Sugar Ester Synthesis by Thermostable Lipase from ... - Springer Link

Appl Biochem Biotechnol (2012) 166:1969–1982 DOI 10.1007/s12010-012-9624-9

Sugar Ester Synthesis by Thermostable Lipase from Streptomyces thermocarboxydus ME168 Aran H-Kittikun & Poonsuk Prasertsan & Wolfgang Zimmermann & Phisit Seesuriyachan & Thanongsak Chaiyaso

Received: 28 June 2011 / Accepted: 20 February 2012 / Published online: 21 March 2012 # Springer Science+Business Media, LLC 2012

Abstract The extracellular lipase from Streptomyces thermocarboxydus ME168 was purified to 9.5-fold with 20% yield, following concentration by acetone precipitation, ion exchange chromatography (Resource Q) and gel filtration chromatography (Superdex 200), respectively. The purified enzyme had an apparent molecular mass of 21 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The N-terminal sequence of the lipase was ASDFDDQILG and was different from most other reported lipase. The enzyme showed maximum activity at 50 °C with the half-life of 180 min at 65 °C. It showed high stability at a broad pH range of 5.5–9.5 and was thermostable at the temperature range of 25–60 °C. The Km and Vmax were 0.28 mM and 1,428 U/mg, respectively, using p-nitrophenyl palmitate as substrate. It was active toward p-nitrophenyl ester with medium to long acyl chain (C8–C16). Lipase activity was inhibited by Zn2+, dithiothreitol (DTT), EDTA and some organic solvents, e.g., ethanol, acetone, dioxane, acetronitrile, tertbutanol and pyridine. Immobilized crude lipase of S. thermocarboxydus ME168 on celite could be used to synthesize sugar esters from glucose and vinyl acetate, vinyl butyrate or vinyl caproate in tert-butanol:pyridine (55:45 v/v) at 45 °C with conversion yields of 93, 67 and 55%, respectively.

P. Seesuriyachan : T. Chaiyaso Division of Biotechnology, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai 50100, Thailand A. H-Kittikun (*) : P. Prasertsan Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai 90112, Thailand e-mail: [email protected] W. Zimmermann Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, University of Leipzig, 04103 Leipzig, Germany

1970

Appl Biochem Biotechnol (2012) 166:1969–1982

Keywords Streptomyces thermocarboxydus . Lipase . Sugar esters . Vinyl acetate . Vinyl butyrate

Introduction Lipases (triacylglycerol acylhydrolases; EC 3.1.1.3) are the most important class of hydrolytic enzymes that catalyze both hydrolysis and ester synthesis [28]. Lipases have unique characteristics, e.g., substrate specificity, stereospecificity and regiospecificity, abilities to catalyze heterogenous reactions at the interface of water-soluble and water-insoluble systems and in organic solvents as well as temperature and pH stability. Lipases are produced by animals, plants and microorganisms but the majority of lipases used in biotechnological purposes, e.g., fat and oils processing, food processing detergents, cosmetics and pharmaceuticals, have been isolated from bacteria and fungi [10, 16, 26, 34]. Most research concentrated on the extracellular lipases produced by different microorganisms, e.g., Aspergillus terreus [10], Rhizopus delemar [2], Bacillus stearothermophilus MC7 [15], B. circulans [14], Acinetobacter sp. RAG-1 [31], Microbacterium sp. 7-1 W [12], Bacillus sp. H-257 [13], Geobacillus sp. TW1 [22], Streptomyces rimmosus R6-554 W [1] and S. cinnamomeus [32]. Sugar fatty acid esters are composted of mono- or oligosaccharides esterified with fatty acids of various chain lengths [24]. They are non-ionic surfactants and can be employed in foods, detergents and cosmetics as well as in the pharmaceutical and biomedical industries [23, 24]. These compounds can be synthesized from renewable resources and are non-toxic, non-allergic and biodegradable [8]. The chemical synthesis of sugar esters is hampered by the severe reaction conditions resulting in high energy consumption, formation of undesirable products and use of toxic chemicals [23, 25]. Alternatively, sugar esters can be synthesized using commercial lipases in organic solvent. The enzymatic method allows obtaining pure products due to the specificity of the enzyme. The catalysis by enzyme is conducted under mild reaction conditions, which minimize side reactions compared to the chemical reaction [20]. Though using commercial lipases such as Candida antarctica lipase B (Novozyme435) and Mucor miehei lipase (Lipozyme IM) could produce high conversion yield of sugar esters, the production cost is really high [5]. So, exploration of new sources of thermostable lipase for sugar ester synthesis is necessary. A few Streptomyces lipases have been characterized [1, 32]. This suggests that there will be more viability of the lipase from Streptomyces. However, those lipases did not report any activity on sugar ester synthesis. In this study, the thermostable lipase from Streptomyces thermocarboxydus ME168 was purified, characterized and firstly investigated on sugar ester synthesis using vinyl ester as acyl donor. The lipase from S. thermocarboxydus ME168 showed a good conversion yield compared to the commercial immobilized lipase.

Materials and Methods Chemicals Resource Q (1 ml) and Superdex 200 were purchased from Pharmacia (Sweden). The substrates for lipase: p-nitrophenyl palmitate (pNPP), p-nitrophenyl acetate (pNPA), p-nitrophenyl butyrate (pNPB), p-nitrophenyl caprylate (pNPC), phenylmethylsulfonyl fluoride (PMSF) and


1971

p-hydroxymercuribenzoate (pHMB) were purchased from Sigma (Heidenheim, Germany). Other analytical reagent grade chemicals were obtained from commercial sources. Microorganism and Lipase Production S. thermocarboxydus ME168 was obtained from the culture collection of the Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, University of Leipzig, Leipzig Germany. For lipase production, S. thermocarboxydus ME168 was cultivated in M65 medium composed of 1.0% malt extract, 0.4% yeast extract, 0.4% glucose and supplemented with 10% (v/v) of 10% (w/v) olive oil in 10% (w/v) gum arabic solution, pH 7.2 and incubated in a shaker with 175 rpm at 45 °C. After 72-h cultivation, the culture broth was centrifuged at 4,000 rpm (1,800g) at 4 °C for 30 min, and the cell free supernatant was used as extracellular lipase. Lipase Activity Assay Lipase activity was measured spectrophotometrically by hydrolysis of pNPP in 50 mM Tris– HCl buffer at pH 8.5, contained Triton X-100 (0.4% w/v) and gum arabic (0.1% w/v) following the method of Kademi et al. [14]. One unit of enzyme was the amount of enzyme liberating 1 μmol of p-nitrophenol (pNP) per minute. The molar adsorption coefficient of pNP was 17,230 cm2/mg at pH 8.5. For immobilized lipase, the lipase activity was assayed by cupric acetate method [20]. Protein Assay Protein was measured using the method of Bradford with bovine serum albumin (BSA) as a standard [3]. For the eluates from the chromatographic columns, protein concentration was determined by measuring the optical density at 280 nm. Purification of Lipase The purification method consisted of sequential concentration, precipitation, ion exchange chromatography (Resource Q) and gel filtration chromatography (Superdex 200). After 72-h cultivation, the supernatant (1,650 ml) was obtained by centrifugation of culture broth at 4,000 rpm for 30 min. The supernatant was ten times concentrated by evaporation using rotary vacuum evaporator at 40 °C. The concentrated supernatant was then precipitated by addition of 1.5 volumes of chilled acetone. The precipitate was recovered by centrifugation at 4,000 rpm. Then, the precipitate was dissolved in 0.1 M Tris–HCl buffer at pH 7.5, dialyzed against 50 mM Tris–HCl buffer at pH 7.5 overnight and stored at −20 °C prior to chromatography. The enzyme solution was applied on Resource Q 1-ml column equilibrated with 0.1 M Tris–HCl buffer at pH 7.5. The column was washed with the same buffer to wash unbound proteins. The bound proteins were eluted with 40 ml NaCl gradient (0–1 M) in the same buffer with a flow rate of 1 ml/min. The fraction volume of 1.0 ml was collected. The active fractions were pooled, concentrated, dialyzed against 50 mM Tris–HCl buffer at pH 7.5 and used for Superdex 200 gel filtration chromatography. The fractions containing enzyme were applied to a Superdex 200 column (1.6×70 cm) previously equilibrated with 0.15 M NaCl in 0.1 M Tris–HCl buffer at pH 7.5. Then, enzyme

1972


was eluted with the same buffer with a flow rate of 1.5 ml/min. A fraction volume of 5.0 ml was collected. The active fractions were pooled and used for sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme characterization. SDS-PAGE was performed on 15% polyacrylamide gel with 0.1% sodium dodecyl sulfate (SDS) to establish the purity of protein as described by Laemmli [18]. SDS-PAGE was run along the standard: β-galactosidase (116 kDa), BSA (66.2 kDa), ovalbumin (45 kDa), lactate dehydrogenase (35 kDa), restriction endonuclease Bsp98l (25 kDa), β-lactoglobulin (18.4 kDa) and lysozyme (14.4 kDa). N-terminal Amino Acid Sequence For N-terminal amino acid sequencing, the purified protein after SDS-PAGE (without reducing agent) was electrotransferred onto a polyvinylidene difluoride (PVDF) membrane with the TransBlot SD Semi-Dry Electrophoretic Transfer Cell from Bio-Rad according to the manufacturer's instruction. The PVDF membrane was rinsed with water and then stained by Coomassie Brilliant Blue R-250. The stained component was used for the N-terminal sequencing. Amino acid analysis was performed by Edman degradation using a protein sequencer (Procise 492HT; Applied Biosystems). The sequenator contained a C18 reverse phase column (PTH; 5 μm; 220×2.1 mm). Characterization of Lipase Effect of Temperature The effect of temperature on lipase activity was carried out by assaying the enzyme activity at different temperatures in the range of 35–75 °C at pH 8.5 using 50 mM Tris–HCl buffer. The thermostability of purified lipase was studied by incubation of the enzyme at different temperatures from 35 to 80 °C for 180 min before assay. Effect of pH The effect of pH on lipase activity was carried out at pH in the range of 6.0–11.0 using 50 mM different buffers: acetate buffer (pH 4.0–5.5), phosphate buffer (pH 6.0–6.5), Tris–HCl buffer (pH 7.0–8.5) and glycine–NaOH buffer (pH 9.0–11.0). For pH stability study, one volume of purified lipase was mixed with three volumes of the above buffers and incubated from 0 to 180 min before assay. Kinetic Parameters The Michaelis–Menten kinetic parameters, Km and Vmax, of purified lipase were carried out with increasing concentration of pNPP from 0.05 mM to 0.5 mM in 50 mM Tris–HCl buffer pH 8.5. The Km and Vmax were determined by Lineweaver–Burk plot. Substrate Specificity Since the pNPA and pNPB were not stable at pH above 8.5, all substrate specificity assays of the purified lipase were studied by hydrolysis of p-nitrophenyl esters of varying acyl chain lengths from C2–C16 at pH 7.5 and 45 °C. The molar adsorption coefficient of pNP was 12,442 cm2/mg at pH 7.5.


1973

Effect of Organic Solvents The effect of organic solvents on lipase activity was studied by incubation of 0.5 ml purified lipase in 0.5 ml of miscible or immiscible organic solvents at 45 °C for 60 min before assay. Effect of Metal Ions, Inhibitors and Other Compounds The effect of various metal ions, oxidizing and reducing agents, and chelating agents on enzyme activity was studied at concentrations of 1.0 mM and 10.0 mM at 45 °C and pH 8.5. Immobilization of Crude Enzyme The ten-time concentrated supernatant was dialyzed against 50 mM Tris–HCl buffer at pH 7.5 overnight and reconcentrated with a rotary vacuum evaporator. The celite was added (25 mg/mg protein) followed by the addition of chilled acetone 25% by volume and stirred by a magnetic stirrer for 60 min at 4 °C. Then, four volumes of chilled acetone was added and stirred for 5 min. The precipitate was recovered by filtration and washed three times with 20 ml chilled acetone. The immobilized crude enzyme was dried in dessicator until used [5]. Sugar Ester Synthesis The substrate mixture for sugar ester synthesis contained 0.3 of various vinyl esters and sugars in 3.0 ml of tert-butanol:pyridine (55:45 v/v) and 100 U (608 mg) of immobilized crude lipase or pure celite which was separately incubated to equilibrium water activity (aw) to 0.07 in LiBr-saturated solution for 3 days. The reaction was started by addition of immobilized crude lipase or pure celite (as control) into the substrate solution. Molecular sieve 4°A (0.5 g) was added to control water content during the reaction. The reaction was carried out by stirring (400 rpm) at 45 °C for 72 h in a screw-capped vial [5]. The water content in the reaction liquid phase before and after addition of molecular sieves was 0.75 and 0.50% (w/w) which was determined by a Metreohm 831 KF coulometric Karl Fisher apparatus. The solubility of the sugar in tert-butanol:pyridine (55:45 v/v) was determined by GC followed the method of Degn [5]. Sugar Ester Analysis Qualitative analysis of sugar ester was carried out by thin layer chromatography (TLC). Ten microliters of reaction mixture was spotted on silica gel 60 TLC plate (4×10 cm). The plates were developed in a mobile phase of chloroform:methanol:formic acid (50:10:1 v/v/v). The sample components were visualized by spraying with sulfuric acid reagent (sulfuric acid: methanol 50:50 v/v) followed by heating at 150 °C for 10 min. The sugar and derivative compounds occurred as black spots at different Rf values. The products were also analyzed by gas chromatography following the method of Degn et al. [6]. For purification of glucose ester, the reaction mixture was filtered through glass wool to remove immobilized lipase and molecular sieves. The organic solvent was evaporated under reduced pressure, and hexane was added to precipitate the product. The remaining glucose was removed by washing the precipitate several times with distilled water followed by drying in a dessicator for 24 h. The precipitate (100 mg) was dissolved in 0.5 ml pyridine and subjected to the preparative TLC using plates of 0.5 mm thickness and the same mobile solvent as described above. The glucose ester band was scraped off the plates and transferred

1974


to the microcolumn and eluted with methanol. The purified glucose ester was analyzed by 1 H-NMR and LC-MS. 1 H NMR spectra of glucose was recorded on a Varian Unity Inova 500 MHz spectrometer. Samples of 20 mg were prepared in 0.5 ml DMSO-d6. The mass spectrometry was performed on a Micromass LCT LC-MS iontrap. The expected mass of the glucose ester (C12H22O7 +Na+) was observed.

Results and Discussion Purification of Lipase The extracellular lipase from S. thermocarboxydus ME168 was purified using three-step procedures, and the purification profile was summarized in Table 1. The concentrated supernatant was precipitated by chilled acetone (1.5 times). Resource Q was used as anion exchange chromatography and the enzyme was eluted at 0.6 M NaCl. The Superdex 200 gel filtration chromatography was used for the final step of purification. The lipase peak was eluted in the void volume (data not shown). This suggested that the enzyme might be hydrophobic in native form because of its aggregated molecules [19]. Formation of aggregates between lipase and lipophilic molecules such as lipopolysaccharides, fatty acids and glycerides was observed in many lipases like B. stearothermophilus MC7 [34], B. thermoleovorans ID-1 [19] and B. subtilis 168 [21]. The purified enzyme was obtained in overall yield of 20.3% and exhibited the increase of 9.6-fold in the specific activity compared to the cell-free supernatant. The specific activity of purified enzyme was relatively low due to the aggregration-related problem. However, the purified enzyme showed single band on SDS-PAGE (Fig. 1). The molecular mass of purified lipase from S. thermocarboxydus ME168 was determined by SDS-PAGE under reducing condition that showed the single band protein of a molecular mass of 21 kDa. The molecular mass of other Streptomyces lipases were reported to be 27.5 kDa for S. rimosus [1] and 50 kDa for S. cinnamomeus [32]. N-terminal Determination The N-terminal sequence of the purified lipase from S. thermocaroxydus ME168 was determined to be ASDFDDQILG. This sequence showed low homology with other Table 1 Summary of purification steps for lipase from Streptomyces thermocarboxydus ME168 Purification step

Volume (ml)

Total activity (U)a

Total protein (mg)

Specific activity (U/mg)

Yield (%)

Purification factor

Crude supernatant Concentration

1,650.0

1,346.0

313.0

4.3

100.0

1.0

185.0

1,345.0

308.0

4.4

99.9

1.0

Acetone precipitation

120.0

1,071.0

217.0

4.9

79.6

1.1

Resource Q

204.0

632.0

59.0

10.7

47.0

2.5

Superdex 200

570.0

273.0

6.6

41.4

20.3

9.6

a

Activity was measured with pNPP as substrate at pH 8.5


1975

Fig. 1 Sodium dodecyl sulphate polyacrylamide gel electrophoresis pattern of the purified lipase from Streptomyces thermocarboxydus ME168. SDS-PAGE was conducted in 15% gel and the protein was stained with Coomassie Brilliant Blue R250. Lane 1 protein markers are β-galactosidase (116 kDa), BSA (66.2 kDa), ovalbumin (45 kDa), lactate dehydrogenase (35 kDa), restriction endonuclease Bsp98l (25 kDa), β-lactoglobulin (18.4 kDa) and lysozyme (14.4 kDa). Lane 2 purified enzyme after gel filtration

Streptomyces lipases [1, 32]. The main reason can be attributed to the fact that known microbial lipase sequences share only 40% overall similarity [33]. However, analysis of a much longer sequence (~200 amino acids) is required before the accurately sequence homology can be determined. Effect of pH and Temperature on Enzyme Activity and Stability The purified lipase was active toward pNPP in a wide pH range (4.0–11.0). The optimum activity was achieved at pH 8.5. The enzyme was not active at acidic pH (pH 4.0–5.5), and the activity was dramatically decreased between pH 9.0 and 11.0. This lipase showed good stability for 3 h in the broad pH range of 4.0 to 10.0 at 45 °C but the stability decreased rapidly at pH over 10.5 (Fig. 2). The purified lipase showed optimum activity toward pNPP at 50 °C, pH 8.5, and it was stable at this temperature with the residual activity of 92% after 3 h. The half life of the enzyme was more than 3 h at 65 °C, but after incubation at 80 °C for 3 h, it was completely inactivated (Fig. 3). The half life of the lipases from S. rimosus was 30 min at 65 °C [1] while B. stearothermophilus P1 was 2 h at 65 °C [30], B. stearothermophilus L1 was 30 min at 62 °C [17] and B. thermocatenulatus was 30 min at 60 °C [27]. Kinetic Parameters The values of Km and Vmax of the purified lipase from S. thermocarboxydus ME168 using pNPP as substrate calculated from the Lineweaver–Burk plot were 0.28 mM and 1,428 U/mg, respectively (Fig. 4). Most industrially used enzymes have Km in the range of 10−1 to 10−5 M when acting on biotechnologically important substrates [9].

1976


Fig. 2 Effect of pH on Streptomyces thermocarboxydus ME168 lipase activity (filled circle) and stability (filled square). The activity of the purified enzyme was assayed using pNPP as substrate with different buffers: acetate buffer (pH 4.0–5.5), phosphate buffer (pH 6.0–6.5), Tris–HCl buffer (pH 7.0–8.5) and glycine–NaOH buffer (pH 9.0–11.0)

Substrate Specificity Enzyme specificity was studied with p-nitrophenyl esters of various acyl chain lengths. Among the substrates tested, pNPP (C16) was the best substrate for the lipase from S. thermocarboxydus ME168, pNPB (C4) and pNPC (C8) were also good substrates, whereas very short chain esters, pNPA (C2) were poor substrates. Similar specificity has been reported for B. stearothermophilus MC7 lipase which preferred long chain fatty acid than short chain fatty acid [15]. Other reports showed that microbial lipases had preference for pNP esters with medium chain (C8 and C10) as substrate [1, 11].

Fig. 3 Effect of temperature on Streptomyces thermocarboxydus ME168 lipase activity (filled circle) and stability (filled square). The activity of the purified enzyme was assayed using pNPP as substrate in 50 mM Tris–HCl buffer pH 8.5 with different temperatures


1977

Fig. 4 Lineweaver–Burk plot of the purified lipase from Streptomyces thermocarboxydus ME168 using pNPP as substrate

Effect of Organic Solvents The stability and activity of enzyme in organic solvents are important for the application in organic synthesis reaction [31]. The stability of purified lipase from S. thermocarboxydus ME168 was studied in both water-miscible and water-immiscible organic solvents. This lipase was tolerant to some organic solvents (Table 2), e.g., dimethylsulfoxide (DMSO), methanol and dimethylformamide (DMF). However, acetone, tert-butanol, pyridine and acetronitrile caused significant deactivation, with less than 20% of initial activity remaining, while 95% ethanol resulted in complete loss of the lipase activity. The extracellular lipase Table 2 Effect of various organic solvents on the activity of the purified lipase from Streptomyces thermocarboxydus ME168 Organic solvent

Relative activity (%)a

Dimethylsulfoxide (DMSO) Methanol

54 85

Dimethylformamide (DMF)

62

Acetronitrile 95% Ethanol

2 0

Acetone

18

Pyridine

1

tert-Butanol 2-Methyl-2-butanol Hexane a

The activity was expressed as a percentage of the activity of untreated purified lipase

2 56 45

1978


from Acinetobacter sp. RAG-1 (LipA) was completely inactivated by pyridine at a concentration of 30% (v/v) in 1 h [8]. The 90% ethanol and butanol were reported to inactivate lipase from Aspergillus carneus after 24-h incubation [26]. Effect of Metal Ions and Other Reagents Various metal ions and other reagents were studied for their effects on the activity of the purified lipase from S. thermocarboxydus ME168 at concentrations 1.0 and 10.0 mM, 45 °C for 60 min. The ions of K+ and Ca2+ were found to stimulate lipase activity. In contrast, this lipase was sensitive to Zn2+ and Fe3+ (59 and 36% inhibition at 10 mM). These metal ions could inhibit at the catalytic site of enzyme directly, specific for lipase formation of complexes between metal ions and ionized fatty acids and changing their solubility and behavior at the interface [20]. Ethylenediamine tetraacetic acid (EDTA) as chelating agent reduced the enzyme activity with 49% inhibition at 10 mM. The reducing agent, dithiothreitol (DTT), reduced lipase activity to 52%, indicating that the presence of disulfide bond in the molecule, which may stabilize the active conformation of lipase [1]. In contrast, this enzyme was quite stable with oxidizing agents. It retained 82 and 99% activity with 10.0 mM ammonium persulphate and potassium iodide, respectively. The serine inhibitor, PMSF did not have any effect on the lipase activity even if at the concentration of 10.0 mM. All known lipases have serine in their active site but some lipases are resistant to inactivation by serine inhibitors, e.g., lipase from S. rimosus [1], Geobacillus sp. TW1 lipase [22] and Mucor hiemalis f. hiemalis [11]. The reagent-masking SH-group, pHMB, did not strongly inhibit lipase activity suggesting that free thiol group might be not essential for S. thermocarboxydus ME168 lipase activity. Lipase from S. rimosus was also not inactivated by pHMB at the concentration of 2.0 mM [1]. Application of Immobilized Crude Lipase from S. thermocarboxydus ME168 for Sugar Ester Synthesis The immobilized crude lipase from S. thermocarboxydus ME168 was tested for its ability to synthesize sugar ester. The TLC analysis of products (Fig. 5) showed that this immobilized crude lipase was able to synthesis sugar esters from glucose and maltose with vinyl acetate, vinyl butyrate and vinyl caproate. The identity of the purified product from the reaction with glucose and vinyl caproate was performed by 1H-NMR, and LC-MS showed that glucose caproate obtained from glucose and vinyl caproate catalyzed by immobilized crude lipase from S. thermocarboxydus ME168 was 6-O-caproate-D-glucopyranose monoester. The position of the ester linkage was clearly seen by the observed down field shift for C6 and H6 of the glucose moiety compared to free glucose. The mass of the purified product was confirmed by LC-MS and found that the expected mass of C12H22O7Na (M+Na+) was 301.7 (calculated was 301.3). Effect of Acyl Acceptors on Sugar Ester Synthesis Various kinds of sugar were used as acyl acceptors for the synthesis of sugar ester using vinyl caproate as acyl donor (Table 3). Glucose, fructose and α-methyl-D-glucose were converted to sugar esters with conversion yield more than 50%. This suggests that these sugars showed good solubility (48, 49 and 55 mM) in the mixture of tert-butanol:pyridine (55:45 v/v). Similar results have been reported that glucose and α-methyl-D-glucose were the good acyl acceptors for sugar ester synthesis from palm fatty acid distillates [4]. In


1979

Fig. 5 TLC analysis of sugar ester from glucose and maltose with three vinyl esters catalyzed by immobilized crude lipase of Streptomyces thermocarboxydus ME168 on celite. Lane 1 vinyl caproate, lane 2 vinyl caprylate and lane 3 vinyl palmitate

contrast, galactose and maltose showed low conversion yields because these sugars showed very low solubility of 27 and 12 mM, respectively. The oligosaccharide, maltotriose, was also investigated on sugar ester synthesis but no product was observed. It seemed to be that when the degree of polymerization of the sugar increased, the conversion yield dramatically decreased. This could be the result of low solubility of sugar in organic solvent when increasing degree of polymerization [6]. Effect of Chain Length of Acyl Donors on Sugar Ester Synthesis The efficiency of different chain lengths of vinyl esters on the formation of glucose ester by immobilized crude lipase from S. thermocarboxydus ME168 was studied. The conversion yield of glucose ester was decreased when the number of carbon atoms in vinyl esters increased. The conversion yields of 93, 67, 55 and 5% were obtained when using vinyl acetate, vinyl butyrate, vinyl caproate and vinyl palmitate as acyl donors, respectively. However, the conversion yield of glucose ester at 90, 89, 89 and 88% was obtained by Table 3 Effect of acyl acceptors on sugar ester synthesis from vinyl caproate catalyzed by immobilized crude lipase of Streptomyces thermocarboxydus ME168 Acyl acceptor

Conversion (%)

Xylose

32.9±0.12

Glucose

55.4±0.26

Galactose

4.2±0.04

Fructose

53.8±0.21

α-Methyl-D-glucose

78.6±0.21

Maltose Maltotriose

6.9±0.01 No reaction

1980


Table 4 Effect of chain length of acyl donors on glucose ester synthesis from vinyl caproate catalyzed by immobilized crude lipase of Streptomyces thermocarboxydus ME168 and Candida antarcitica lipase B (Novozyme435) Acyl donor

Conversion (%) ME168

Novozyme435

Vinyl acetate

93.40±0.23

90.21±0.44

Vinyl butyrate

66.67±0.25

89.16±0.16

Vinyl caproate

55.3±0.13

88.72±0.03

Vinyl palmitate

4.98±0.04

88.10±0.29

Novozyme 435 (Table 4). These results were contrary to the results obtained by Pseudomonas sp. lipase [29] and C. antarctica lipase B [19] which showed increasing conversion yield when an acyl group of donors was lengthened. Effect of Organic Solvents on Sugar Ester Synthesis The conversion yield of glucose to glucose caproate was investigated in various organic solvents (Table 5). Acetone and butanone showed very low conversion yield and no product was observed with hexane. In contrast, the highest conversion yield of 55% was obtained when the mixture of tert-butanol:pyridine (55:45 v/v) was used. A similar result was obtained by C. antarctica lipase [7]. Although, tert-butanol and pyridine inactivated the free lipase from S. thermocarboxydus ME168, this problem did not occur in the immobilized lipase. It might be that the immobilization of enzyme may protect the enzyme from solvent denaturation. Moreover, it helps in maintaining homogeneity of enzymes in the reaction media since it avoids aggregation of enzyme particles [35].

Conclusion The results obtained from our study showed that S. thermocarboxydus ME168 produced thermostable lipase. This strain grew at moderately high temperature, and its lipase also showed good thermostability. This lipase was able to catalyze the synthesis of sugar ester from glucose, fructose and various vinyl esters. So, this lipase is quite attractive for biotechnological applications based on its thermostability and ester synthesis activity.

Table 5 Effect of organic solvents on glucose ester synthesis from vinyl caproate catalyzed by immobilized crude lipase of Streptomyces thermocarboxydus ME168 Organic solvent

Conversion (%)

Hexane

No reaction

Acetone Butanone

1.5±0.03 4.2±0.18

tert-Butanol:DMSO (95:5 v/v)

16.3±0.20

tert-Butanol:pyridine (55:45 v/v)

54.7±0.72

tert-Butanol:pyridine (2:1 v/v)

43.3±0.48


1981

Acknowledgments The financial support by the Royal Golden Jubilee Program (RGJ, Thailand) to Thanongsak Chaiyaso was highly appreciated. The partial support from German Academic Exchange Service (DAAD, Germany) was also gratefully acknowledged.

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