ELSEVIER

Isolation of a novel carboxylesterase from Bacillus coagulans with high enantioselectivity toward racemic esters of 1,2=O=isopropylideneglycerol Francesco Molinari,* Oreste Brenna,? Mario Valenti,* Fabrizio Aragozzini”

“Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche Sezione Microbiologia Industriale, UniversitG degli Studi di Milano, Milano, Italia ?Dipartimento di Scienze e Tecnologie Agro-Alimentari, Ambientali e Microbiologiche, UniversitLi degli Studi de1 Molise, Campobasso, Italia

An intracellular carboxylesterase from Bacillus coagulans NUMB 9365 with high stereospecjfic hydrolytic activity toward racemic esters of 1,2-0-isopropylideneglycerol was purified and characterized. The microor- ganism contained wo intracellular carboxylesterases designated as carboxylesterase A and B. Purification was achieved in three steps: precipitation with ammonium sulfate from the crude cellular lysate, ion exchange, and geljiltration chromatography. Carboxylesterase A is stereoselective, has a molecular weight of 70-73 kDa with an isoelectric point of 4.7, and a maximum activity (assayed on a-naphthylcaprylate) at pH 7.0 and 65°C. The enzyme showed good a&&y toward (R)-benzoyl-1,2-isopropylideneglycerol (I$,, = 1.05 mM) and its activity on this substrate was competitively inhibited by (s)-benzoyl-1,2-isopropylideneglycerol. The purified enzyme yielded (s)-1.2-0-isopropylideneglycerol with high enantiomeric excesses (e.e.) by resolution of various racemic esters (97% e.e starting from benzoate ester). 0 1996 by Elsevier Science Inc.

Keywords: Carboxylesterase: Bacillus coapulans: enantioselective hydrolysis; enzyme purification; biotransformation; isopropylideneglycerol

Introduction

(s)-1,2-0-isopropylideneglycerol is an important chiral building block for the synthesis of many optically active compounds such as beta-blockers, prostaglandins, and leu- kotrienes.’ Its chemical synthesis is too costly for industrial applications since it requires chiral starting materials, mul- tistep reactions, and expensive separation procedures. Bio- logical methods to obtain enantiomerically pure (s)-1 ,2-0- isopropylideneglycerol have thus received increasing atten- tion.?-’ We previously described the microbial hydrolysis of benzoyl-(R,s)-1,2-O-isopropylideneglycerol by Bacillus

Address reprint requests to Professor Fabrizio Aragozzini, Dipartimento di Scienze e Tecnologi Alimentari e Microbiologiche, UniversitL degli Studi di Milano, via Celoria 2, 20133, Milano, Italy Received 27 September 1995; revised 30 January 1996; accepted 6 Feb- ruary 1996

coagulans. Resting cells of the microorganism hydrolyzed the racemic ester producing the (s)-alcohol with a 45% yield and 88% enantiomeric excess (e.e.)s (Figure I).

In the present work, we report the purification and char- acterization of the esterase/lipase responsible for this trans- formation. The enantios&ective hydrolytic activity of the purified enzyme toward different racemic esters of 1,2-iso- propylideneglycerol was also investigated.

Materials and methods

Materials

The chemicals in this study were reagent grade. 1,2-O-isopropy- lideneglycerol esters were prepared as described by Aragozzini et a1.8 The chromogenic reactive Fast Garnet GBS salt was from Sigma Chemical (St. Louis, MO). Carrier ampholites, Sepharose CL-6B and Sephadex G-75 were obtained from Pharmacia Bio- tech (Uppsala , Sweden). Chromatographic accessories were: peri- staltic pump, Watson-Marlow 101 v; detector, Gilson 112 UVNIS; microfraction collector, Gilson 203; and recorder, Gilson Nl.

Enzyme and Microbial Technology 19:551-556, 1996 Q 1996 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

0141-0229/96/$15.00 PII SO141-0229(96)00066-X

Papers

Figure 1 Enantioselective hydrolysis of racemic esters of 1,2- 0-isopropylideneglycerol

Polyacrylamide gel electrophoresis (PAGE) and isoelectric focus- ing were performed using a LKB apparatus (generator 2197, mul- tiphor 2117, multitemp thermostat II 2119). Protein was stained with Coomassie brilliant blue R 250 (BioRad, Richmond, CA). A pH-stat apparatus (PHM 64, titrator TIT 60, servograph REC 6 1, autoburette ABU 12: Radiometer, Milan, Italy) was used to mea- sure the hydrolytic activity of the enzyme.

Microorganism

Bacillus coagulans NCIMB 9365 was maintained and cultured as described by Aragozzini et aL8

Purification procedure

STEP 1 (cell disruption). Washed cells (22 g) were suspended in 480 ml of 0.1 M phosphate buffer pH 6.8 and treated with lysozyme (2 mg ml-‘) for 60 min at 37°C followed by RNase treatment (0.02 mg ml-‘) for 1 h. The suspension was centrifuged (5,000 g for 30 min) to remove cell debris. The supematant was retained.

STEP 2 (ammonium sulfate precipitation). Ammonium sulfate was added to the cell-free supematant to a final concentration of 40%. The mixture was centrifuged and the precipitate (ppt40) was collected. The ammonium sulfate concentration of the recovered supematant was further increased to 80% saturation. The precipi- tate (ppt80) was recovered by centrifugation (5,000 g for 30 min). The precipitates, ppt40 and ppt80, were independently dissolved in 20 mM NaH,PO,/NaOH buffer pH 6.0. The resulting solutions were desalted on a Sephadex G25 column equilibrated and eluted with the same buffer containing 0.01% sodium azide.

STEP 3 (ion-exchange chromatography). DEAE-Sepharose ion-exchange chromatography was performed on a 20 x 2 cm glass column containing 30 cm3 of gel. Desalted ppt40 (35 ml) was loaded and the column was washed with 20 mM NaH,PO,/NaOH buffer pH 6.0 containing 0.01% sodium azide. Elution was carried out applying a linear gradient of sodium chloride (from 0 to 0.3 M,

total volume 300 ml) at a flow rate of 45 ml h-l. Fractions of 4 ml were collected. Pooled enzyme A fractions (32 ml) were concen- trated to 3.5 ml by ultrafiltration (cut-off 10,000).

STEP 4 (gel filtration chromatography). Portions of 2 ml of the previously concentrated solution were subjected to gel filtration on a semipreparative 95212 Amicon column (1 m x 16 mm) contain- ing 16.5 g of Sephadex G75 gel preswollen with 50 mM phosphate buffer pH 7 containing 0.1 M NaCl. The column was connected with a 213 1 Microperpex peristaltic pump and standard chromato- graphic accessories. Samples were applied and eluted with the same buffer with a flowrate of 15 ml h-l. The elution time was

compared with times obtained from eluting proteins of known MW (bovine serum albumin, 67.000 Da; ovalbumin. 43,000 Da; chy- motrypsin. 25,000 Da; and ribonuclease A. 13.700 Da).

Protein determination

Protein concentrations were measured by the Biuret reaction” by comparison with a standard curve generated using bovine serum albumin.

Enzyme assays

Esterase activity was assayed using c1- and P-naphthylacetate, (Y- and P-naphthylbutyrate, and cy-naphthylcaprylate as substrates by measuring the absorbance (560 nm) relative to the chromophores originated by reaction of the hydrolyzed substrate with Fast Garnet GBS salt. One unit (U) is defined as the amount of enzyme that catalyzes the hydrolysis of 1 pmol of naphthyl ester in 20 min at 45°C. Absorbances were converted into wmol products by using calibration curves. The substrate solutions were prepared by add- ing the naphthyl derivatives in 1 ml of acetone to 99 ml buffer solution (0.1 M Tri-HCl pH 7). The reaction mixtures were ob- tained by adding 1.5 ml of the substrate solution to 1 ml of the enzymatic fraction in 0.1 M Tris-HCl pH 7. The same substrates and chromogenic compound were used to stain bands with esterase activity on polyacrylamide gel following electrophoresis. The ther- mal stability was evaluated by incubating the enzyme in 2 ml of buffer (0.1 M Tris-HCl pH 7) at various temperatures. After cool- ing in ice-water, the remaining activity was determined at 30°C.

1,2-0-isopropylideneglycerol ester hydrolysis and determination of enantioselectivity

A solution (0.5 ml) of racemic 1.2-O-isopropylideneglycerol es- ters in acetone (20 mg ml-‘) was treated with 0.3 ml of a solution of bis(2-ethylhexyl) sulfosuccinate sodium salt (AOT) in acetone (1 mg ml-‘). After evaporation of acetone under a nitrogen stream, 0.2 ml of crude lysate (protein content 17.9 mg) or purified samples (protein content ranging from 0.12-2.2 mg) were added together with 4.8 ml of 0.1 M phosphate buffer pH 6.8. The reac- tion mixture was incubated at 45°C for 12-24 h. The hydrolysis molar conversions were evaluated by gas chromatography (GC, Carbowax 1540) of the free alcohol using n-octanol as internal standard. To determine the e.e. of the obtained alcohol, it was first extracted with ethyl acetate from the NaCl-saturated reaction mix- ture and then esterified with R-(+)-cw-methoxy-a-trifluoro- methylphenylacetic acid. lo The diastereomeric esters were then analyzed by capillary GC (SE 52).

Gel electrophoresis

Polyacrylamide gel electrophoresis (PAGE) was performed as de- scribed by Moore” at room temperature using 120 V in 25 mM Tris, 200 mM glycine pH 8.9. The migration marker was bromo- phenol blue. To locate esterase activity in the gel, a strip was cut and soaked in a solution of the substrate (20 mg of the naphthyl- derivatives in 99 ml of buffer) and the chromogen. The enzymes with esterase activity were separated from the electrophoretic gels by removing and eluting with 0.2 M phosphate buffer pH 6.8 the area of gel containing the enzyme.

Sodium dodecyl sulfate (SDS-PAGE) of the purified enzyme was done according to the methods of Laemmli12 using 15% gels at pH 8.8. The same procedure has been used to set up a calibration curve with marker proteins from the LMW Electrophoresis Cali- bration Kit supplied by Pharmacia.

552 Enzyme Microb. Technol., 1996, vol. 19, November 15

Isoelectric focusing

Isoelectric focusing was performed using 7% polyacrylamide gels. The mixture to be polymerized (60 ml) contained Ampholine pH 3.5-10 (0.5 ml), Ampholine pH 4-6 (2 ml), and Ampholine pH 5-7 (0.5 ml). Electrode solutions were 1 M lysine and 1 M phos- phoric acid. Samples (15 p,l) were applied. The gels were run at 30 W for 150 min with the maximum voltage set at 1,000 V.

K,,, and Ki determination

K, of the enzyme toward (R-benzoyl-1,2-O-isopropylideneglyc- erol was determined at 50 5 O.l”C using a pH-stat apparatus. NaOH ( 10 mM) was used to titrate the product obtained by hydro- lysis of various amounts of (a-benzoyl-1.2-O-isopropylidene- glycerol dissolved in 1.95 ml of 50 mM KC1 containing 50 p,l of enzymatic solution and 0.1 mg of AOT. The Michaelis constant was obtained from Lineweaver-Burk analysis of the data using the least squares method. Ki determination was achieved using the same procedure but adding different amounts of (s)-benzoyl-1.2- O-isopropylideneglycerol as inhibitor.

Results

Purification of the intracellular enzyme

Bacillus coagulans NCIMB 9365 was grown in a 10-l Che- map fermentation apparatus, cells were centrifuged, resus- pended in phosphate buffer pH 7, and disrupted by treat- ment with lysozyme and RNase. The resulting lysate con- taining cellular debris was recentrifuged. Nondenaturing PAGE of the crude lysate showed two bands of esterase activity assayed using a-naphthylacetate as substrate (lane 1, Figure 2).

Figure 2 Esterase-stained PAGE. Cell-free extract, lane 1; ppt80, lane 2; ppt40, lane 3; and enzyme A after gel filtration chromatography, lane 4

The crude lysate was fractionated by precipitation at 40% (fraction named ppt40) and 80% (ppt80) ammonium sulfate concentration and the two fractions desalted with Sephadex G25. In both the fractions, two bands containing esterase activity were observed. The enzyme relative to band A, hereafter called enzyme A, was mostly present in the ppt40 fraction (lane 3, Figure 2) while band B was more evident in the ppt80 fraction (lane 2. Figure 2).

The bands relative to carboxylesterase A and B were eluted from the electrophoretic gel and tested for enantio- selective hydrolysis of benzoyl-(R,s)-1,2--O-isopropyli- deneglycerol. Enzyme B showed very low activity and ste- reospecificity while enzyme A hydrolyzed the racemic mix- ture with 95% e.e. Consequently enzyme A, mainly contained in ppt40 fraction, was separated by ion-exchange chromatography on a DEAE Sepharose CL-6B column equilibrated with 20 mM phosphate buffer pH 6. Elution was performed with increasing amounts of NaCl (Figure 3).

Enzymes A and B were eluted in two fractions. The enzyme in the first one showed an electrophoretic band coincident with the one of esterase B while the enzyme in the second one is coincident with band A.

a-Naphthylbutyrate. B-naphthylbutyrate, and a-naph- thylcaprylate were also tested as substrates. The acetate and butyrate esters were hydrolyzed by both the enzymes while o-naphthylcaprylate was hydrolyzed only by enzyme A. The fractions containing enzyme A showed a markedly higher activity toward cr-naphthylcaprylate (0.7 U ml-‘) than toward acetate (0.2 U ml-‘).

Fractions containing carboxylesterase A #showed several bands on Coomassie-stained PAGE. The enzyme was there- fore further purified by gel filtration on a Sephadex G-75 column equilibrated with 20 mM phosphate buffer pH 7 containing 0.1 M NaCl. Enzyme A fractions were pooled and concentrated. PAGE showed one band of esterase ac- tivity (lane 4, Figure 2) and only one other notable band on Coomassie-stained gels with an estimate of the purity of the carboxylesterase A preparation at 70-75%.

NaCl concentration [M]

* “,

i i i 0.3

a I N&l ’ k I 0,2

0,l i

+ \

I t

O,l

. T l

Figure 3 Separation of carboxylesterases A and B by DEAE Sepharose CL-66 chromatography with a NaCl concentration gradient (U = amount of enzyme that catalyzes the hydrolysis of 1 umol of a-naphthylacetate in 20 min at 48°C

Enantioselectivity carboxylesterase from Bacillus coagulans: F. Molinari et al.

Enzyme Microb. Technol., 1996, vol. 19, November 15 553

Papers

A summary of the purification of carboxylesterase A is reported in Table 1. Activity was determined using a-naph- thylcaprylate as substrate. Enzyme A was purified approxi- mately 34-fold with a recovery of 34% with ion-exchange chromatography. Only a 38-fold enhancement of purity and a recovery of 23% of the total activity were obtained after additional purification with gel filtration chromatography.

Hydrolysis of 1,2-0-isopropylideneglycerol esters

Table 2 shows the hydrolytic activity of the enzymatic frac- tions toward benzoyl-1,2-O-isopropylideneglycerol at dif- ferent stages of purification. In a subsequent set of experi- ments, a study was performed on the hydrolysis of racemic esters of 1,2-0-isopropylideneglycerol with aliphatic acyl chains of different length catalyzed by carboxylesterase A contained in the fraction from gel filtration (Table 3).

Tests performed using whole cells of BaciEZus coagulans had previously shown that hydrolysis of acetate, propionate, and caprylate esters of 1,2-O-isopropylideneglycerol pro- ceeded with low enantioselectivity (5-10%). Carboxylester- ase A does not hydrolyze the acetate esters while higher molar conversions and higher e.e. were observed with longer acyl chains.

Characterization of the carboxylesterase A and B

Carboxylesterases A and B had apparent molecular weights of 73 and 60 kDa, respectively, on SDS-PAGE. The relative mobility of carboxylesterase A on Sephadex G-75 gel fil- tration medium was consistent with a protein of 70 kDa. The isoelectric point of the two esterases was determined as 4.75 for enzyme A and 4.60 for enzyme B.

The esterases subjected to SDS-PAGE in the presence or absence of B-mercaptoethanol showed similar profiles rul- ing out the presence of subunits bridged by disulfide bonds. Hydrolysis of o-naphthylcaprylate by enzyme A was in- spected in the pH range between 5.5 and 11.0 at 50°C. It showed a broad symmetrical peak with the highest activity at pH 9.0.

Influence of temperature was investigated using phos- phate buffer at pH 7.0 and 9.0 (Figure 4).

The absolute maximal activity was detected at 65°C in pH 7.0 buffer whereas in the pH 9.0 buffer, the higher activity was observed at 50°C.

Table 2 Hydrolysis of racemic benzoyl-1,2-O-isopropyli- deneglycerol at different purification steps of the enzymes con- tained in 19. coagolans

Purification step Molar conversion Walcohol

(%) (e.e.)

Crude extract 40% Ammonium sulfate

(ppt40) Fractions of enzyme A

from DEAE Sepharose Fractions of enzyme B

from DEAE Sepharose Fractions of enzyme A

from Sephadex G-75

59 84

45 92-93

32 94-95

6 0

24 97-98

The thermostability was then investigated incubating the enzyme at pH 7.0 at different temperatures and following the timecourse of activity toward o-naphthylcaprylate at 65°C and pH 7.0. In the range between 45-55”C, no dra- matic changes in the activity were observed for 48 h while at 60°C the half-life of the enzyme is only 30 min.

Incubation of partially purified carboxylesterase A with 1 mM phenylmethylsulfonyl fluoride (PMSF) and mercuric chloride at 45°C for 30 min resulted in 100% and 20% inhibition, respectively. Total inhibition by PMSF is a char- acteristic feature of many serine esterases while the low inhibition following mercuric chloride treatment seems to indicate that thiol groups, if present, are not essential for activity. Enzyme B is partially inhibited by PMSF (40%) and mercuric chloride (85%).

The Lineweaver-Burk plots with R-benzoyl-1,2-O-iso- propylideneglycerol as substrate indicate that carboxylester- ase A obeys Michaelis-Menten kinetics with a Km of 1.05 mM for the partially purified enzyme acting on R-benzoyl- 1,2-o-isopropylideneglycerol (which after hydrolysis gives the s-alcohol).

The inhibition effects of s-benzoylisopropylideneglyce- rol on enzymatic R-ester hydrolysis was investigated using inhibitor concentrations ranging from 0.214.85 mM. The compound was found to be a competitive inhibitor with a K, value of 1.45 mh4.

Discussion

The use of whole cells as biocatalysts in stereoselective synthesis is often defective. Incomplete enantioselectivity attained with whole cells could be due to the activity of a single enzyme with low stereospecificity or different en- zymes with different or even opposite stereospecificity. In the latter cases, an improvement in the enantioselectivity may be achieved by using purified enzymes. It has been reported that resting cells of Bacillus coagulans NCIMB 9365 hydrolyzed benzoyl-(a$)-1,2-O-isopropylideneglyc- erol with an 88% e.e (s-alcohol) while very low stereo- specificity was observed with an aliphatic acyl chain.8

In the present study, an improvement in the enantiose- lective hydrolysis of racemic 1,2-O-isopropylideneglycerol esters was achieved by using a partially purified carboxyl- esterase from Bacillus coagulans NCIMB 9365. Two intra- cellular esterases were detected and called A and B. Only the first one showed stereoselective hydrolytic activity to- ward racemic esters of 1,2-0-isopropylideneglycerol. They are distinguishable on the basis of different substrate speci-

Table 3 Enantioselective hydrolysis of different racemic esters 1,2-0-isopropylideneglycerol catalyzed by carboxylesterase A

Substrate Molar conversion k&alcohol

(%) (e.e.)

Acetate 0 I Propionate 15 80 Butyrate 24 94 Caprylate 23 94

554 Enzyme Microb. Technol., 1996, vol. 19, November 15

Enantioselectivity carboxylesterase from Bacillus coagulans: F. Molinari et al.

Table 1 Purification of carboxylesterase A from Bacillus coagulans

Purification step Activity Protein (U ml-‘)

Specific activity (mg ml-‘) (U mg-‘) Yield (%I

Purification (fold)

Crude extract 2 89.3 0.02 100 1

40% Ammonium sulfate (ppt40) 1.2 11.0 0.11 58 6

DEAE Sepharose 0.7 1.0 0.70 34 34

Sephadex G-75 0.5 0.6 0.83 23 38

ficity since c-w-naphthylcaprylate is hydrolyzed only by es- terase A.

The stereoselective carboxylesterase A is expressed in- tracellularly (unlike most industrial Bacihs enzymes). Its partial purification was accomplished with standard proce- dures. With the partially purified enzyme, the hydrolysis of benzoyl-(R,s)-1,2-O-isopropylideneglycerol proceeds with higher e.e. (97-98%) than that observed for the whole cells; moreover, the purified carboxylesterase produces the (s)- alcohol with good optical purity also starting from racemic aliphatic esters (80% e.e. for propionate, 94% e.e for buty- rate, and 94% e.e. for caprylate). Enantiomeric excesses increase by using enzymes with a higher degree of purifi- cation. It is noteworthy that simple precipitation of the cell lysate with 40% ammonium sulfate gives an enzymatic mix- ture able to hydrolyze the racemic benzoyl ester with a significant increase in the enantioselectivity (92-93% e.e) with respect to the whole cells.

The occurrence of a second carboxylesterase with non- stereospecific activity on racemic 1,2-O-isopropylidene- glycerol and different inhibition characteristics suggests a potential to achieve higher enantioselectivities by using whole cells. Enzyme B synthesis could be inhibited by se- lecting appropriate conditions for the microorganism growth or its activity can be selectively blocked by suitable inhibitors. The presence of two different intracellular ester- ases where one is unable to hydrolyze esters with the acyl portion of four or more carbon atoms is similar to that observed for B. subtiliy I3 i . .

/ \

80

70

60 -

50 -

40 \

*pH 9.0 +pH 7.0 \ 30

45 50 55 60 65 70

temperature f”C)

Figure 4 Temperature-activity profile for purified carboxyles- terase A at pH 7.0 and 9.0 using a-naphthylcaprylate as sub- strate (% activity referred to maximum activity observed at 65°C and pH 7.0)

The use of Bacillus carboxylesterase for the production of valuable chiral building blocks seems to be a promising approach. The stereospecific hydrolysis of racemic menthyl acetate was achieved using B. subtilis. ‘+I5 Resolution of R,s-naproxen enantiomers by hydrolysis of racemic ethyl ester was obtained by employing high stereospecific car- boxylesterase from a Bacillus sp.i6 while an extracellular esterase from a B. sphaericus strain hydrolyzed ethyl 2-hy- droxyalkanoates with excellent enantioselectivity.”

The results obtained with the isolated enzyme from Ba- cillus coaguluns NCIMB 9365 are remarkable. This enzyme could be applied to the enantioselective hydrolysis of race- mic 1,2-0-isopropylideneglycerol esters to produce the im- portant chiral intermediate (s)-1,2-O-isopropylideneglyc- erol with high optical purity.

Acknowledgments

The authors with to thank Prof. M. Duranti for helpful ad- vice and discussion.

References

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

Jurczak. J., Pikul, S., and Bauer, T. (R) and (s)-O-isopropylidene- glyceraldehyde in stereoselective organic synthesis. Tetrahedron 1986, 42,447-188 Ladner, W. E. and Whitesides, G. M. Lipase-catalyzed hydrolysis as a route to esters of chiral epoxy alcohols. 1. Am. Chem. Sot. 1984, 106,7250-725 1 Breitgoff, D., Laumen, K., and Schneider. M. P. Enzymatic differ- entiation of the enantiotopic hydroxymethyl groups of glycerol. Synthesis of chiral building blocks. J. Chem. Sot. Chem. Commun. 1986, 1523-1524 Fuganti, C., Grasselli, P., Servi, S., Lazzarini, A., and Casati, P. Penicillinacylase and cY<hymotrypsin catalyzed hydrolysis of phen- ylacetate and phenylpropionate esters of 2,2-dimethyl-1,3-dioxo- lane4methanols. J. Chem. Sot. Chem. Commun. 1987. 538-539 Aragozzini, F., Maconi, E., Potenza, D., and Scolastico, C. Enan- tioselective microbial reduction of monoesters of 1,3- dihydroxypropanone: Synthesis of (R) and (s)--1.2~O-isopropyli- deneglycerol. Synthesis 1989, 225-227 Maconi. E., Gualandtis, R., and Aragozzini, F. Hydrolytic microbial resolution of 1,2_O_isopropylideneglycerol: Improvements by us- ing esters with homologous fatty acids. Biotechnol. Len. 1990. 12, 415-418

Bosetti, A.. Bianchi, D., Cesti, P.. and Bolini, P. Lipase catalyzed resolution of isopropylidene glycerol: Effect of co-solvents on en- antioselectivity. Biocatalysis 1994. 9, 71-77

Aragozzini. F., Maconi, E., and Scolastico, S. Enantioselective hy- drolysis of benzoyl 1,2-&isopropylideneglycerol by Bacillus co- agulans. Appl. Microbial. Biotechnol. 1991, 34, 433-435 Gomall, A. G., Bordawill, C. J., and David, M. M. Determination of serum proteins by means of the Biuret reaction. J Biol. Chem. 1949, 177, 75 l-766

Dale, J. A., Dull, D. L., and Mosher, H. S. o-Methoxy-o-trifluoro-

Enzyme Microb. Technol., 1996, vol. 19, November 15 555

Papers

methylphenylacetic acid, a versatile reagent for the determination of enantiomeric composition of alcohols and amines. J. Org. Chenr. 1969, 34, 2534-2539

11. Moore, W. E. C., Hash, D. E.. Holdemann, L. V., and Cato, E. P. Polyacrylamide slab gel electrophoresis of soluble proteins for stud- ies of bacterial flores. Appl. Environ. Microbid 1980, 39, 900

12. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nurure 1970, 227, 680-685

13. Higerd, T. and Spizizen, J. Isolation of two acetyl esterases from extracts of Bncillus subtilis. J. Bacreriol. 1976, 114, I 18&l 192

14. Brookes, I. K., Lilly, M. P., and Drozd, L. A. Stereospecific hydro- lysis of d,l-menthyl acetate by Bacillus subtilis: Mass-transfer in-

teractions in a liquid-liquid system. En,-vme Microb. 7’rchrrol. 1986, f&53-57

15. Brookes, I. K., Lilly, M. P., and Drozd. L. A. Use of immobilized Bacillus subtilis for the stereospecific hydrolysis of d,l-menthyl acetate. Enzyme Microb. Technol. 1987. 9, 217-220

16. Quax, W. J. and Broekhuizen, C. P. Development of a new Bucillu.s carboxyl esterase for use in the resolution of chiral drugs, Appl. Microbid. Biotechnoi. 1994, 41, 425-43 1

17. Jackson, M. A., Labeda, D. P., and Becker, L. A. Enantioselective hydrolysis of ethyl 2-hydroxyalkanoates by an extracellular esterase from a B. sphaericrts strain. Enzyme Microb. Technol. 1995, 17, 175-179

556 Enzyme Microb. Technol., 1996, vol. 19, November 15