RESUMEN

To utilize Pichia pastoris to produce S-adenosyl-L-methionine (SAM), an intracellular expression vector harboring S. cerevisiae SAM2 was transformed into GS115. A recombinant strain having 2 copies of expression cassette was obtained through G418 resistance screening. This strain had higher SAM synthetase activity and higher SAM production capacity than the original strain, when cultured in medium containing methanol and methionine. The carbon source and nitrogen source of medium was optimized. The results showed SAM production by this strain was closely related to carbon metabolism. With supplementation of 0.2% glycerol every day from the beginning of 3rd day, this strain produced 1.58 g/L SAM when cultured in a medium containing 0.75% L-methionine and optimized carbon and nitrogen source after 6 days.

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RESUMEN

It is important to select favorable reaction media for use of enzymes in organic synthesis. In the presence of PEG with a certain molecular weight, toluene was able to dissolve horseradish peroxidase and the latter maintained a high activity. The HRP reaction conditions, such as ratio of PEG/toluene, water content, pH, and substrate concentration were investigated. It was found that the activity of HRP increased when PEG concentration was low and water content was high. In such a system, the enzyme functioned best at pH 7.0,the concentration of H(2)O(2) and guaiacol being 20 mmol/L and 0.1 mol/L respectively. Under the same condition, no activity was detected in the PEG / ethanol and PEG / dioxane systems. However, in PEG / toluene system, HRP maintained a higher activity and the activity was ten-fold more than that in toluene alone.

RESUMEN

Modification of enzymes can enhance their activities in organic solvents. Horseradish peroxidase (HRP) was modified with methoxy-PEG 5000 which was linked onto HRP peptidal free aminos or onto the aminos introduced through the oxidation of sugar side chains of HRP. It was found the enzyme with PEG linked to peptide chain expressed a nearly same activity as the native HRP in aqueous system. However, PEG modified HRP by oxidation of carbohydrate chains remained only about one third activity of the native HRP. The same results were observed in reversed micelles and in dioxane of less than 30%. Apparently, both PEG modified HRP had the higher activities in high concentration of dioxane. Especially, the activities of the modified HRP in toluene were both enhanced up to nearly 3 fold. When HRP was modified with PEG through the amino groups of peptide chain, it was more stable than the native enzyme in water, dioxane and toluene. However, the stability of PEG-modified HRP by carbohydrate chains was higher in water and lower in both dioxane and toluene in comparison with native HRP.

RESUMEN

Effects of water content (W(0)) and surfactants on the activity of HRP in CTAB/isooctane-n-pentanol reversed micelles were investigated. Based on UV-Vis spectroanalysis and the activity of HRP at various W(0) and CTAB content, it was found that the active-site of HRP was mainly influenced by reversed micelles. However, CTAB had no effect on the active-site of HRP. In addition, the stabilities and Soret band of HRP-H(2)O(2) complexes in reversed micelles were compared to that in aqueous system. The complex HRP-II and HRP-III did disappear more rapidly in reversed micelles than in aqueous system. This demonstrated the enzyme molecules were destabilized in reversed micelles.

RESUMEN

Polyvinyl alcohol (PVA) was selected to develop a new type of membrane for biosensor. The PVA membrane was first activated by cyanogens bromide, followed by coupling with ethyl diamine. After treated by glutaraldehyde, glucose oxidase and lactate oxidase were immobilized onto the membrane respectively. Coupled with oxygen electrode, the enzyme membrane was used to construct glucose and lactate biosensor respectively. The linear range was 5 mg/dl-800 mg/dl for glucose and 1mg/dl-35 mg/dl for lactate, and the response time of these biosensors was 10 seconds.