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With the development of the research on biotechnology and modern pharmacy, the application of enzyme drugs have grown rapidly and enzyme drugs have become an important branch of biopharmaceutics. In this article, some new varieties of therapeutic enzymes, enzyme targets, mechanisms and new technologies of application in therapeutic enzymes were reviewed, and the direction of development of therapeutic enzymes were discussed.

The potential effects of recombinant shark hepatical stimulator analogue (r-sHSA) in liver disease have been revealed in our previous studies. In order to further evaluate its clinic application, we carried out high cell-density fermentation in 5 L fermentor to get enough products. Based on the trials in shaking flask, we optimized the parameters for 5 L fermentor, including medium composition, medium supplement, inducer concentration and induction time, etc. In detail, the improved LB medium (0.97% glycerol, 0.91% yeast extract, 0.72% tryptone, 0.782% KH2PO4, 0.267% K2HPO4.3H2O, 0.062% MgSO4.7H2O, 0.5% NaCl, pH 7.0) is chosen to cultivate the engineering bacteria with the constant fermentation condition (pH 7.0, and the dissolved oxygen concentration is about 25%-30%). When bacterial culture reaches exponential phase, the modified feeding medium (620 g/L glycerol, 94.8 g/L tryptone, 3.3 mL/L trace elements, and 7.5 g/L MgSO4.7H2O) is then supplied through the method of exponential fed-batch mode. After the optical density (OD600) of engineering bacterial culture reaches to 23, the ultimately concentration of 0.5 mmol/L IPTG is added to induce the expression of r-sHSA for 6 h. Results show that the amount of r-sHSA production is (2.662 +/- 0.041) g/L, which is about 13.7 folds of the one optimized before.

This study was intended to establish a method of preparation of recombinant human insulin, with (His)6-Arg-Arg-human proinsulin (RRhPI) expressed by Escherichia coli. After DEAE-Sepharose Fast Flow ion-exchange chromatography, Sephadex G-25 chromatography and refolding, enzyme cleavage and Superdex 75 size exclusion chromatography,the RRhPI expressed by Escherichia coli in inclusion body form was converted to human insulin. The obtained recombinant human insulin was analyzed by SDS-PAGE, HPLC, amino acid composition analysis and bioidentity test (mouse convulsion test). The results indicate that our obtained preparation is highly purified, active recombinant human insulin.

A uridine phosphorylase(UPase) was isolation from Enterobacter aerogenes EAM-Z1 and purified by means of ammonium sulfate precipitation, DEAE-cellulose, Phenyl-Sepharose, DEAE-Sepharose, FPLC ion exchange, and Sephacryl S-200 column chromatography. The purified UPase showed homogeneity on the native polacrylamide gel electrophoresis. The UPase is a trimer of 43 kD subunits. Fifteen residues from the amino terminal end of UPase were identified as MRMVDLIATKRDGGE. The isoelectric point was pH 4.46. Michaelis constant for uridine was 0.29 mmol/L. The UPase has a maximal activity at a pH value of 7.8 and 50 degrees C. The UPase could catalyses the phosphorolysis of uridine, thymidine, 5-Fluorouridine, 5-Fluoro-2'-deoxyuridine, uracil-beta-D-arbinofuranoside, and could also catalyse the synthesis of 5-Fluorouridine, a better prodrug form of the anticancer drug 5-fluorouracil, from 5-fluorouracil and uridine, and 47% uridine was converted to 5-Fluoro-uridine.

Conditions for biotransformation and purification of FUR were investigated. The result showed that when the cell concentration of E. aerogenes was 10% (w/v), the temperature and pH were 7.8 and 60 degrees C respectively, 59.7% UR was converted to FUR. It also demonstrated that the ideal procedure for the purification of FUR is silica gel column chromatography by two elution systems (S1:CHCl3:CH3OH:H2O = 61:13:1, and S2; CH3COOCH2CH3:CH3OH:CH3COOH:H2O = 12:3:3:2) with the purity and yield of 98.0% and 86.0% respectively.