Activities of Candida rugosa lipase and other esterolytic enzymes coated on glass beads and suspended in substrate and water vapor: Enzymes in thin liquid films* Neil W. Ross and Henry Schneider Division o f B i o l o g i c a l S c i e n c e s , N a t i o n a l R e s e a r c h C o u n c i l o f C a n a d a , O t t a w a , Ontario, C a n a d a

Candida rugosa lipase was enzymatically active when coated on glass beads and exposed to mixtures o f substrate and water vapor over a range o f relative humidities up to 100%. Evidence was obtained f o r operation o f the enzyme in a thin liquid fihn o f concentrated buffer on the surJbce o f the glass beads. Formation o f the thin film was associated with hygroscopicity o f the barfer used to suspend the enzyme in preparation o f the enzyme-coated beads. At some buffer concentrations estimated to be on the bead surface, the enzyme was partially soluble and both soluble and insoluble forms were enzymatically active. The vapor mode o f operation over a range o f relative humidities had comparatively small effects on kinetic constants fi~r hydrolysis o f ethyl acetate, which were also similar to those in phosphate buffer. The extent o f reaction occurred in the order hydrolysis > alcoholysis > ester interchange > esterification. Reaction preference between alcoholysis and hydrolysis changed as acyl chain length o f sabstrate increased with C. rugosa lipase, as well as with Rhizopus arrhizus lipase and porcine liver esterase, with details depending on the enzyme. The vapor mode approach has the potential o f being, used with a wide variety o f substrates, as shown by the ability to obtain hydrolysis at 30~C with sabstrate vapor pressures as low as 0.08 mm Hg attd with substrate.s with boiling points as high as 206°C.

Keywords: Enzyme: lipase: Candida rugo.~a: cstcrasc: hydrolysis: water activity: thin tilm: vapor: gas

Introduction E n z y m e s have traditionally been employed in aqueous solution in either a dissolved or a suspended form. Recently, the range of solvents that can be used for e n z y m e reactions has been expanded by the demonstration that some enzymes function when suspended in nonaqueous solvents that contain relatively low concentrations of water, t-3 An additional expansion of the range of conditions under which enzymes can be used depends on the ability of some to function when suspended as solids in mixtures of water and substrate vapor. At least one system functions with vacuum-

*Issued as NRCC Number 31930 Address reprint requests to Dr. Schneider at the Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada KIA 0R6 Received 8 May 1990: reviscd 19 September 1990

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dried enzyme in the absence of added water: hydrogenase-catalysed conversion o f o r t h o to para hydrogen 4 and exchange between H 2 and 2H 2 to form H2H. 5 However, most systems studied had water vapor present, the concentration of which could have large effects. Those using well-defined systems and relative humidities less than 100% include the oxidation of methanol vapor by Pichia p a s t o r i s methanol oxidase, 6 reaction of a number of enzymes in the glycolytic pathway with solid substrates, 7 hydrolysis of urea by urease, ~ and reaction of volatile haptens with antibodies. 9 There is an earlier literature dealing with the effects of relative humidities less than 100% on e n z y m e reactions in seeds and foods. 10-1z The potentials of systems where e n z y m e s are suspended in mixtures of substrate and water vapor are relatively unexplored, in contrast to those where enzymes are placed directly in aqueous or nonaqueous solvents. The approach could have benefits for reac-

~; 1991

Butterworth-Heinemann

Gas-solid lipase reactions: N. W. Ross and H. Schneider tions involving volatiles. Mass transfer would be more rapid than in solution. The relatively small amounts of solvent involved could facilitate product purification as well as disposal of residual waste. Furthermore, substrate use might not be limited by water solubility, while volatile toxic or inhibitory products might be removable continuously. The use of enzymes suspended in substrate and water vapor bears analogy to the use of inorganic catalysts in heterogeneous vapor phase reactions. ~3 The present study investigated factors in the reactions in substrate and water vapor of Candida rugosa lipase and other ester hydrolases coated on glass beads. An esterase system was chosen for study, in part, because of the volatility of esters and the interest in esterase-mediated production of synthons (re(. 14 and references cited therein).

Methods

Materials Lipase from Candida rugosa (formerly Candida cylindracea; 93.1 × 103 units mg-~ protein, 23 × 103 units m g i solid), Rhizopus arrhizus lipase (431 × 103 units mg-~ protein), porcine liver esterase (160 units mg -~ protein), Rhizopus protease (0.58 units mg-J protein), bovine pancreatic trypsin (I0 x 10-~ units mg -~ protein), bovine pancreatic chymotrypsin (55 units mgprotein), and glass beads (212-300 p.m in diameter) were purchased from Sigma (St. Louis, MO, USA). The beads were acid etched prior to use by steeping in 10% nitric acid for 2 h followed by extensive washing with distilled water and air drying. Ethyl propionate was obtained from Sigma and other esters from Aldrich Co. (Milwaukee, WI, USA).

Enzyme treatment In an initial screening for enzymes that could hydrolyse esters when presented as vapors, enzymes supplied as solids were used as such. Porcine liver esterase and R. arrhizus lipase, which were obtained as suspensions in buffer containing (NH4)2SO 4, were dried onto acidetched glass beads (3/zg protein mg ~ beads and 0.32 ~g protein mg -l beads, respectively) as follows. (NH4)2SO 4 was removed by two cycles of dilution and concentration with 50 mM potassium phosphate buffer pH 7.0 using a Centricon microconcentrator (Amicon Canada Ltd., Oakville, Canada). The enzyme solutions were then mixed with glass beads and air dried at room temperature until free-flowing. For detailed studies with C. rugosa lipase, the enzyme was coated onto acid-etched glass beads by dissolving 2.7 mg protein in 2.86 ml of 50 mM potassium phosphate buffer pH 8.0, mixing with 5.72 g acidetched glass beads, and drying as described above.

vapor mode approach. A weighed amount of enzyme or enzyme-coated glass beads was placed in a 400-/~1 centrifuge tube which was inserted into a 14-ml serum vial that contained 0.5 ml of the saturated salt solution used to control relative humidity. The vial was sealed with a butyl rubber septum and the enzyme preequilibrated with water vapor by storage at the incubation temperature for 30 to 60 min. The reaction was initiated by addition of substrate to the saturated salt solution through the rubber cap using a microliter syringe. The amounts of substrate used and incubation temperatures are specified in Results. Aliquots of the liquid or vapor phases were removed through the cap with a syringe for product analysis by gas chromatography. The solutions used to control relative humidity consisted of water saturated with KNO 3, KCI, NaCI, and NaBr(2H20), which provided relative humidities at 30°C of 92%, 84%, 75%, and 56%, and at 25°C of 94%, 84%, 75%, and 58%, respectively.~5 To examine the solubility of C. rugosa lipase in concentrated potassium phosphate buffer, 1 mg of enzyme was suspended in 100/.d of 2.6 and 3.6 M K2HPO 4 adjusted to pH 8.0, centrifuged (2 min at 10,000g), and the supernatant filtered through a 0.45-/~m membrane. To measure the activity of the soluble and supernatant fractions against ethyl acetate vapor, the same procedure was used as in the paragraph above, except that the enzyme-coated beads were replaced with an aliquot of the supernatant fraction or the pellet, and relative humidity was controlled by 0.5 ml of the phosphate buffer used to precipitate the lipase. The amount of ethyl acetate added was i /xl and incubation was for 5 h at 30°C. For kinetic studies of ethyl acetate hydrolysis by C. rugosa lipase when suspended in substrate and water vapor, 50 mg of enzyme-coated glass beads (4/~g protein) was used with incubation times of 6 to 8 h at 25°C. A temperature of 25°C was used instead of 30°C, as for most other experiments, to avoid temperature-induced effects on shifting reaction mixtures between incubator and room temperature. For kinetic studies in bulk aqueous solution, 50 mg of enzyme-coated beads was added directly to 0.5 ml of 0.02, 2.6, or 3.6 M potassium phosphate pH 8.0 buffer and incubated as above. Ester interchange experiments differed by the inclusion of a small vial containing a nonvolatile solvent, 100/xl methyl cellosolve, which was used to assist in trapping products. The reaction was initiated by adding substrates to the aqueous phase. Total product formation was determined from the sum of products in the aqueous and organic phases. Water uptake by the beads -+ buffer --+ enzyme was determined by the Karl Fischer titration method 16after exposure to controlled relative humidities for 24 h.

Enzyme stability in vapor mode Assay procedures Unless indicated otherwise, the same general procedure was used to measure enzyme activity using the

Beads coated with C. rugosa lipase were incubated at 30°C with ethyl acetate at relative humidities of 56% to 100% for 5 days, air dried for 8 days, and then reassayed

Enzyme Microb. Technol., 1991, vol. 13, May

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Papers Table 1 Initial screening of enzymes for hydrolytic activity against ethyl acetate and butyl acetate vapors % Hydrolysisa Enzyme (amount) Lipase, Candida Rugosa (1 mg) Lipase, Rhizopus arrhizus 136 ktg)b Esterase, porcine liver (30 #g)b Protease, Rhizopus (9 mg) Protease, Streptomyces griseus (9 mg) Trypsin (9 mg) Chymotrypsin (9 mg)

Relative humidity (%)

Ethyl acetate

Butyl acetate

90

90

95

83

15

70

83

3

8

90

22

90

0.9

83 100

0.7 0

0 1

a 20 h incubation at 37°C b Dried on glass beads

at 92% relative humidity. The incubations with substrate employed 1 tzl of ethyl acetate.

Distribution o f ethyl acetate between liquid and vapor phases Partitioning of ethyl acetate between vapor and salt solutions in the sealed 14-ml vials was determined by measuring the amount of ester remaining in the aqueous phase after adding a known amount to the system and allowing equilibration to occur. The ester was added directly to 0.5 ml of saturated salt solution placed on the bottom of the vial and the vial then incubated at 25°C for 4 h with intermittent shaking. The amounts of ester used are given in Results. The vapor pressure of ethyl acetate corresponding to the amount present in the vapor space was computed from the ideal gas law PV = nRT, where P = partial pressure of the gas (mm Hg), V = volume (liters), n = numbcr of moles, R = gas constant (62.40 liter, mm H g ' degree- ~" mole ~); and T = temperature (°K).

Analytical methods Substrates and products were analyzed with a HP-5890 gas chromatograph (Hewlett-Packard) fitted with a DBWAX column using helium as the carrier gas.

Results Enzyme screen In an initial step, selected esterases, lipases, and proteases were screened at several relative humidities to identify those with the ability to hydrolyse esters in an apparent vapor-solid mode (Table 1). With the exception o f c h y m o t r y p s i n , several enzymes yielded positive results with either ethyl acetate, butyl acetate, or both at relative humidities less than 100%. Chymotrypsin

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was the only enzyme that required a relative humidity of 100% to manifest activity. Candida rugosa lipase exhibited extensive hydrolysis of both substrates and was chosen for detailed study.

State o f enzyme on surface o f glass beads Water was taken up by the beads -+ buffcr +- C. rugosa lipase, and the amount decreased as relative humidity decrcased (Table 2). While the beads alone took up water, more was taken up when buffer was present, and the addition of e n z y m e caused a further small increase in uptake. The ratio of weights of water to enzyme, 37.9 to 3.4 g water/g cnzyme, decreased as relative humidity decreased, but all values exceeded the 0.38 g water/g protein expected for hydration of proteins with a monolayer of water. ~7'~ Notably, at 100% and 92% relative humidity, the ratio of water to enzyme excecdcd the monolayer value by factors of 100 and 33, rcspcctively. Thc relatively high ratios of water to enzyme, in particular at the higher relativc humidities, suggested that the bead-associated e n z y m e was in a thin film of buffer and that the concentration of buffer increased as rclative humidity decreased. The view that the surface of the beads is covered by a concentrated buffcr solution was supported by cohesion of the beads after expcriments in which they wcrc exposed to rclative humidities of 75% to l(X)%. The high water uptake reflects the hygroscopicity of the buffer used to suspend the enzyme prior to drying on the beads, which was deliquescent at relative humidities of 75% and higher. On the basis of the amount of water and potassium phosphate present, the buffcr concentrations at 100cA- and 92% relative humidity were estimated to contain 2.6 and 3.6 M dibasic potassium phosphate, respectively. High salt concentrations can precipitate proteins from solution; hence, the state of the e n z y m e in buffer concentrations expected in the film of buffcr on the bead surface was of interest. The state of the enzyme was investigated in phosphate buffers of concentrations computed to be those on the beads at 92% and 100% relative humidities. The e n z y m c was only partially soluble in thesc buffers, and both soluble and insoluble forms hydrolyscd ethyl acetate (Table 3). Enzyme activity in the precipitate was due largely to the insoluble e n z y m e and not to entrained supernatant. Pellet volumes were 15% and 8% that of the supernatant with 2.6 and 3.6 M buffers, respectively, and the small amounts of supernatant entrained in the precipitate were insufficient to account for the level of enzyme activity in the pellets.

Kinetics o f ' C . r u g o s a lipase localized on glass beads A comparison was carried out of the kinetics of ethyl acetate hydrolysis by C. rugosa lipase coated onto glass beads (1) when suspended in substrate vapor and (2) when suspended in dilute and conccntrated phosphate buffer solutions. To facilitate comparison of ki-

Gas-sofid lipase reactions: N. W. Ross and H. Schneider Table 2

Water uptake by glass beads coated with C. rugosa lipase at several relative humidities at 30°C Ratio of water uptake to buffer or enzyme weight

Water uptake (p,g H20 mg ~ beads)

Relative humidity

Beads only

Beads & buffer alon&

100 92 84 75 56

1.75 0.89

Activities of Candida rugosa lipase and other esterolytic enzymes coated on glass beads and suspended in substrate and water vapor: enzymes in thin liquid films.

Candida rugosa lipase was enzymatically active when coated on glass beads and exposed to mixtures of substrate and water vapor over a range of relativ...
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