71D lost its activity in the alkaline pH range completely, while N71S did not lose as much as that of N71D. Except for Asn-71, the random mutagenesis to other residues of the xylanase was also studied. The alkali-tolerant mechanism of the xylanase was analyzed by their charged character, ionized state, and the hydrogen bond network of the residues surrounding the two catalytic residues on the basis of homology modeling of the mutated xylanases.

Aims: The aim of this study was to investigate the properties of temperate bacteriophage of Lactobacillus fermentum, based on its morphology, restriction patterns, protein profile and the impact on the growth of host strain. Methods and Results: With Mitomycin C, seven temperate phages were induced from Lactobacilli derived from Chinese yogurt. The temperate phages induced belong to the most common Bradley' s group B, having hexagonal head and long, noncontractile tail. They were furthermore confirmed to be the same bacteriophage by identical restriction patterns. SDS–PAGE profile showed that the phage studied had one major structure protein about 31Æ9 kDa. The presence of the prophage influenced the cell shape and colony size of its lysogenic strain. Conclusions: The phage obtained had similar, but not complete identical properties with other L. fermentum phages reported. It influenced the growth behaviour of its lysogenic strain. Significance and Impact of the Study: This study provides some information about bacteriophages occurring in the Chinese yoghurt manufacture and contributes to our knowledge on the bacteriophage diversity in the dairy industry.

For the purpose of producing pyruvate from dl-lactate by enzymatic methods, four microorganism strains that produce lactate oxidase (LOD) were screened and isolated from many soil samples. Among them, strain SM-6, which showed high potential for pyruvate production,was chosen for further research. Physiological studies and 16S rDNArelationship reveal that SM-6 belongs to Pseudomonas putida. The optimized pH and temperature of the enzyme-catalyzed reaction were pH 7.2, and 39℃, respectively. Low-concentration EDTA (1mM) could improve the stability of pyruvate and conversion ratio of lactate oxidase. Vmax and Km value for dl-lactate were 2.46 mol/(min mg) protein and 9.53mM, respectively. On preparation scale, cell-free extract from SM-6, containing 300mg/l of crude enzyme (4037U/ml lactate oxidase), could convert 66% of 116mM of dl-lactate into 76.6mM pyruvate in 18h, and 82% of substrate was transformed after 48 h, giving 95.0mM (10.5 mg/ml) of pyruvate. The ratio of product to biocatalyst was 34.8:1 (g/g).

To produce an industrial strain of Saccharomyces cerevisiae that metabolizes xylose, we constructed a rDNA integration vector and YIp integration vector, containing the xylose-utilizing genes, XYL1 and XYL2, which encode xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis, and XKS1, which encodes xylulokinase (XK) from S. cerevisiae, with the G418 resistance gene KanMX as a dominant selectable marker. The rDNA results in integration of multiple copies of the target genes. The industrial stain of S. cerevisiae NAN-27 was transformed with the two integration vectors to produce two recombinant strains, S. cerevisiae NAN-127 and NAN-123. Upon transformation, multiple copies of the xylose-utilizing genes were integrated into the genome rDNA locus of S. cerevisiae. Strain NAN-127 consumed twice as much xylose and produced 39% more ethanol than the parent strain, while NAN-123 consumed 10% more xylose and produced 10% more ethanol than the parent strain over 94 h.