L-Speci®c ꢀ-Amino Acid Benzamidase from B. bassiana
671
®ltration, dried, and crystallized from 95% ethanol. Products from proline and pipecolinic acid did
not precipitate in crystalline form and were isolated by extraction with CH2Cl2 and evaporation of
the extract to give a solid residue which was then crystallized. All products gave satisfactory
spectroscopic data and melting points in agreement with those reported [20].
N-Benzoyltyrosine was prepared by a modi®cation of the above procedure in which methanol
(10 cm3) was added to the reaction mixture following the 2 h stirring period at room temperature.
The mixture was then stirred at room temperature overnight and subsequent steps performed as
described above.
Biotransformations with B. bassiana
A growth medium (1 dm3) composed of glucose (10 g) and corn steep liquor (20 g) in distilled water,
adjusted to pH 4.85 with 1 M NaOH, was distributed in 5 1 dm3 Erlenmeyer ¯asks which were
stoppered with foam plugs and sterilized by autoclaving at 121ꢀC for 20 min. The ¯asks were
allowed to cool and then inoculated under sterile conditions with B. bassiana taken from a 3-day old
agar slope. The ¯asks were allowed to stand overnight at 27ꢀC and then placed on a rotary shaker
(100orbit) at 180 rpm, 27ꢀC. After three days, a solution of the appropriate substrate (0.5 g) in 95%
ethanol (10 cm3) was added, and growth was allowed to continue for a further 3 days. The fungal
mass was removed by ®ltration, and the ®ltrate was adjusted to pH 3 and continuously extracted with
CH2Cl2 for 4 days. The extract was then evaporated to give a residue that was examined by TLC and
chiral stationary phase HPLC and subjected to column chromatography using ethyl acetate/methanol
solvent mixtures containing 2% acetic acid. The solvent composition was changed from 0%
methanol to 40% methanol in 10% steps, and elution with the ®nal mixture was continued until all
product was removed from the column. All products had physical constants (melting points and
optical rotation data) as reported [20] and exhibited spectroscopic data consistent with their
structures.
Acknowledgements
This work was supported by the Natural Sciences and Engineering Research Council of Canada
References
[1] Faber K (1997) Biotransformations in Organic Chemistry, 3rd edn. Springer, Berlin, p 50
[2] Miyazawa T (1999) Amino Acids 16: 191
[3] Drauz, K, Waldmann H (eds) (1995) Enzyme Catalysis in Organic Synthesis VCH, Weinheim,
p 379
[4] Kamphuis J, Boesten, WHJ, Kaptein B, Hermes HFM, Sonke T, Broxterman QB, van den Tweel
WJJ, Schoemaker HE (1992) The Production and Uses of Optically Pure Natural and Unnatural
Amino Acids. In: Collins AN, Sheldrake GN, Crosby J (eds) Chirality in Industry. Wiley,
Chichester, p 187
[5] Wong CH, Whitesides GM (1994) Enzymes in Synthetic Organic Chemistry. Pergamon, Oxford,
p 42
È
[6] Glanzer BI, Faber K, Griengl H (1987) Tetrahedron 43: 771
[7] Chenault HK, Dahmer J, Whitesides GM (1989) J Am Chem Soc 111: 6354
[8] Yang YB, Lin CS, Tseng CP, Wang YJ, Tsai YC (1991) Appl Environ Microbiol 57: 1259
[9] Neuberg C, Mandl I (1950) Enzymologia 14: 128
[10] Kameda Y, Toyoura E, Kimura Y (1958) Nature 181: 1225
[11] Matsumura S, Shin T, Murao S, Sakaguchi M, Kawano T (1985) Agric Biol Chem 49: 3643