Evaluation of penetrating keratoplasties with long-term cryopreserved corneas using simplified method.

Since March 1986, the authors have studied and simplified the corneal cryopreservation technique using a self-made QL Bio-freezer. On the basis of successful animal experiments, penetrating keratoplasties with human cadaver corneas cryopreserved 9 to 571 days (averaging 131 days) were performed in 60 patients (61 eyes). The patients were followed up for 1.5 to 50 months. 41 of the corneal grafts remained clear, 9 became translucent and 11 cloudy. A total of 46 eyes had their visual acuity improved significantly. The results show that clinical use of corneal cryopreservation by the simplified two-step method is noteworthy.

[Asymmetric biosynthesis of chiral styrene oxide].

Used styrene as the sole carbon and energy source, 12 strains of aerobic bacterial and two strains of fungi were screened from a series soil samples. These strains were able to convert styrene to styrene oxide. HPLC and chiral GC method were used for determination the substrate, product and e.e value of culture broth. There were obvious effect on the biomass and epoxidase activity under various conditions in the fermentation. Styrene epoxidation of the strain PS-1206 was studied in different reaction time. 35% of conversion yield and 80% e.e value of (R)-styrene oxide could been obtained at the optimum conditions.

[Cloning, expression and preliminary application of a alpha-hydroxynitrile lyase from cassave].

alpha-Hydroxynitrile lyase (ME-HNLs, E.C. 4.1.2.3.37) from the cyanogenic crop cassava(Manihot esculentz, Crantz) catalyze the condensation of hydrocyanic acid and aldehydes or ketone into (s)-cyanohydrins, which are valuable starting material for various optically active compounds, such as pharmaceuticals and agrochemicals. The cDNA of a ME-HNL were obtained by RT-PCR and cloned. The sequencing result for the cDNA showed that the sequence encoded for the ME-HNL was inconsistent with all those which are published, such as hnl10, hnl24, hnl4. The full sequence analysis demonstrated that the cloned cDNA was about 75.2%, 79.8%, 99.2% homologous to other three reported HNL genes from cassava, respectively, among which the last was the same to the cloned gene except the five base substitution at the site 142, 337, 476, 634 and 636, respectively. The two base substitutions lead to change the amino acid sequence, i.e., Ser113-->Gly113, Phe158-->Tyr158. To construct the recombinant plasmid pET30a-hnl, the cDNA was inserted into an expression vector pET30a. After transformation of pET30a-hnl and induction with IPTG, the ME-HNL was efficiently expressed in E. coli. BL21 (DE3) and reached over 2100 units/L of culture with the specific activity 8.5 u/mg protein. By one simple treatment, incubating 10 minutes at 70 degrees C, the recombinant ME-HNL may be used as an catalyst for production of (S)-mandelonitrile with enantiomeric excess of 95.2% and 98.2% yield.

[Studies on asymmetric synthesis of R-phenylethanolamine by whole cells of Arachnia sp. P163].

Effects of various factors on asymmetric synthesis of R-phenylaminoethanol from aminoacetophenone by the whole cells of Arachnia sp. P163 producing alcohol dehydrogenase for phenylethanol amine was investigated. It found that, although the reduction was inhibited by the substrate and the product, but it has the very high stereoselectivity. The reduction was normaly carried out with 2% glucose for reproduction of coenzyme in the reaction system without oxygen. The conversion yield and ee value of the product achieved 65% and 100%, respectively.