After being pretreated with a sublethal dose of either oxidative or hyperosmotic stress, Saccharomyces cerevisiae cells could withstand a subsequent higher dose of the same stress. Cross-adaptation also existed between the two stresses. Especially wild-type cells pretreated with 1%KCl (a hyperosmotic stress generating agent) and hyperosmotic-resistance mutant cells could significantly resist to lethal concentration of H2O2 (10mM). The addition of N-acetylcysteine (NAC) (30 mg 1-1) and MnSO4 (4mM) showed a strong reversion to hyperosmotic stress, which implicated that antioxidants participated in yeast adaptation to hyperosmosis. The two sublethal dose of stresses treatment, especially hyperosmosis, increased the level of GSH, CAT, SOD and total antioxidant capability (T-AOC) in yeast cells, which indicated adaptation between oxidative and hyperosmotic stresses was accompanied by the production of above several antioxidants. Furthermore, at least seven new proteins were induced by both oxidative and osmotic treatment.

A cDNA encoding for laccase was isolated from the ligninolytic fungus Trametes versicolor by RNA-PCR. The cDNA corresponds to the gene Lcc1, which encodes a laccase isoenzyme of 498 amino acid residues preceded by a 22-residue signal peptide. The Lcc1 cDNA was cloned into the vectors pMETA and pMETαA and expressed in Pichia methanolica. The laccase activity obtained with the Saccharomyces cerevisiae α-factor signal peptide was found to be twofold higher than that obtained with the native secretion signal peptide. The extracellular laccase activity in recombinants with the α-factor signal peptide was 9.79Uml−1. The presence of 0.2 mM copper was necessary for optimal activity of laccase. The expression level was favoured by lower cultivation temperature. The identity of the recombinant protein was further confirmed by immunodetection using Western blot analysis. As expected, the molecular mass of the mature laccase was 64.0kDa, similar to that of the native form.

A novel fibrinolytic enzyme from Rhizopus chinensis 12 was purified through ammonium sulfate precipitation, hydrophobic interaction, ionic exchange, and gel filtration chromatography. The purification protocol resulted in a 893-fold purification of the enzyme, with a final yield of 42.6%. The apparent molecular weight of the enzyme was 18.0kDa, determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and 16.6kDa by gel filtration chromatography, which revealed a monomeric form of the enzyme. The isoelectric point of the enzyme estimated by isoelectric focusing electrophoresis was 8.5±0.1. The enzyme hydrolyzed fibrin. It cleaved the α, β, and γ chains of fibrinogen simultaneously, and it also hydrolyzed casein and N-succinyl-Ala-Ala-Pro-PhepNA. The enzyme had an optimal temperature of 45℃, and an optimal pH of 10.5. EDTA, PCMB, and PMSF inhibited the activity of the enzyme, and SBTI, Lys, TPCK, and Aprotinine had no obvious inhibition, which suggested that the activity center of the enzyme had hydrosulfuryl and metal. The first 12 amino acids of the N-terminal sequence of the enzyme were S-V-S-E-I-Q-LM-H-N-L-G and had no homology with that of other fibrinolytic enzyme from other microbes.

The bioconversion of methyl-testosterone to methandienone in a biphasic system was studied using freeze-dried, thawed and growing Arthrobacter simplex AS 1.94* cells as biocatalyst. The biphasic system consists of an organic phase [30% (v/v)] with steroid 10g/L and an aqueous phase [70% (v/v)] containing the cells (5g.d.c.w/L). Carbon tetrachloride and Tween-80 were used as organic solvent and surfactant, respectively, and menadione was added as an external electron acceptor. The factors affecting the conversion rate in the two-phase system were investigated. The results showed that menadione was necessary in this system. Small amounts of product added to the reaction system can increase the dehydrogenation rate. Higher activities were obtained with thawed cells as compared to the freeze-dried and growing cells. Under the optimal operation conditions, more than 95% (w/w) of added methyl-testosterone could be converted to methandienone within 48h. Also, an easy way to recover the product in a high purity is proposed.

Alpha amylase gene from Bacillus licheniformis was mutated by site-directed mutagenesis to improve its acid stability. The mutant gene was expression in Bacillus subtilis under the control of the promoter of sacB gene which was followed by either the a-amylase leader peptide of Bacillus licheniformis or the signal peptide sequence of sacB gene of Bacillus subtilis. Both peptides efficiently directed the secretion of a-amylase from the recombinant B. subtilis cells. The extracellular a-amylase activities in two recombinants were 1001 and 2012 U ml-1, respectively. The purity of the recombinant product was confirmed by SDSPAGE.