CLEA-Based Immobilization of Methylotropic ... - ACS Publications

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CLEA-Based Immobilization of Methylotropic Yeast Alcohol Oxidase: Influence on Storage Stability and Reaction Efficiency † ‡,§ ,† Ruta ̅ Gruškiene,̇ Visvaldas Kairys, and Inga Matijošyte*̇ †

Institute of Biotechnology, Sector of Applied Biocatalysis, and ‡Institute of Biotechnology, Department of Bioinformatics, Vilnius University, V. A. Graičiu̅no str. 8, LT-02241 Vilnius, Lithuania § Faculty of Chemistry, Department of Applied Chemistry, Vilnius University, Naugarduko str. 24, Vilnius LT-03225, Lithuania ABSTRACT: There is still a great demand for sustainable oxidative systems for the conversion of alcohol to carbonyl compound, a pivotal reaction in daily organic synthesis, in the fine chemical and pharmaceutical industries. In this study we have focused on the reclamation of a biocatalytic process viable for laboratory and large scale applications. In the past, alcohol oxidase (AO), which is known to catalyze oxidation of short aliphatic alcohols, was trialed to perform oxidations, but its poor stability has limited the comprehensive applications of this enzyme. An immobilization technique by forming the cross-linked enzyme aggregates (CLEA) of this enzyme was selected as a bioengineering tool to overcome the stasis of AO application. It was demonstrated that inhibition of AO by hydrogen peroxide can be neglected due to the presence of catalase in both forms of enzyme: free and immobilized. However, AO was inactivated by the second reaction product, aldehyde. Two stages of inactivation were distinguished in the case of 1-propanol oxidation: reversible competitive inhibition followed by irreversible inactivation. Immobilization has greatly increased the storage stability of AOafter 12 weeks more than 50% of enzyme activity remained at 8 and 22 °C temperature. It was demonstrated that the application of biocatalysis for alcohol oxidation processes is not limited by the stability of enzymes, but rather due to lack of engineering solutions, and further development strategies should be directed toward creating engineering systems with an immediate elimination of the formed reaction productaldehyde.

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INTRODUCTION Oxidation of alcohols is a pivotal reaction in organic chemistry, supplying the key intermediates with functional groups such as aldehyde, ketones, and carboxylic acids, which can be further applied for functionalization or manufacturing of building blocks. Conventional chemical oxidation methods have many well-known shortcomings related to ecological, economical, and technical issues. In the search for alternatives, heavy-metal-free systems are a choice for green oxidation processes.1−4 However, bio-oxidation systems show even higher chemo-, regio-, and stereoselectivity, and, importantly, occur under mild conditions, e.g. low temperature, neutral pH, no pressure, and no toxic waste and byproducts.5,6 Products obtained by microorganisms and enzymes are considered to be natural. This is very important for the synthesis of ketones and aldehydes, which are the main products in the flavor and fragrance industries, whereas consumers have a strong preference for natural additives over chemically synthesized ones.2 During recent years, the interest in alcohol oxidation systems was on the increase, resulting in the development of a number of enzymatic oxidations of primary and secondary alcohols with novel redox systems.3,7 In nature the direct conversion of alcohols to their corresponding carbonyl compounds is catalyzed by two different enzymes: alcohol dehydrogenase (ADH, EC 1.1.1.1) and alcohol oxidase (AOX or AO, EC 1.1.3.x).8 However, ADH can catalyze the reverse reaction. Compared to ADH, AO is often of eukaryotic origin and is much less abundant in nature; AO requires flavin-based cofactors, while ADH requires expensive NAD-based cofactors; AO enzymes are more complex, and their structural and functional characteristics are © XXXX American Chemical Society

less explored. The important difference between ADH and AO is the manner in which the cofactor is regenerated: in dehydrogenase the regeneration occurs via hydrogenation of another organic substrate, while reoxidation of AO proceeds via hydrogenation of O2 which is reduced to H2O2. Hence, AO is easier to use, since O2 is a cheap substrate to use for cofactor regeneration, thus making it much more attractive for various oxidations. Based on substrate specificity, alcohol oxidases are categorized broadly into four different groups: (i) short chain alcohol oxidase (SCAO or AO); (ii) long chain alcohol oxidase (LCAO); (iii) aromatic alcohol oxidase (AAO); and (iv) secondary alcohol oxidase (SAO).8 AO is highly attractive for industrial applications due to its easy availability and wide substrate specificity, and due to it being extracellular.9 For decades, AO has been studied in the regulation system of its gene in methylotrophic yeasts10,11 and as the main component of an alcohol sensor.12−15 These and recent unveilings of AO for possible applications in different areas designate this enzyme as being a very attractive biocatalyst for organic synthesis.8 However, the operational and storage stability of AO is limiting the substantial AO application. It was reported that AO is relatively susceptible to the reaction products: hydrogen peroxide (H2O2) and aldehyde.16−19 For this instance, immobilization could be used to stabilize the quaternary structure of complex multimeric AO to prevent it from subunit dissociation and to protect it from suicide inactivation by the oxidation products. Several different Received: September 17, 2015

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DOI: 10.1021/acs.oprd.5b00291 Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development

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Figure 1. Stereoview homology model of P. pastoris AO1 built based on the Pleurotus eryngii aryl-alcohol oxidase template (PDB ID: 3fim). The FAD cofactor from the template is superposed on the model and is shown as colored balls-and-sticks. The Lys and Arg residues are depicted as green and purple sticks, respectively. Most lysines and arginines are on the surface, but several can be seen inside of the protein. The poorly modeled residues 478−555 and the C-terminal 637−663 are not shown. These residue stretches, which are likely to be on the surface, contain 6 Lys and 2 Arg residues. The AO1 and AO2 have the same total number of Lys+Arg residues, but the individual numbers for Lys and Arg are only slightly different because of three K to R or R to K interchanges. For this reason, the model of AO2 is not shown. The figure was generated using UCSF Chimera.21

Table 1. Summary of Production Parameters for AO−CLEA Investigated parameter Precipitant Amount of precipitant, % Precipitation time, h Cross-linker Amount of cross-linker Cross-linking time, h

Trial values

Optimal values

ammonium sulfate, isopropanol, acetone, dimethoxy ethane, 2-(2-methoxyethoxy)ethanol, tert-butanol, dimethyl sulfoxide, dimethylformamide, diethylenglycol, acetonitrile, PEG-600, ethyl lactate, 1,4-dioxane, tetrahydrofuran, 2butanone. 33, 50, 60, 67, 71, 83, 90

isopropanol

0.5, 1, 2, 3, 4, 5, 16, 24 glutaraldehyde (GA) 1,3,5-triacryloyl hexahydrotriazine (TAT) GA: 1, 5, 10, 25, 50, 75 mM TAT: 0.05, 0.1, 0.25, 0.5, 0.75, 1.0% 1, 2, 3, 4, 5, 16, 24

2 glutaraldehyde

67

5 mM 24

structure, making it difficult for all subunits to interact with the planar surface of a carrier. Thus, the CLEA method does not require a carrier or a support and increases the possibility of multipoint covalent attachment via the whole surface of protein. AO on its surface has a number of lysine and arginine residues (Figure 1), which are essential for cross-linking, the latter affecting the immobilization procedure efficiency and, furthermore, the stability of the immobilized derivatives. The strategy for immobilization of cross-linked enzyme aggregates was constituted as usual, followed by two stages: precipitation and cross-linking. To the best of our knowledge, AO was never immobilized by the CLEA method before; thus, there was a need to establish the optimal precipitation and cross-linking parameters. The overview of the tested and optimized parameters is presented in Table 1. Isopropanol with a saturation of 67% was assessed to be the best precipitant, if the precipitation was performed for 2 h at 8 °C. Five mM of GA was found to be a better cross-linker than hexahydrotriazine (TAT), with the optimized 24 h cross-linking time at 8 °C. The concentration of the cross-linker and the cross-linking time are the key factors for immobilization: too large amount of the cross-linker, or performing cross-linking for too long could cause enzyme inactivation. Very good immobilization results were obtained at the above-mentioned conditions: the immobilization yield was estimated to be 100−105% with a purification factor (P.F.) of 1.5−2.0. The latter refers to the ratio of the specific activity of redissolved aggregates obtained using a particular precipitant, and the specific activity of the sample of free enzyme that they were derived from. It is a measure of how effective the precipitation was for a given enzyme sample protein using different precipitants.

immobilization techniques for AO immobilization have been investigated, including covalent attachment to glyoxal, epoxy, or glutaraldehyde activated supports, entrapment within porous materials, and adsorption onto PEI-coated supports and polyurethane foam.20 This study deals with AO (EC 1.1.3.13) immobilization by the carrier-free CLEA (cross-linked enzyme aggregate) method. The CLEA technology is a simple and effective method for enzyme immobilization that offers several advantages, such as high productivity, facile reusability and recovery, enhanced thermal and mechanical stability, and partial enzyme purification. We have investigated the stabilization of AO by the CLEA method, which, to the best of our knowledge up to date, was applied for AO immobilization for the first time, and which was shown to be a proper technique, suited particularly for that purpose, due to AO’s structural complexity. Moreover, despite the increase of storage stability, immobilization allowed overcoming the challenge of oxidation reactionsenzyme inhibition by reaction products, which was investigated and will be discussed. The limitations of the biocatalytic alcohol oxidation method catalyzed by AO were identified, and it was demonstrated that the process is not limited by the stability of enzymes.

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RESULTS AND DISCUSSION Immobilization of AO from P. pastoris. Immobilization of AO from P. pastoris by the CLEA method was chosen for study for several reasons. It is considered that AO is an oligomeric enzyme; the quaternary structure usually consists of 4 to 8 identical subunits arranged in a spatial quasi-cubic fashion.20 Our studied AO has eight units in the quaternary B



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Figure 2. pH (A) and temperature (B) stability of nonpurified free, partially purified free, and immobilized alcohol oxidase. (A) The incubation was performed in 0.04 M Britton-Robinson buffer at pH from 2 up to 12 for 30 min at 30 °C. (B) The incubation was performed in 0.1 M phosphate buffer pH 7.3 for 30 min, varying the temperature in the range 22−70 °C. The experiments were done in triplicate, and the percentage error in each set of reading was within 5%.

Stability of AO−CLEA. Extracellular AO may comprise up to 60−80% of total cellular protein.16 For evaluation and comparison we have also examined the stability of the free and partially purified enzymes. Henceforth, temperature and pH stability were evaluated for the immobilized enzyme labeled as AO−CLEA for the soluble enzyme labeled as free-AO and for partially purified free-AO. It was assumed that the purified AO was more sensitive to the oxidative environment, and its natural protection was lost during purification. The survey indicated that the biochemical properties of soluble AO from P. pastoris had topt 30 °C and pHopt 7.3. In the current study, the pH stability test was performed during 30 min incubation at 30 °C. The obtained results demonstrated that the immobilization of enzyme improved the AO pH stability profile by almost 2 pH units toward higher acidity and only slightly toward higher

alkalinity (Figure 2A). Both soluble and immobilized enzymes had a broad pH stability interval, and their stability for pH was not affected by the purification. Next, the temperature stability test was performed for 30 min in the range 22−70 °C. A broad stability profile with similar trends was observed for the soluble and partially purified enzyme with the relative activity of 90−100% at 22−43 °C (Figure 2B). AO−CLEA had a slight decrease of activity at 30 °C and maximum activity at 40 °C. All three derivatives showed a drop of activity above 43 °C, and almost no activity was observed at 60 and 70 °C. There is a tendency that AOs from various yeast strains have different embodied biochemical properties; for example, AO from P. pastoris IFP206 has topt 37 °C and pHopt 7.5.17 Alcohol oxidases are usually active over the C



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Figure 3. Stability course of alcohol oxidase for 12 weeks storage. The incubation was performed in 0.1 M phosphate buffer pH 7.3 at 8 and 22 °C. The experiments were done in triplicate, and the percentage error in each set of readings was within 5%.

Oxidation by AO−CLEA. Generally, the immobilized enzymes are described by the operational stability, which is an important parameter for practical applications. For its determination, the immobilized derivative is repeatedly exposed to new portions of the reaction solution of the targeted reaction, and this procedure is repeated several to several tens of cycles. In our study, the operational stability of AO−CLEA was examined by oxidation of 1-propanol and 2-methoxyethanol. However, already after 4 cycles, the duration of each being 1 h, the relative aldehyde content of both substrates was only 40−45% (Figure 5). In addition, a more focused approach to the reaction profile for 1-propanol oxidation (Figure 6) was not very reassuring: the formation of the product was relatively

pH range 6−9, and the widely reported topt values are 25−30 °C and 37 °C, with deviations up to 50 °C.8 Figure 3 shows the stability course for the AO storage at 8 and 22 °C during 12 weeks. Soluble AO and partially purified AO were less stable: 50% activity was already lost after 2−3 weeks storage, and less than 10% of activity was left after 12 weeks storage. In contrast, AO−CLEA remained highly active even after 12 weeks: 70% and 56% at 8 and 22 °C, respectively. Notably, the samples were prepared in phosphate buffer pH 7.3 without any addition of stabilizers. It seems that the conformational change during the immobilization was not followed by FAD dissociation; the latter event is known to occur much more intensely during the storage at room temperature. SDS-PAGE of AO−CLEA, soluble AO, and partially purified soluble AO was also carried out (Figure 4). There was no significant difference between the free-AO and the partially purified free-AO. Besides, the immobilization helped to polish off the redundant proteins from the immobilized derivatives.

Figure 5. Reusability of AO−CLEA. Reaction of each cycle was performed for 1 h at 22 °C. After each cycle, AO−CLEA was centrifuged for 15 min at 12 500 rpm at 4 °C. The pellets were washed with 0.1 M phosphate buffer (pH 7.3) and applied again to the new portion of reaction solution. The experiments were done in triplicate, and the percentage error in each set of reading was within 5%.

Figure 4. SDS-PAGE of AO−CLEA, free-AO, and partially purified AO. D



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Figure 6. Reaction profile for oxidation of 1-propanol. Oxidation was performed under the following conditions: 100 mM 1-propanol solution in 0.1 M phosphate buffer (pH 7.3), 20 U of soluble AO or AO−CLEA, 10 mL of reaction volume, 180 min of reaction time, 30 °C temperature. The experiments were done in triplicate, and the percentage error in each set of reading was within 5%.

Figure 7. Decomposition of H2O2 by AO−CLEA. Hydrogen peroxide decomposition was followed on time polarographically by a Clark electrode under the following conditions: 30−180 μM initial concentration of H2O2 in 0.1 M phosphate buffer pH 7.3, 3.0 mL reaction volume, 0.2 U of AO− CLEA, 25 °C temperature. The experiments were done in triplicate, and the percentage error in each set of readings was within 5%.

high catalase activity in the soluble AO solution and the partially purified soluble AO. AO and catalase were not separated during the precipitation procedure. The catalase activity was also found in AO−CLEA, and the immobilization efficiency for catalase was calculated to be 35%. The formation of CLEA enables catalase to be also incorporated into the structured protein cluster. By this, catalase is steadily involved in the H2O2 decomposition during the application of CLEA (Figure 7). Therefore, the inhibition of AO by hydrogen peroxide can be neglected when the enzyme samples are subjected to the oxidation reactions. Another cause for slow down of the oxidation reaction is assumed to be the inactivation of enzyme by the other product of the oxidation reactionthe aldehyde. The formed aldehyde

slow, and it almost stopped after 2 h, reaching about 20% oxidation efficiency. It has been proposed that AO may be inhibited by the reaction products, both H2O2 and aldehyde. For example, it was reported that AO’s from Kloeckera sp. and Hansenula polymorpha DL-1 were rapidly and irreversibly inactivated by H2O2.18 Therefore, AO is usually used in conjunction with catalase in order to steady the decomposition of formed H2O2 into water and molecular oxygen. We have observed that addition of catalase had no effect on the catalytic activity of AO from P. pastoris. The presence of catalase in P. pastoris has already been confirmed, with the purified catalase being characterized by 223 kDa Mw, optimum pH of 6.7−7.0, and 0.036 M KM.22 Thus, the assumption of the presence of catalase in our AO samples was verified. Indeed, we found a E



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Figure 8. Effect of enzyme inactivation during oxidation of 1-propanol. Oxidation of 1-propanol was followed for 150 min reaction time under the following conditions: 100 mM 1-propanol solution in 0.1 M phosphate buffer (pH 7.3), 20 U of soluble AO or AO−CLEA, 10 mL of reaction volume, 30 °C temperature. Afterward 0.5 mL of reaction solution was taken and applied to 3 mL of an air-saturated 3% methanol solution in 0.1 M phosphate buffer (pH 7.3) at 25 °C. The experiments were done in triplicate, and the percentage error in each set of readings was within 5%.

Figure 9. Effect of propionaldehyde concentration on the enzymatic activity of AO and AO−CLEA. Twenty U of AO or AO−CLEA was incubated with 10 mL of 1-propanal at concentrations of 2 mM, 25 mM, and 50 mM in 0.1 M phosphate buffer (pH 7.3) at 30 °C. After incubation for the times 10, 25, 40, and 65 min, 0.5 mL of incubation solution was taken and used to initiate the oxidation of methanol. The experiments were done in triplicate, and the percentage error in each set of reading was within 5%.

aldehyde was formed, which inhibited the enzymatic activity of AO. Similar trends were observed for the soluble AO and the immobilized AO (the results not shown). These results can be simply interpreted as follows: the cross-linking step of immobilization tied up the amino groups on the outer surface of the protein, while Lys and/or Arg groups located inside of the protein remained unaffected. However, the aldehyde group leaving from the binding pocket after the catalysis reaction could easily react with the Lys and Arg side chains located in the inner part of protein. This causes reduced substrate

may react with functional protein groups such as lysine and/or arginine amino groups which are abundant on the AO surface (Figure 1). Hence, the inactivation of enzyme by the formed aldehyde group may easily take place during the oxidation process. This was explored by taking samples from the 1propanol oxidation reaction after various reaction times and initiating the oxidation of 1-methanol. The efficiency of 1methanol oxidation catalyzed by AO−CLEA and by AO was plotted versus reaction time of 1-propanol oxidation (Figure 8). Figure 8 shows that during the reaction a certain amount of F



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CONCLUSIONS Alcohol oxidase from the methylotrophic yeasts Pichia pastoris was examined for its inhibition by the oxidation reaction productshydrogen peroxide and aldehyde. It was found that inactivation by hydrogen peroxide was not essential due to the high catalase activity in the AO samples in soluble or immobilized forms. In contrast, we found a significant inactivation of enzyme by aldehyde, which is the targeted molecule of oxidation reactions and is inevitably formed during the reaction. A two-step inactivation process was distinguished: a reversible competitive inhibition followed by an irreversible inactivation. The strategy is that the protection of functional groups via immobilization occurs when the aldehyde groups of the crosslinker react with the amino groups of the enzyme. Thus, the amino groups of the enzyme are protected and cannot react with aldehyde formed during the oxidation reaction. However, our study indicated that immobilization did not settle out fully the issue of enzyme inactivation by aldehyde. The cross-linking procedure does not engage all the functional amino groups of the protein. The cross-linking during the immobilization ties up only the functional groups on the outer surface of the protein molecule. The functional amino groups in the inner side of the protein are unaffected, and they can easily interfere with the product of the oxidation reaction by lowering the accessibility of the substrate to the enzyme active site. This may also cause diffusion limitations and the decrease of catalysis efficiency. The strategy to use the CLEA-based immobilization lead to better results compared to the immobilization on a support: the latter method bridges only functional groups on the very surface of the enzyme and a support. Moreover, the stability of the enzyme due to immobilization has increased considerably. Further, the improvement strategies should be directed toward engineering systems with an immediate elimination of the formed reaction product from the reaction solution in order to consolidate the application of AO for oxidation reactions.

accessibility to the active site. For AO−CLEA this effect is much more triggering because of the complexity of the eight subunit AO protein. The immobilization makes the overall structure even more complex and, furthermore, introduces additional diffusion limitations. Therefore, the obtained results for AO−CLEA were not better than for soluble AO, as expected. For a further study of AO inhibition by aldehyde, propionaldehyde (1-propanal) was selected as a model compound at 2 mM, 25 mM, and 50 mM concentrations. The reaction profile for methanol oxidation with aldehyde and enzyme samples, which were incubated before the reaction for increasing lengths of time at 30 °C, is presented in Figure 9. Inhibition of AO with propionaldehyde resulted in a gradual inactivation of the enzyme, and the obtained data demonstrated the direct correlation between the decrease of activity and the amount of aldehyde. The aldehyde incubated samples displayed lower activity in comparison with the nonincubated samples. Propionaldehyde is a small and volatile molecule; therefore, the partially purified AO was incubated with 50 mM propionaldehyde until enzymatic activity was completely lost, and afterward the incubation solution was directly processed by the rotavap in order to remove the propionaldehyde from the solution. Indeed, 21% of initial activity was recovered even though 0.5% of initial propionaldehyde concentration was still present in the reaction solution. Subsequently, it was determined that the inactivation by aldehyde proceeded faster at higher temperatures. Twenty U of AO or AO−CLEA was incubated with 10 mL of 1-propanal at 2 mM concentration in 0.1 M phosphate buffer (pH 7.3) for 10−65 min at 20, 30, and 40 °C. After the incubation, 0.5 mL of incubation solution was taken and used to initiate the oxidation of methanol. The enzyme inhibition by the reaction product was evaluated by measuring the consumption of oxygen. For example, after 25 min incubation with 2 mM 1propanal, the AO−CLEA retained 50% and 25% of its activity at 30 and 40 °C, respectively (data not shown). However, almost 80−90% of the activity remained at 20 °C. The results of experiments with propionaldehyde may be interpreted as a competitive inhibition by the product (rapid equilibrium) followed by a slower process of the covalent interaction between the aldehyde and the enzyme. The competitive inhibition is responsible for the initial rapid drop of activity. Inactivation of AO by aldehyde was also evidenced by Nichols and Cromartie (1980) when inactivation of AO from Candida boidinni was observed during oxidation of propargyl alcohol.19 Aldehyde-based deactivation also represents a problem in biotransformations catalyzed by lipases, thiamine-diphosphate dependent enzymes, and 2-deoxy-Dribose 5-phosphate aldolases.23−25 Furthermore, KM values of free and immobilized AO for 1-propanol were determined as 7.3 mM and 8.9 mM, respectively. This indicated that the immobilized enzyme had a slightly weaker affinity toward 1propanol than the free enzyme. The immobilization of enzyme has not influenced the affinity orientation between the active site and the substrate; however, it had an impact on the accessibility and the binding between the enzyme and the substrate. The cross-linking during the immobilization engages only the functional groups on the outer surface of the protein molecule.

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EXPERIMENTAL SECTION General. The substrates and reagents were of analytical grade and obtained from Fluka and Sigma-Aldrich. From the yeast collection Pichia pastoris wt was cultured and kindly gifted by the Eukaryote Gene Engineering Laboratory of the Institute of Biotechnology, Vilnius University. An Agilent 8453 UV−vis spectrophotometer was used for optical absorbance measurements. Catalase activity was measured as described in the liretature.26 Hydrogen peroxide decomposition was followed polarographically by a Clark electrode. Denaturing SDS-PAGE was carried out according to the method of Laemmli.27 KM value was estimated by Lineweaver−Burk plot method28 at 25 °C temperature varying the substrate concentration from 0.25 mM to 100 mM. All experiments were done in triplicate and the percentage error in each set of reading was within 5%. Homology modeling. HHpred server29 was used to perform a search for the best homologous template for P. pastoris alcohol oxidases 1 and 2 (UniProt ID: P04842 and C4R702). The best aligned templates belonging to Agaricus meleagris pyranose dehydrogenase (PDB ID: 4h7u; identity with P. pastoris AO 26%) and Pleurotus eryngii aryl-alcohol oxidase (PDB ID: 3fim; identity 27%) were used to generate P. pastoris AO structures. The structures of P. pastoris AO1 and AO2 based on the two templates were built each separately using MODELER.30 Due to the high similarity between P. G



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For estimation of storage stability, samples of AO and AO− CLEA in 0.1 M phosphate buffer (pH 7.3) were kept at two different temperatures 8 and 22 °C for 12 weeks. After each week the enzyme activity was assayed (section Alcohol oxidase activity assay). Assay for aldehyde determination (Purpald method). The quantitative amount of aldehyde was determined by Purpald method which was adopted from Avigad.32 Purpald (4amino-5-hydrazino-3-merkapto-1,2,4-triazole) in the interaction with aldehyde yields a colored 6-substituted S-triazolo-[4,3-b]S-tetrazine-3-thiol. Reagent solutions of 1% (w/v) Purpald in 1 N NaOH and 0.2% (w/v) NaBH4 in 1N NaOH have to be prepared freshly for the day’s use. For calibration curve a series of 0.6 mL samples containing 15−380 nmol of 1propionaldehyde were delivered into test tubes. After the addition of 1.5 mL of Purpald solution to each sample, the tubes were incubated in a shaker at 180 rpm at room temperature for 30 min. 1.5 mL of 0.2% (w/v) NaBH4 alkaline solution was then added with mixing to each tube to stop the reaction and the absorbance was measured spectrophotometrically at 542 nm. The formed chromogen is stable for at least 1 h. The reference sample was tested analogously. The experiments were performed in triplicate. Uptake of oxygen. Consumption of oxygen was measured using Clark-type electrode connected to a YSI 5300A biological oxygen monitor. The YSI 5300A Clark-type electrode consists of a platinum cathode and a silver anode, both in contact with an electrolyte KCl solution. The electrode was placed and fixed on the top of the magnetically stirred sample chamber. Airsaturated medium of 3 mL 3% methanol in 0.1 M phosphate buffer (pH 7.3) was introduced into the electrode chamber (total volume of 10 mL) and incubated until a steady baseline was obtained on the chart. Then solution of AO was added into the chamber through the port on the top of the chamber using a syringe with an extra long needle and the oxygen consumption was followed on time. All of the measurements were carried out at 25 °C. Reusability of AO−CLEA. To evaluate the reusability of AO−CLEA, 3 unit of AO−CLEA were added in 1.5 mL of substrate (100 mM of 1-propanol or 100 mM of 2methoxyethanol). Reaction was performed for 1 h at 22 °C temperature. AO−CLEA was centrifuged after each reaction cycle for 15 min at 12 500 rpm at 4 °C temperature. The pellets were washed with 0.1 M phosphate buffer (pH 7.3) and applied again to the new portion of reaction solution. This procedure was repeated for four cycles. The oxidation efficiency was evaluated by using the Purpald method. The relative amount of aldehyde was calculated by taking the amount of aldehyde of the first cycle as 100%. Oxidation of 1-propanol. The oxidation reaction was made in screw-capped flasks in order to prevent the loss of volatile substrates and products. 100 mM 1-propanol solution was prepared in the reaction flask of 10 mL volume containing 0.1 M phosphate buffer (pH 7.3). The reaction was initiated by adding 20 U of soluble AO or AO−CLEA. The oxidation was performed in environmental shaker-incubator ES-20 (Biosan) with shaking at 180 rpm for 180 min at 30 °C temperature. The oxidation efficiency was followed on time by using the Purpald method. Inhibition of AO and AO−CLEA by aldehydes. Influence of the formed oxidation product on inhibition. Twenty U of AO or AO−CLEA were applied for 1-propanol oxidation (see section Oxidation of 1-propanol). At a given

pastoris AO1 and AO2 (97% identity) their models were also found to be very similar. Except for two poorly modeled relatively small regions, consisting of residues 478−555 and 637−663, models derived from the two templates were consistent with each other and were used for the analysis of the distribution of Lys and Arg residues within the protein. Preparation of cell-free AO samples. The yeast cells were disrupted by treatment with glass beads: 1 g of wet biomass was dissolved in 4 mL of 0.1 M KH2PO4−KOH (pH 7.3) buffer and 5 mL of glass beads were added. The tube was shaken vigorously for 0.5 min on a mixer and then cooled in ice for 0.5 min. The procedure was repeated 15 times. Then the solution was centrifuged at 10000 rpm for 10 min at 4 °C in order to remove unbroken cells and pellets. The supernatant liquid with AO was decanted and was used as an enzyme source (AO solution). Partial AO purification. AO in the prepared cell-free supernatant sample was precipitated by adding 2-propanol with volume ratio 1:2 at 8 °C temperature. After 1 h precipitation, the solution was centrifuged for 30 min at 10000 rpm at 4 °C temperature. The pellets were redissolved in 0.1 M phosphate buffer pH 7.3 and used for the experiments. Preparation of AO−CLEA at the defined optimal conditions. For the preparation of CLEA, alcohol oxidase was precipitated from the supernatant by adding 2-propanol with volume ratio 1:2 at 8 °C temperature. After 2 h precipitation procedure, the enzyme aggregates were cross-linked with glutaraldehyde (GA) solution to reach a final concentration of 5 mM. The mixture was shaken at 400 rpm at 8 °C for 24 h. After cross-linking, particles were centrifuged at 12 500 rpm at 4 °C for 30 min and washed 5 times with Milli-Q water to remove the excess of glutaraldehyde. The pellets were redissolved in 0.1 M phosphate buffer pH 7.3 and used for experiments. Protein concentration assay. The protein concentration was determined as described (www.piercenet.com/products) measuring the absorbance of the bicinchoninic acid (BCA) after 30 min incubation with enzyme. Bovine serum albumin (2 mg/mL) was used as a standard for the calibration curve. Alcohol oxidase activity assay. The alcohol oxidase activity was assayed in 2 mL of reaction mixture containing 1.5 mL of 3% methanol solution in 0.1 M KH2PO4−KOH (pH 7.3) buffer and 0.5 mL of AO solution at 30 °C temperature. After 15 min the reaction was stopped by adding 0.14 mL of 4 N HCl solution. The amount of produced formaldehyde was measured spectrophotometrically at 412 nm, by the Hantzsh reaction.31 One unit (U) of AO activity was defined as the amount of enzyme that oxidizes 1 μmol of methanol per minute at 30 °C temperature and pH 7.3. Stability of AO and AO−CLEA. For the study on pH stability, the Britton-Robinson buffer consisted of equally mixed 0.04 M acetic, 0.04 M boric and 0.04 M phosphoric acid was used with altered pH between pH 2 and 12. Samples of AO and AO−CLEA were incubated for 30 min at 30 °C. After incubation pH value of each sample was restored to pH 7.3 and the enzyme activity was assayed (section Alcohol oxidase activity assay). For the study on temperature stability, samples of AO and AO−CLEA were incubated in 0.1 M phosphate buffer (pH 7.3) for 30 min at temperatures in a range from 22 to 70 °C. After incubation the temperature of each sample was committed to 30 °C and enzymatic activity was assayed (section Alcohol oxidase activity assay). H



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time 0.5 mL of reaction solution was taken and used to initiate the oxidation of methanol (see section Uptake of oxygen). The enzyme inhibition by reaction product was evaluated by measuring the consumption of oxygen (see section 2.10). Influence of aldehyde concentration on inhibition. Twenty U of AO or AO−CLEA were incubated with 10 mL of 1-propanal at concentrations of 2 mM, 25 mM and 50 mM in 0.1 M phosphate buffer (pH 7.3) at 30 °C temperature. After incubation for the time of 10, 25, 40, and 65 min, 0.5 mL of incubation solution was taken and used to initiate the oxidation of methanol. The enzyme inhibition by reaction product was evaluated by measuring the consumption of oxygen (section Uptake of oxygen).

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +370 5 2404679. Fax: +370 5 2602116. E-mail address: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Evaldas Č iplys (Department of Eukaryote Gene Engineering, Institute of Biotechnology, Vilnius University) for supply of Pichia pastoris biomass. The authors also thank Č eslovas Venclovas (Department of Bioinformatics, Institute of Biotechnology, Vilnius University) for useful discussions.

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REFERENCES

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