Activation and Stabilization of Penicillin V Acylase from Streptomyces lavendulae in the Presence of Glycerol and Glycols Miguel Arroyo,* Raquel Torres-Guzma ´ n, Isabel de la Mata, M. Pilar Castillo ´ n, and Carmen Acebal Departamento de Bioquı ´mica y Biologı ´a Molecular I, Facultad de Ciencias Biolo ´gicas, Universidad Complutense de Madrid, Spain Penicillin V acylase (EC 3.5.1.11) from Streptomyces lavendulae showed both enhanced activity and stability in mixed water/glycerol and water/glycols solvents. The catalytic activity was increased up to a critical concentration of these cosolvents, but further addition of the latter led to a gradual protein deactivation. The highest stabilizing effect was achieved in the presence of glycerol. Thermal stability was increased proportionally to the concentration of glycerol and glycols in the reaction mixture only if the amount added is below the threshold concentration. Reaction conditions that allow simultaneously enhanced activity and stability in the hydrolysis of penicillin V catalyzed by penicillin V acylase from S. lavendulae could be established. Introduction Nonaqueous enzymology has greatly expanded the potential scope and economic impact of biocatalysis, opening a new field in the biotechnological applications of enzymes (Gupta, 1992). Enzymatic activity sometimes may be improved as a result of the incorporation of a small amount of a water-miscible organic solvent in the reaction mixture (Tan and Lovrien, 1972; Batra and Gupta, 1994). However, aqueous-organic monophasic systems have not become very popular because many water-miscible organic solvents can act as denaturing agents for proteins at moderate concentrations (Mozhaev et al., 1989). Despite this, there are some polyhydric cosolvents that may improve enzyme stability. These cosolvents such as glycerol and glycols, are well-known protective agents against thermal denaturation, and they are less deleterious at high concentrations in the reaction medium (Mozhaev et al., 1989). In particular, glycerol is known to be a strong stabilizing agent of biomolecules (Gekko and Timasheff, 1981a) and is widely employed for storing proteins and enzymes. In 1991, Khmelnitsky and co-workers introduced a new parameter called denaturation capacity (DC), which indicates the protein denaturing strength of an organic solvent on a scale from 0 to 100. In the DC scale of Khmelnitsky, glycerol and ethylene glycol have very low DC values (20.2 and 18.7, respectively), meaning that these solvents exert the least denaturing effect on proteins. In this work, we have systematically studied the effect of increasing the con- centrations of glycerol, ethylene glycol, diethylene glycol, and triethylene glycol on the activity and stability of penicillin V acylase (PVA) from Streptomyces lavendulae. This enzyme catalyzes the hydrolysis of penicillin V to yield 6-amino penicillanic acid (6-APA), an important key intermediate of semisynthetic penicillins (Shewale and Sudakaran, 1997). The use of water/glycerol and water/ glycol mixtures can enhance the hydrolysis rate of penicillin V catalyzed by PVA, in addition to improving its thermal stability. The present work describes a useful medium in the manufacture of 6-APA employing PVA from S. lavendulae. Experimental Section Enzyme Production and Partial Purification. Penicillin V acylase (EC 3.5.1.11) was produced from S. lavendulae (ATCC 13664) by fermentation (Torres et al., 1999), and it was purified 30-fold with a yield of 26.4% as reported elsewhere (Torres et al., 1998). Enzyme Activity. The potassium salt of phenoxy methyl penicillin (PVK) from Sigma (St. Louis, MO) was used as substrate in the enzyme assays. A 50 µL sample of the purified PVA solution was mixed with 85 µL of water, 15 µL of 1 M potassium phosphate buffer (pH ) 8.0), and 150 µL of a PVK solution (45 mg/mL) in water. The reaction mixture was incubated for 30 min at 40 °C under gentle shaking. The reaction was stopped by addition of 0.9 mL of 20% (v/v) acetic acid solution. The reaction mixture was centrifuged, and a 0.9 mL aliquot was processed for estimation of 6-APA by addition of 300 µL of a 0.5% (p/v) solution of p-dimethylamino benzal- dehyde (Sigma Chemical Co., St. Louis, MO) in methanol (Shewale et al., 1987). One activity unit is defined as the amount of enzyme producing 1 µmol/min of 6-APA under the assay conditions. Previous kinetic analyses were performed to make sure that the activity was linear in the assay conditions for 30 min (in buffer and in the presence of cosolvents) using the saturating concentration of penicillin V described above. We employed an enzyme solution with an activity of 1.0 unit/mg protein in all the assays. Effect of Glycerol and Glycols on Enzyme Activ- ity. The effect of glycerol, ethylene glycol, diethylene glycol, and triethylene glycol (Merck, Germany) on penicillin V acylase activity at different solvent concen- trations was analyzed. The solvents were incorporated into 50 µL of enzyme solution prior to the addition of penicillin V. The volume of water was corrected for the added solvent so that the volume remained 150 µL. The enzyme activity, in contact with the solvent, was mea- sured after the addition of 150 µL of PVK solution, so * Tel: (+34) 91-394 41 50. Fax: (+34) 91-394 46 72. 368 Biotechnol. Prog. 2000, 16, 368-371 10.1021/bp000023h CCC: $19.00 © 2000 American Chemical Society and American Institute of Chemical Engineers Published on Web 05/16/2000