[Effect of ZnSO4 on L-lactic acid production by Rhizopus oryzae].

OBJECTIVE: To improve the yield and quality of L-lactic acid by Rhizopus oryzae, we aim to understand the relationship between inorganic salts utilization and the L-lactic acid metabolism of the strain RLC41-6, through systematic analysis of the effects of zinc ion concentration on the production of L-lactic acid and the Lactic Dehydrogenase (LDH) activity. METHODS: Rhizopus oryzae was cultured at 36 degrees C for 36h with different quantity of ZnSO4 in fermentation medium. The fermentation products were monitored by reversed-phase high performance liquid chromatography (RP-HPLC), LDH isoenzyme composition in the cell was analysed by non-denatured polyacrylamide gel electrophoresis (PAGE). RESULTS: Our results showed that the concentration of ZnSO4 in medium could modulate the expression of LDH isoenzyme except LDH1, especially stimulated the expression of LDH4 and LDH5. When initial concentration of ZnSO4 is above 0.02%, the LDH4 and LDH5 reached the highest level. However, the activity of LDH was inhibited by higher concentration zinc ion in extracellular environment. When ZnSO4 concentration is 0.02%, LDH activity reaches its maximum 200U/mL, the HPLC assay showed only L-lactic in the fermentation products (137 g/L), while the conversion rate of glucose to lactic acid is 91%. CONCLUSION: Zinc ion can regulate the metabolic processes of Rhizopus oryzae and modulate the types of the final fermentation products. An optimal concentration of ZnSO4 can not only facilitate the LDH expression but also prevent pyruvate from transformation into the malic acid and fumaric acid during the metabolism process, thereby enhance the metabolism of glucose to lactic acid of Rhizopus oryzae.

[Production of ligninase by co-fermentation of Coprinus comatus and Trichoderma reesei].

In order to enhance the utilization efficiency, reduce the environmental pollution of traditional chemical treatment and the agriculture waste incineration; we studied the ligninase production by Coprinus comatus, Aspergillus niger and Trichoderma reesei through the plate screening. The results showed that C. comatus mixed culture with T. reesei have a good compatibility and higher yields of Laccase. On the basis of this pre-experiment, we studied the optimal conditions of mixed culture for enzyme production. Under the optimal conditions: the inoculation proportion of C. comatus and T. reesei (5:2), the interval of time (12 h), the temperature 260C, the shake rotation speed 150 r/min, fermented for 3 days, the Laccase activity reached 3267.1 U/mL, increased by 106% contrasted with single culture of C. commatus.

[Progress in study on microbial enzymes for the metabolism of environmental refractory organic compounds].

With the rapid development of socialization and industrialization, more and more pollutes were produced and discharged into natural environment. It is harmful to human health and life. These pollutes included refractory degradation organic compounds like PAHs, RDX, HMX, CL-20, PCBs and alkanes and their relative substances. Various compounds exist in nature with long life span. They are the most hazardous than other organics. The impact of pollutes can be treated by microorganisms. Results showed that it is an effective way for bioremediation of these pollutes with microbial metabolism or cometabolism. A few key enzymes, mainly oxidative and reductive enzymes, connected with the first step of initial degradation. Normally, enzymes grouped with other active fraction on the cell membrane are composed of one oxidative and reductive system for substrates oxidation. The metabolic intermediates can be used with TCA by microorganisms. The pathways of metabolism and the key enzymes were summarized. The further research topics should be focused on microorganism screen and its relative enzyme, pathway and mechanism of metabolism or cometabolism for such compounds degradation, and the result was hoped for the environmental protection.

[Investigation of fucoidanase by FTIR spectra].

Fucoidanase was isolated and purified from marine fungus LD8 by solid state fermentation, extraction with citric acid buffer, acetone precipitatation and column chromatography on Sephadex G-100. A single band on PAGE shows that pretty pure fucoidanase has been obtained. FT-IR spectra and its derivation, self-deconvolution and curve-fitting methods were used to analyze the secondary structure of the fucoidanase. Composite bands of the amide I and amide III were studied by using Fourier self-deconvolution (FSD) with an enhancement factor of K = 2.2 and a half width of 20.2 cm(-1). The relative average fractions of alpha-helix, beta-sheet, random coil, beta-turn are 11.5%, 58.6%, 14.5% and 15.9%, respectively, according to amide I region, while the content of alpha-helix is 12%, beta-sheet 57.3%, random coil 14.5%, and beta-turn 16.3% on amide III region. In other words, both the conclusions were exactly consistent. All the above results show that beta-sheet was the dominant component, which is about 58%, and that beta-turn is about 15%, random coil 15%, and alpha-helix 12% at room temperature.

[Production and properties of chitinase from Beauveria bassiana Bb174 in solid state fermentation].

This paper studied the chitinase production of Beauveria bassiana Bb174 under solid state fermentation condition. The optimal medium consisted of wheat bran and silkworm chrysalis at the ratio of 4:1, supplemented with 1 g peptone L(-1) as nitrogen source and some other mineral nutrients. The enzyme activity reached 126 units per gram dry medium after cultured for 2 days at 28 degrees C and natural pH by inoculated 3 ml spore suspension into this medium. The optimal temperature and pH for chintinase production were 40 degrees C and 5.0, respectively. The temperature to lose 50% activity of the enzyme was 48 degrees C after incubated at 30-70 degrees C for 1 h. The enzyme was stable at 30-40 degrees C and pH 4-6, and the Km and Vmax values were 0.52 mg x ml(-1) and 0.7 deltaE680 x h(-1), respectively.

[Assessment of the Escherichia coli Tat protein translocation system with fluorescent proteins].

The possibility of using fluorescent proteins as probes to study the twin-arginine translocation (Tat) system was assessed in Escherichia coli. When fused to the twin-arginine signal peptide of trimethylamine N-oxide reductase, the DsRed2 red fluorescent protein from the Discosoma sp. was successfully synthesized and folded in E. coli cells. However, RR-DsRed2 aggregated inside the cells. Therefore, although DsRed2 has been engineered from DsRed for faster maturation and lower non-specific aggregation, it is still not compatible with Tat-dependent translocation. In contrast, the jellyfish green fluorescent protein (GFP) was efficiently exported into periplasm even when the RR motif was changed to KR or RK. These results show that GFP can be used as an efficient reporter protein to study Tat system, but DsRed2 is not suitable for such purpose because of its aggregation property. In addition, when the protein concentration was similar, the fluorescence intensity of KR-GFP and RK-GFP decreased compared with RR-GFP, which would suggest that the twin-arginine signal peptide is not only essential for mediating protein translocation, but also important for the folding of down-stream protein.

[Mutagenesis of ion beam implantation and identification of two new rifampicin resistance determining sites in rpoB gene in Escherichia coli].

The rifampicin resistance mutations induced by low energy nitrogen ion implantation had been investigated in this study referred to the study of 60Co-gamma ray irradiation. It is suggested that low energy nitrogen ion beam implantation might generate a lower damage but get higher mutation rates than 60Co-gamma ray irradiation did. The frequencies showed the preference of base substitutions induced by N+ ion beam are CG--> TA transition, GC-->AT transition, AT--> GC transition and AT-->TA transversion which occupy 87.5% (77/88) of the total mutation of base substitutions. Two new rif-determining sites had also been identified in rpoB gene in Escherichia coli. The first new site located in 1551. When dG1551 was replaced by dC, it resulted in Gln517 was substituted by a histidine. The other new site located in 1692. When dC1692 was replaced by dT, it resulted in a Pro564 is changed into a leucine. The synonymous mutation of the second site had been reported but the nucleotide substitution, dC-->dT, had not been identified before.