Biotechnology Letters 26: 133–136, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.
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Activity of immobilized papain dehydrated by n-propanol in low-water media Theerapong Theppakorn1 , Pawinee Kanasawud1,∗ & Peter J. Halling2 1 Department of
Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand Pure and Applied Chemistry, University of Strathclyde, Glasgow G1 1XL, UK ∗ Author for correspondence (Fax: +66 53 892277; E-mail:
[email protected]) 2 Department of
Received 28 August 2003; Revisions requested 3 September 2003/26 September 2003; Revisions received 23 September 2003/10 November 2003; Accepted 11 November 2003
Key words: immobilized papain, low-water media, propanol-rinsed enzyme preparations
Abstract A propanol-rinsed enzyme preparations (PREP) of papain showed an activity of 59 nmol min−1 (mg powder)−1 in tert-butanol at the optimal water activity of 0.2. The immobilized papain was stable in aqueous media for 3 d at 4 ◦ C. Solid-state buffers (bases and their HCl salts) suspended in the organic medium decreased the initial rate in all cases tested. The operational stability during Z-Gly-Phe-NH2 synthesis was improved when solid cysteine was added, doubling the yield after 24 h.
Introduction Two important parameters that affect the catalytic rate of enzymes operating in non-aqueous media are the form of the biocatalyst and its method of preparation. Freeze-dried enzyme powders are commonly used due to their ease of preparation from commercially supplied enzymes but they exhibit low activities (Triantafyllou et al. 1997, Persson et al. 2002). Enzyme immobilization on to solid supports, which can improve enzymatic activity, can be carried out very simply by adsorption from an aqueous solution, followed by filtration to recover the immobilized enzyme. The wet support may further be dried by dehydration over molecular sieve, under vacuum or in a fluidized bed dryer. However, a better method of dehydration is by rinsing with a suitable solvent that is completely miscible with water (Partridge et al. 1996). Dehydration of the immobilized enzyme by rinsing with n-propanol provides a very high activity biocatalyst. The products are named propanol-rinsed enzyme preparations (PREPs) (Partridge et al. 1998). The method has been used successfully with subtilisin Carlsberg and α-chymotrypsin giving 1000-fold greater cata-
lytic activity than freeze-dried powder (Partridge et al. 1998). An important enzyme that has been widely studied in non-aqueous media is papain. It is a versatile protease for the synthesis of peptides due to its broad substrate specificity (Stevenson & Storer 1991). In the present paper, we report on the catalytic activity of spray-dried papain adsorbed on to silica gel, using the PREP drying method. Materials and methods Materials Substrates N-benzyloxycarbonyl-L-glycine (Z-GlyOH) and L-phenylalanine amide (H-Phe-NH2) were purchased from Bachem, St Helens, UK. Silica gel S0507, arginine, glycine, lysine and its hydrochloride were purchased from Sigma. Betaine, carnitine and its hydrochloride were obtained from Merck. HPLCgrade tert-butanol, n-propanol and acetonitrile were purchased from Fluka.
134 Preparation of spray-dried papain Fresh papaya latex was agitated and then centrifuged to separate the insoluble material. To remove some of the small molecular weight material, the supernatant was placed in an ultrafiltration unit of regenerated cellulose (MW cut-off 10 000, Millipore). The filtration process was carried out at 4 ◦ C until the volume had been reduced to 25% of the original. A sample of the concentrate was dried to determine solid content, which was then adjusted to 15% (w/v) with water. This solution was spray-dried, giving some 9 g powder from 100 ml solution. Preparation of immobilized papain Spray-dried papain was dissolved at 2 mg ml−1 in 30 mM citrate and 140 mM sodium phosphate buffer, pH 6.4, containing as activating agents 100 mM cysteine and 1 mM EDTA disodium salt (freshly prepared). Of this solution, 20 ml was mixed with 1 g silica gel and shaken for 1 h at 25 ◦ C. The silica was allowed to settle out and a sample of the supernatant was assayed for protein (Bradford reagent). Then 17 ml of the clear supernatant was removed, and replaced with an equal volume of the aqueous buffer without activating agents. The stock aqueous suspension of immobilized enzyme was stored at 4 ◦ C with 0.02% (w/v) NaN3 until required. Propanol-rinsed enzyme preparation of papain (PREP) The aqueous suspension of immobilized papain was agitated and 1 ml (50 mg support) was taken and allowed to settle out in an Eppendorf tube. Aqueous buffer was then removed and the supported enzyme was dried by rinsing with n-propanol (6 × 1 ml) having the same water activity (aw ) as that in the required reaction system. The papain PREP was then immediately used to catalyze reactions in organic media without further delay. Assay for catalytic rate of papain PREP in organic solvent The papain PREP (50 mg) was suspended in 2.5 ml tert-butanol in a 15 ml vial, then the reaction was initiated by adding 2.5 ml tert-butanol containing 25 mM of N-benzyloxycarbonyl-L-glycine (Z-GlyOH) and L-phenylalanine amide (Phe-NH2). The mixture was then incubated at 40 ◦ C with constant re-
Fig. 1. Effect of water activity (aw ) on the catalytic rate of papain PREP. Water was added to tert-butanol to give the required aw values.
ciprocal shaking (1000 min−1 ). Samples of the reaction mixture were filtered to remove suspended solids (0.2 µm nylon, Whatman) before analyzing by HPLC with C-18 ODS2 reverse phase column and a mobile phase consisting of acetonitrile/0.01% aqueous trifluoroacetic acid (45:55 v/v).
Results and discussion Effect of aw on the catalytic activity of papain PREPs Papain was immobilized by adsorption on to silica gel and then dehydrated by rinsing with n-propanol. The papain PREP was strongly affected by water in the reaction mixture (Figure 1.). The maximum activity (59 nmol min−1 mg preparation−1) for the synthesis reaction was obtained at aw 0.2. This is a relatively low optimum water activity for enzyme action in an organic medium. Some hydration is normally essential for good enzymatic activity, while the decline at higher aw is probably because mass action effects make synthesis less favorable, as reported in many other cases. Stability of immobilized papain The stability of immobilized papain stored in aqueous buffer was monitored by assaying samples for catalytic activity after transfer to tert-butanol at aw of 0.2. Figure 2 shows that stability was good for about 3 d. Since the enzyme was immobilized by adsorption, desorption of protein from the support could contribute to loss of activity. Additionally, oxidation of the active site thiol may be important, since there was no re-activation treatment before use in low-water media.
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Fig. 2. Stability of papain adsorbed on to silica gel. The immobilized papain was stored in citrate/phosphate buffer pH 6.4 at 4 ◦ C. The reaction rates were investigated at 40 ◦ C in the reaction system of tert-butanol, aw 0.2, using the PREP method for drying the immobilized papain from the aqueous phase. The initial activity of the immobilized enzyme was 59 nmol min−1 mg preparation−1 . Table 1. Catalytic rate of papain PREPs in the presence of solid-state buffers (bases and their hydrochloride salts). Reactions were performed at 40 ◦ C with tert-butanol at aw of 0.2 containing 10 mg ml−1 of each solid-state buffer pair (no buffer in the control). Buffers
Catalytic rate (nmol min−1 mg preparation−1 )
No buffer (control) Arginine/arginine.HCl Betaine/betaine.HCl Carnitine/carnitine.HCl Glycine/glycine.HCl Lysine/lysine.HCl Lysine.HCl/lysine.2HCl
59 22 34 16 26 27 4
Effect of solid-state buffers on the rate of papain PREP catalysis The papain PREP has a pH-memory in organic media arising from the last aqueous buffer that was used (Theppakorn et al. 2003). In addition, a number of solid-state buffers able to exchange Na+ and H+ (pairs of acids and their sodium salts) had no affect on its activity. Here we have studied the effect of the other class of solid-state buffers, bases and their hydrochloride salts, on the catalytic rate of papain PREPs. This class of solid-state buffers successfully controlled the protonation state of immobilized subtilisin Carlsberg by exchanging H+ and Cl− with the basic (e.g. amino) groups on enzymes (Zacharis et al. 1997).
Fig. 3. Time course of the synthesis of Z-Gly-Phe-NH2 in tert-butanol aw 0.2 at 40 ◦ C. The reaction yield was investigated in the presence and absence of solid cysteine. The cysteine added in the reaction was 20–400 (% w/w) relative to the immobilized papain: control (), 20 (), 50 (), 100 (), 200 () and 400 ().
Table 1 shows that all buffer pairs tested decreased the catalytic rate when compared to the control, but the rate found depended upon the solid buffer used. Hence neither these basic (H+ and Cl− ) buffers nor the H+ /Na+ buffers tested before can improve the catalytic activity of papain. One possible reason for this difference from other enzymes is the reaction of the solid buffers with the substrates of the papain reaction, which are themselves an acid (Z-Gly) and base (Phe-NH2). Solid-state cysteine increased the operational stability of papain PREPs Papain can be inactivated reversibly or irreversibly by oxidation of the active site thiol, hence may be protected by addition of mild reducing agents for optimal activity in aqueous media. To test whether papain undergoes such inactivation during use in tert-butanol, solid cysteine was added to the reaction mixture (Figure 3). There was only a slight increase in the initial rate, probably because the papain was immobilized from a buffer containing activating agents (cysteine and EDTA). However, the presence of cysteine had a much clearer effect as the reaction proceeds, reducing the decline in rate, so that yields after 24 h were approximately doubled (Figure 3). The cysteine may function either to continuously re-activate the papain, or to react with oxygen that is otherwise responsible for inactivation.
136 Conclusion
References
A stock suspension of papain adsorbed on to silica gel retained 95% activity for 3 d after immobilization. The immobilized papain dried by the PREP method provided activity of 59 nmol min−1 mg preparation−1 in tert-butanol at the optimal water activity of 0.2. Solid-state buffer pairs (a base and its hydrochloride salt) decreased the catalytic rate when compared to the control reaction. Addition of solid cysteine clearly increased the yield of peptide product, probably by preventing enzyme inactivation during the synthetic reaction. Such addition should always be considered when using cysteine protease-catalyzed reactions in non-aqueous media.
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Acknowledgement We thank the Thailand Research Fund for supporting T.T. via the Royal Golden Jubilee Ph.D. program.
