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Surfactants from xylan: Production of n-octyl xylosides using a highly thermostable xylanase from Thermotoga neapolitana
Process Biochemistry 45 (2010) 700–705
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Surfactants from xylan: Production of n-octyl xylosides using a highly thermostable xylanase from Thermotoga neapolitana Gashaw Mamo *, Sangita Kasture 1, Reza Faryar, Suhaila Hashim, Rajni Hatti-Kaul Department of Biotechnology, Lund University, Getingevagen 60, Box 124, SE-221 00 Lund, Sweden
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 September 2009 Received in revised form 5 January 2010 Accepted 6 January 2010
A highly thermostable recombinant xylanase from Thermotoga neapolitana was used as a catalyst for single-step synthesis of n-octyl xylobioside and n-octyl xylotrioside from xylan and n-octanol. Effect of xylan concentration, enzyme dose, reaction water content, reaction temperature and initial pH on the yield of these surfactants has been studied. The optimal conditions for n-octyl xylobioside and n-octyl xylotrioside synthesis are found to be different. The maximum n-octyl xylobioside and n-octyl xylotrioside yield were 120 and 38 mg/g of xylan, respectively. The n-octyl xylobioside yield achieved in this study was better than the yield achieved in all other enzymatic synthesis studies reported so far except what is achieved with the use of supercritical fluoroform under high pressure. The n-octyl xylotrioside yield is the highest ever achieved through enzymatic synthesis. An integrated system of production and recovery of n-octyl xylosides has improved the yield of n-octyl xylobioside and n-octyl xylotrioside by a factor of 1.7 and 2.4, respectively. ß 2010 Elsevier Ltd. All rights reserved.
Keywords: Octyl xylosides Nonionic surfactants Thermotoga neapolitana Xylan Family 10 xylanase Integrated product removal
1. Introduction Alkyl glycosides are a group of nonionic surfactants synthesized from naturally occurring renewable resources of fatty alcohols and sugars. Owing to their excellent surfactant properties, alkaline pH stability and biodegradability [1–4], alkyl glycosides have made their way into several applications, especially where the conventional petrochemical based surfactants are not desirable, such as in foods, hygiene, pharmaceuticals and cosmetics industries [3,5]. Their biocompatibility further makes them preferable in research with biological systems, e.g. for application in membrane protein extraction [6,7]. Production and applications of alkyl glycosides are expected to grow significantly mainly due to the demand for safe and eco-friendly products, and possibility of producing new surfactants with novel properties. Synthesis of alkyl glycosides from sugar and fatty alcohol as raw materials has been achieved by chemical and enzymatic routes. The latter has the advantage of providing a cleaner process involving fewer steps due to the high selectivity of enzymes, and is accomplished under mild reaction conditions without the need for toxic reagents [8,9]. Glycosyl hydrolases have been the main group
* Corresponding author. Tel.: +46 46 2224741; fax: +46 46 2224713. E-mail address: [email protected] (G. Mamo). 1 Current address: Biopharmaceutical Research Center, Panacea Biotech Ltd., B-1, E-12 Mohan Co-operative Industrial Estate, Mathura Road, New Delhi 110 044, India. 1359-5113/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.01.005
of enzymes used as catalysts for the synthesis of alkyl glycosides via reverse hydrolysis or transglycosylation mechanisms [8]. The enzymes are required to be active in the low water environment necessary for minimizing hydrolysis of the product, and high temperature for facilitating mixing of the hydrophilic and lipophilic components of the reaction. With these prerequisites, enzymes from (hyper-)thermophiles have proven to be the most effective biocatalysts, which besides their thermostability are known also to exhibit higher activity and stability at low water activity [10,11]. The majority of alkyl glycosides known are based on hexoses or their dimers as glycosyl moiety. As carbohydrates are structurally diverse, the use of different sugar groups as surfactant components would potentially provide molecules with novel properties. With increasing development in the production of second-generation bioethanol from glucose obtained from cellulose, the option of valorising other sugars from residual polysaccharides is attractive. Hemicelluloses constitute up to one-third of most plant materials and contain pentoses as primary building blocks. Xylose, the monomer of the dominant hemicellulose xylan, has been used in surfactant synthesis and the surfactants have indeed proven to have excellent properties [2,4,12]. Alkyl b-D-xylobiosides have shown interesting foaming properties and biodegradability [2]. Similarly, 1-O-alkenoyl-D-xylose and alkenyl-D-xylosides display a remarkable foaming ability at low concentrations and have demonstrated outstanding surface-active properties such as reduced surface tension, which is lower than that generally obtained for sugar esters [4]. Despite the obvious advantages,
G. Mamo et al. / Process Biochemistry 45 (2010) 700–705
enzymatic synthesis of pentose based surfactants is less explored. Most of the enzymatic transxylosylation studies reported so far have used b-xylosidases, while only a few reports have involved endo-xylanases [13–16]. Microbial species belonging to the order Thermotogales produce a variety of highly thermostable glycosyl hydrolases, which would have potential for synthetic applications. One of the members is the hyperthermophilic bacterium Thermotoga neapolitana with an optimum growth temperature of 80 8C. The biocatalytic potential of the organism was demonstrated by transglycosylation of 1hexanol, 9-fluorene methanol, 1,4-butanediol and geraniol using a crude cell homogenate [16]. T. neapolitana produces a thermostable family 10 endo-xylanase [17], which is used in this study for the synthesis of xylose based surfactants from xylan and n-octanol.
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for 10 min after which the reaction was stopped by adding DNS reagent. One unit of xylanase activity was defined as the amount of enzyme that releases 1 mmol of reducing sugar equivalent to xylose per minute under the experimental conditions. Protein concentration in the enzyme sample was determined by bicinchoninic acid method using bovine serum albumin as standard [20]. 2.4. Xylanase mediated synthesis of n-octyl xylosides from n-octanol and xylan A typical reaction was performed by mixing 0.6 mL of sodium acetate buffer, pH 5.5 containing 50 mg of xylan and T. neapolitana xylanase with 1.4 mL of n-octanol in a 4 mL reaction vial. The final concentration of the buffer in the reaction mixture was 20 mM. Unless otherwise mentioned, the enzyme amount added was 0.1 mL that corresponded to the xylanase activity of 0.5 U. The reaction mixture was incubated with shaking at 70 8C up to 24 h in a heating block (HLC Biotech, Bovenden, Germany). Samples (250 mL) were withdrawn at specific time intervals after briefly vortexing the vials. The samples were centrifuged at 6000 g for 10 min to separate the phases and pellet out insoluble material. The phases were then analysed to determine the concentration of n-octyl xylosides.
2. Materials and methods 2.1. Chemicals
2.5. Purification of alkyl xyloside products and preparation of standards
n-Octanol and birchwood xylan were purchased from Sigma–Aldrich, xylose was from Fluka (Switzerland), while silica gel 60 F254 precoated aluminum plates and acetonitrile were from Merck (Darmstadt, Germany). All the other materials used were of analytical grade
The xylanase catalysed reaction was performed in 200 mL reaction mixture in a 500 mL round bottomed flask at 70 8C in a thermostated oil bath with constant stirring at 700 rpm. The proportion of the reaction mixtures and the reaction conditions were similar to that described above. After running the reaction for 14 h, the aqueous and n-octanol phases were separated and alkyl xylosides were purified from the n-octanol phase. The alcohol was first removed under vacuum and the concentrated sample was loaded on an 80 mL silica column equilibrated with chloroform–methanol–water (65:25:2, v/v/v). The products were eluted using the same solvent system and the eluate was collected in 10 mL fractions that were analysed by thin layer chromatography (TLC). Fractions containing n-octyl monoxyloside, n-octyl xylobioside and n-octyl xylotrioside were pooled separately, dried and used as standards. The purity of these preparations was confirmed using HPLC and mass spectrometry.
2.2. Production and purification of recombinant T. neapolitana xylanase The recombinant E. coli BL21 (DE3) harboring the gene encoding T. neapolitana xylanase was grown in LB medium under conditions described earlier for recombinant Bacillus halodurans xylanase [18]. After cultivation, the cells were harvested by centrifugation for 10 min at 8000 g in a Sorvall refrigerated centrifuge. The cell pellet was resuspended in 10 mM Tris–HCl buffer pH 7.4 and treated with lysozyme (2 mg/mL) at room temperature for 1 h and subsequently subjected to freezing at 20 8C for 2 h and thawing prior to sonication using an ultrasonicator (Dr. Hielscher UP 400s, Germany) with 5 cycles of 1 min, 0.5 cycles/s, and 50% amplitude and 1 min interval between each cycle. The homogenate was centrifuged at 8000 g to remove the cell debris and the supernatant containing the enzyme was heated for 30 min at 80 8C to denature the contaminant host cell proteins. The clear supernatant obtained after centrifugation was used as the source of the enzyme. The specific activity of the enzyme preparation was 36 U/mg of protein. 2.3. Determination of enzyme activity and protein content The hydrolytic activity of T. neapolitana xylanase was determined based on the release of reducing sugars from xylan using the dinitrosalicylic acid (DNS) method [19]. A reaction mixture composed of 1% (w/v) birchwood xylan dissolved in 50 mM Na-acetate buffer, pH 5.5 and appropriately diluted enzyme was incubated at 80 8C
2.6. Semi-continuous process for synthesis of n-octyl xylosides A semi-continuous production and recovery of n-octyl xylosides was set up as shown in Fig. 1. The reaction mixture was composed of 5 g xylan, 350 mL n-octanol, 600 U xylanase, 10 mL of 1 M sodium acetate buffer (pH 5.5) and water to make up the volume to 500 mL. The reaction was performed at 70 8C and continuously stirred using a magnetic stirrer. During the course of the reaction, part of the reaction mixture was continuously pumped at a rate of 0.8 mL/min into a separating funnel to allow phase separation between the n-octanol and aqueous phases. After running the reaction for 3 h the n-octanol phase from the separation funnel was pumped over a Dowex1 50WX2-200(H) column (20 cm height, 1.8 cm internal diameter, 50 mL packed bed volume) pre-equilibrated with water saturated n-octanol. The water phase from the separation funnel as well as the
Fig. 1. Schematic presentation of the integrated system used for production and semi-continuous recovery of n-octyl xylosides. (1) Circulating water bath (70 8C), (2) magnetic plate stirrer, (3) reactor, (4) reaction mixture (5) magnetic stir bar, (6 and 7) pumps, (8) separation funnel, (9) adsorbent column, (10) elution solvent, and (11) product.
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column effluent, were circulated back to the reactor. This was continued for about 6 h after which the bound n-octyl xyloside products were eluted from the column using 50 mL of 99% (v/v) ethanol. The column was then re-equilibrated with noctanol (50 mL) and re-loaded for another 6 h. This was repeated for 3 cycles. The amount of products in the eluted samples was analysed by HPLC and compared to the amount achieved in the batch system (which had exactly the same set up but without the integrated Dowex column). 2.7. Determination of partition coefficient for alkyl xylosides from the reaction mixture The xylanase catalysed reaction between xylan and n-octanol was carried out in a 2 mL reaction mixture in 4 mL screw capped vials as described above. The phases were separated after centrifugation at 6000 g for 10 min and the amount of the alkyl xylosides was quantified by HPLC in both phases. The total concentration of each product in the reaction mixture was determined by diluting the biphasic reaction mixture with acetonitrile that resulted in a single homogenous phase from which samples were taken and analysed by HPLC. The partition coefficient, K of the different n-octyl xylosides was determined as the ratio of the concentration in the organic and aqueous phases at room temperature and pH 5.5. The amount of each product in the interphase was calculated by deducting the sum of its concentration in the top and bottom phase from its total concentration in the reaction mixture. 2.8. Anaylsis of n-octyl xylosides and reducing sugars Thin layer chromatography on silica gel 60 F254 precoated aluminum plates was used for qualitative analysis of the samples. The solvent system used was chloroform–methanol–water (65:25:2, v/v/v). After a single solvent ascent, spots corresponding to n-octyl xyloside products and reducing sugars were detected by spraying with 10% sulfuric acid in methanol and heating at 110 8C until spots were visible. n-Octyl-monoxyloside, -xylobioside and -xylotrioside had Rf values of about 0.73, 0.61 and 0.48, respectively. The concentration of n-octyl xylosides was quantitatively determined by HPLC (LaChrom system with an L-7100 pump, L-750 autosampler and a D-7000 interface system) using reverse phase RP-18 column (250 mm 4.6 mm, Kromasil1 1005C18, Akzo Nobel, Sweden) and a 500 ELSD evaporative light scattering detector (Alltech Associates, Deerfield, MI). The column was maintained at room temperature and the mobile phase used was acetonitrile:water (65:35) at a flow rate of 0.8 mL/min. The retention times of n-octyl-monoxyloside, -xylobioside and xylotrioside were 4.28, 3.25 and 2.55 min, respectively. The identity of the purified n-octyl xyloside products was confirmed by mass spectrometry using a quadrupole time of flight (Q-TOF) hybrid tandem mass spectrometer (API Qstar, MDS Sciex, Ontario, Canada) equipped with an electrospray ionization source (Turboion Spray1).
Table 1 Distribution behavior of the alkyl xylosides in n-octanol-buffer system. Product
Partition coefficient
Relative amount (%) Top phase
Interphase
Bottom phase
n-Octyl xyloside n-Octyl xylobioside n-Octyl xylotrioside
100 8.4 2.1
74 84 66
26 6 1
0 10 33
and in addition, improves xylan solubility. The experiments were carried out with n-octanol as the nucleophile, where n-octylmonoxyloside, -xylobioside and -xylotrioside were produced; their yields depending on the reaction conditions. 3.1. Partition coefficients of n-octyl xylosides in the reaction system The reaction between xylan (25 mg/mL) and n-octanol (70%) catalysed by the recombinant T. neapolitana xylanase in 2 mL volume was run for 9 h. The total amounts of n-octyl-monoxyloside, -xylobioside and -xylotrioside produced were about 0.34, 4.45 and 0.47 mg, respectively. Table 1 shows the distribution coefficient of n-octyl xyloside products in the biphasic system. nOctyl monoxyloside was distributed between the top phase and interphase, and was not detected in the aqueous phase. With increase in the number of sugar units, the relative amounts of the alkyl xylosides partitioned to the aqueous phase increased to 10% and 33% for n-octyl-xylobioside and -xylotrioside, respectively. According to an earlier report, only n-octyl xylobioside and noctyl xylotrioside (and not n-octyl xylomonoside) possess the desirable surfactant properties [2]. Hence, the major focus in this work has been to study the influence of various reaction parameters on the yield and optimize conditions for improved production of these products. 3.2. Influence of reaction parameters on production of n-octylxylobioside and -xylotrioside
3. Results and discussion Glycosyl hydrolase family 10 xylanases possess a retaining mode of reaction mechanism. The enzymes randomly cleave xylan and the covalent intermediate of enzyme-xylooligosaccharides formed reacts with the nucleophiles available to generate the products. Hydrolysis occurs when water is the nucleophile, while the presence of other nucleophiles results in the formation of transglycosylation products, e.g. alkyl xylosides are produced in the presence of an alcohol. The products formed are also subject to further attack by the same enzyme, resulting in other hydrolysis or transglycosylation products. Hence, knowledge of reaction conditions required for optimal production of the desired alkyl xylosides is important in order to control the degradation and maintain high yields of the product(s) of interest. Production of alkyl xylosides using xylan as raw material is attractive from technical as well as economic point of view as both the xylan hydrolysis and transglycosylation are combined in one step hence avoiding the need for prior isolation of hydrolysis products. Preliminary experiments on the reaction between xylan and different alcohols, catalysed by T. neapolitana xylanase, showed the occurrence of transglycosylation reaction up to an alcohol chain length of C12, however the reaction efficiency was found to decrease with increasing alkyl chain length. Investigation on the enzyme stability showed the xylanase to be fairly stable in the presence of n-octanol, retaining about 80% of its original activity after incubation for 8 h at 80 8C in 50 mM acetate buffer, pH 5.5 containing 50% (v/v) n-octanol. This property allows the use of high temperature when performing transglycosylation reaction
In the reaction medium with 70% (v/v) n-octanol, increasing xylan concentration from 5 to 50 mg/mL showed a decrease in the equivalent total product yield from 144 to 58 mg per gram of xylan; the decrease in n-octyl xylobioside concentration being more prominent (Table 2). At xylan concentration of 50 mg/mL, there was practically no water phase which resulted in unsuitable conditions for mixing and mass transfer, which in turn affected the product yield. On the other hand, lower xylan concentration results in a diluted product requiring larger reaction volumes, an undesirable effect for downstream processing of the products. In this study, a xylan concentration of 25 mg/mL that gave the total Table 2 Effect of xylan concentration on yields of n-octyl-xylobioside and -xylotrioside after 9 h of reaction with n-octanol catalysed by T. neapolitana xylanase. The reaction mixture was composed of xylan, 1.4 mL octanol, 0.6 mL buffer and 0.1 U xylanase. Product
Actual yield (mg/mL)
Equivalent yield (mg product/g xylan)
5
n-Octyl-xylobioside n-Octyl-xylotrioside
1.12 0.32
112 32
10
n-Octyl-xylobioside n-Octyl-xylotrioside
1.37 0.38
69 20
25
n-Octyl-xylobioside n-Octyl-xylotrioside
1.46 0.46
58 19
50
n-Octyl-xylobioside n-Octyl-xylotrioside
2.18 0.69
44 14
Xylan concentration (mg/mL)
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actual product yield of 1.92 mg/mL (the sum of n-octyl-xylobioside and -xylotrioside) was used for further studies. The hydrolysis of xylan (25 mg/mL) in the two-phase system was also followed. About 40% of the xylan was converted to reducing sugar during 9 h incubation, which according to TLC analysis contained a significant amount of xylose and xylobiose. Such a significant degree of hydrolysis is not surprising and is due to the high water content of the reaction. The possibility of producing n-octyl xylosides by reacting n-octanol with xylose and xylobiose was investigated but there was no product formation (data not shown). This seems to be in accordance with an earlier report by Matsumura et al. [21] showing that xylotetraose was the shortest xylooligosaccharide accepted by Aureobasidium pullulans xylanase for reaction with n-octanol. A low water activity would favor the reaction equilibrium towards product synthesis [8], however running transglycosylation reactions in water-free media suffer from drawbacks such as insolubility of sugar substrates, inactivation of glycosidases and/or unsuitable enzyme conformation. Accordingly, there was no detectable alkyl xyloside formation in a water-free reaction system. Besides acting as a competing nucleophile for the reaction, water serves as a medium for solubilizing the substrate xylan. Variation of the water content in the range of 10–50% (v/v) resulted in a low yield of alkyl xylosides both at low water content (10%, v/ v) where the xylan solubility is low and also at high water content (40–50%, v/v) that favors product hydrolysis. As shown in Fig. 2, the yield of n-octyl xylobioside increased gradually with time and was highest at a water content of 30% (v/v) while the highest level of n-octyl xylotrioside was reached at 20% (v/v) water and then decreased with further incubation due to hydrolysis to n-octyl xylobioside and xylose. The n-octyl xylobioside yield increased with increasing enzyme concentration up to 0.25 U/mL of the reaction volume. Further increase up to 1 U/mL did not make a significant difference except
Fig. 2. Time profile of the yields of (A) n-octyl xylobioside and (B) n-octyl xylotrioside at initial water content of 10% ( ), 20% (*), 30% (~), 40% (&), and 50%, v/v (^), respectively. Defined amounts of n-octanol were added to a mixture of 50 mg xylan, 0.1 U of T. neapolitana xylanase and sodium acetate buffer, pH 5.5 in a final reaction volume of 2 mL. The mixture was incubated with shaking at 70 8C.
*
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Fig. 3. Effect of recombinant T. neapolitana xylanase concentration on the yields of (A) n-octyl xylobioside and (B) n-octyl xylotrioside with time from xylan and noctanol at 70 8C. The enzyme was used at a concentration of 0.025 U (&), 0.05 U (*), 0.1 U (^), 0.25 U (&), 0.5 U (~) and 1 U/mL (*), respectively, in a reaction mix containing 50 mg of xylan, 1.4 mL of n-octanol and 0.6 mL of sodium acetate buffer, pH 5.5.
that the time required to achieve the maximum yield was shortened to 3–6 h (Fig. 3). At high enzyme dosage (0.5–1 U/mL) and extended incubation, n-octyl xylobioside concentration declined due to its hydrolysis. n-Octyl xylotrioside yield was higher when 0.05 and 0.1 U/mL enzyme was used, while at high enzyme dosage it was completely hydrolysed and hence not detected in the reaction mixture. In general, oligomers with higher degree of polymerization are cleaved more efficiently by xylanases and affinity constants decrease with sugar chain length [22]; higher affinity of a xylanase for n-octyl xylotrioside than n-octyl xylobioside has been demonstrated earlier [21]. This is related to the nature of the active site of the xylanases, which are known to have 3–5 subsites for binding the xylopyranose rings of xylan [23]. For a glycosidic bond to be broken the substrate should bind at least to one + subsite and one subsite. The longer the oligosaccharide, the higher the probability for the oligosaccharides to bind to the + and subsites, facilitating hydrolysis. Although there is no structural information available so far on the binding of alkyl glycosides to the xylanase active site, noctyl xylobioside is a large enough molecule for the sugar groups to bind to the subsites, with consequent hydrolysis of the glycosidic bond. Transglycosylation may also occur where a xylose unit is transferred to xylobiose (produced as the hydrolysis product) to form xylotrioside that in turn is hydrolysed to xylose and xylobiose [15]. Furthermore, family 10 endo-xylanases are known to have low xylosidase activities [24], and can slowly cleave smaller xylooligosaccharides. Fig. 4 shows the effect of temperature on the reaction yield. Higher yields of n-octyl-xylobioside and -xylotrioside were achieved in a temperature range of 70–80 8C. The initial reaction rate was nearly similar in this temperature range; however, a slightly higher yield of n-octyl xylobioside was obtained on prolonged incubation (up to 24 h) at 70 8C. This is probably due to
G. Mamo et al. / Process Biochemistry 45 (2010) 700–705
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Fig. 4. Synthesis of (A) n-octyl xylobioside and (B) n-octyl xylotrioside from xylan and n-octanol catalysed by recombinant T. neapolitana xylanase at 50 8C (^), 60 8C (&), 70 8C (~), 80 8C (*), and 90 8C ( ), respectively. The reaction mixture containing 1.4 mL n-octanol, 50 mg xylan and 0.6 mL sodium acetate buffer, pH 5.5 and 0.1 U xylanase was incubated at different temperatures in a shaking heating block and samples withdrawn at defined time intervals for analysis of the products.
*
higher stability of the enzyme. On the other hand, the maximum yield of n-octyl xylotrioside (over 35 mg/g xylan) was achieved within 9 h. With respect to the initial pH of the aqueous phase of the reaction, n-octyl xylobioside yield increased with time and was highest at pH 5 reaching 100 mg per gram xylan after 24 h of reaction (data not shown). On the other hand, the yield of n-octylxylotrioside increased up to 4 h and was highest at pH 7 (20 mg/g xylan). Subsequently, the n-octyl-xylotrioside underwent hydrolysis and was undetectable after 18 h of reaction at pH 5–6. The extent of hydrolysis was significantly lower at pH 7, which can be attributed to lower enzyme activity under these conditions. 3.3. Production of n-octyl xylooligosaccharides from xylan From the data obtained above it is clear that optimal conditions for synthesis of n-octyl-xylobioside and -xylotrioside
are slightly different. Production of n-octyl xylobioside is optimal in the reaction with initial water content of 30% (v/ v), enzyme dosage of 0.25 U/mL, pH 5 and 70 8C. On the other hand, n-octyl xylotrioside production is more favorable at a lower water content of 20%, enzyme dose of 0.1 U/mL, pH 7 and at 70–80 8C. The reaction was run under optimal conditions for n-octyl xylobioside production. About 100 mg n-octyl xylobioside/g xylan was produced during 18 h reaction and prolonged incubation up to 96 h resulted neither in formation of more surfactant nor product hydrolysis (data not shown). Further addition of equal amount of enzyme after 18 h did not improve the yield but instead led to product hydrolysis, which is in agreement with the effect of the increased enzyme dose shown in Fig. 3 and also indicates that the enzyme denaturation was not the cause for termination of the reaction. As shown in Table 3, comparison of the results obtained in this study with the earlier reports on xylosidation reactions using xylanase from other microbial sources shows that the productivity was higher than all the other systems except for the system using supercritical fluid (SCF) as the reaction medium where either comparable or relatively higher yields were reported depending on the SCF used. The maximum yield of n-octyl xylotrioside (38 mg/g xylan) is the highest ever achieved through enzymatic synthesis. Although the n-octyl xylobioside yield of about 10% (based on xylan amount) appears to be low, the enzymatic process provides several advantages. The endo-xylanase produces the more surface-active b-anomers [15,16], while the chemical synthesis results in anomerically mixed products dominated by a-anomers [2,25], from which the separation of b-anomer products is complex and results in poor yields. Moreover, the chemical methods often result in alkyl monosides [25], which have less desirable surfactant properties than alkyl-xylobioside and -xylotrioside produced by the xylanase [2]. 3.4. Integrating product removal with production Product inhibition is the major bottleneck in several biotechnology processes, limiting their productivity. In the system described above, it was observed that replacing 50% of the product-enriched n-octanol phase with fresh n-octanol after 6 and 12 h of reaction resulted in improvement of the n-octyl xylobioside yield by about 16%. Hence, a preliminary experiment on integrating product capture with the production system was set up as shown in Fig. 1. The reaction was first allowed to run under batch conditions and then the reaction mixture was pumped to a separating funnel for phase separation, after which the n-octanol phase was passed over a column of an ion exchange resin, Dowex 50WX2-200(H). This adsorbent was selected based on our screening studies on adsorption of n-octyl
Table 3 Maximum yield achieved per mL of reaction volume by different transxylosidation studies between 9 and 18 h of reaction. System
Water content (%v/v)
Total reaction volume (mL)
Xylan concentration (mg/mL)
Supercritical fluid
Pressure (kg/cm2)
T. neapolitanaa T. neapolitanab A. pullulans A. pullulans A. pullulans A. pullulans A. pullulansc
30 30 9.8 20 9.8 10 30
2 2 2.55 2.5 2.55 10 10
25 25 19.6 20 19.6 5 5
– – CO2 CHF3 – – –
– – 150 70 – – –
Productivity (mg/g xylan/h) n-Octyl xylobioside
n-Octyl xylotrioside
2.78 2.50 2.45 4.03 0.86 0.89 0.00
0.28 2.11 0.54 0.50 0.00 0.00 0.00
a and b denote systems with maximum production of n-octyl -xylobioside and -xylotrioside, respectively. a Reaction mixture containing 70% (v/v) n-octanol, 50 mg xylan, 0.5 U xylanase, and sodium acetate buffer, pH 5 was incubated at 70 8C for 18 h. b A mixture containing of 80% (v/v) n-octanol, 50 mg xylan, 0.1 U xylanase, and sodium acetate buffer, pH 5.5 was incubated at 70 8C for 9 h. c Only n-octyl-xyloside but no -xylobioside and - xylotrioside was produced.
Reference
This study This study [9] [9] [9] [9] [13]
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Acknowledgment This work was performed within the framework of Greenchem, a research program supported by the Swedish Foundation for Strategic Environmental Research (Mistra). References
Fig. 5. Production of n-octyl xylosides in integrated (&) and batch reactor system ( ). The reaction mixture was composed of xylan (5 g), n-octanol (350 mL), xylanase (1.2 U/mL), 10 mL of 1 M sodium acetate buffer, pH 5.5 and water (to 500 mL). The total concentration for n-octyl monoxyloside (OX1), n-octyl xylobioside (OX2) and n-octyl xylotrioside (OX3) was determined after 24 h reaction time.
xylobioside to various resins (Sahoo et al., unpublished data). As shown in Fig. 5, the yield of the n-octyl xylosides was somewhat higher than that achieved in the batch system, the increase in noctyl xylotrioside yield being about 2.4 fold. Optimization of the integrated system parameters is expected to further increase the yields of n-octyl xylobioside and n-octyl xylotrioside per gram of xylan used. The integrated system has an added advantage of concentrating the n-octyl xylosides and easy separation from noctanol. Currently, recovery of alkyl glycosides from the respective alcohols used as substrates is carried out by distillation, which is a very energy intensive process. In the integrated system described here, the product is stripped from the bulk n-octanol phase by adsorption and is eluted from the adsorbent using ethanol, which is much easier to remove than the longer chain alcohols. 4. Conclusions This study clearly shows the advantages of using a thermostable xylanase for synthesis of interesting surfactant products, n-octyl-xylobioside and -xylotrioside directly from xylan. The use of xylan directly as substrate is attractive as it cuts down the costs for pre-hydrolysis to xylooligosaccharides for further transxylosidation to alkyl xylosides. At the same time, process technologies need to be developed that will overcome the limitations of the enzymatic processes, such as product inhibition and degradation so as to make the processes economically competitive. Pentose based surfactants are valuable products and their production can be integrated as a part of the lignocellulose biorefineries. Inspite of their relatively low yields, the reaction described in this paper results in other major side-products, i.e. xylooligosaccharides that can be recovered from the aqueous phase and used either directly or further valorized to other products thereby increasing the value addition to the raw material.
[1] Sarney DB, Vulfson EN. Application of enzymes to the synthesis of surfactants. Trends Biotechnol 1995;13:164–72. [2] Matsumura S, Ando S, Toshima K, Kawada K. Surface activity, antimicrobial properties and biodegradability of n-alkyl xylosides. J Jpn Oil Chem Soc 1998;47:247–55. [3] Hill K, Rhode O. Sugar based surfactants for consumer products and technical applications. Fett/Lipid 1999;101(1):25–33. [4] Damez C, Bouquillon S, Harakat D, Henin F, Muzart J, Pezron I, et al. Alkenyl and alkenoyl amphiphilic derivatives of D-xylose and their surfactant properties. Carbohydr Res 2007;342:154–62. [5] Pillion DJ, Ahsan F, Arnold JJ, Balusubramanian BM, Piraner O, Meezan E. Synthetic long-chain alkyl maltosides and alkyl sucrose esters as enhancers of nasal insulin absorption. J Pharm Sci 2002;91:1456–62. [6] Le Maire M, Champeil P, Møller JV. Interaction of membrane proteins and lipids with solubilizing detergents. Biochim Biophys Acta 2000;1508:86–111. [7] Garavito RM, Ferguson-Miller S. Detergents as tools in membrane biochemistry. J Biol Chem 2001;276:32403–6. [8] van Rantwijk F, Woudenberg-van Oosterom M, Sheldon RA. Glycosidasecatalysed synthesis of alkyl glycosides. J Mol Catal B Enzym 1999;6:511–32. [9] Nakamura T, Toshima K, Matsumura S. One-step synthesis of n-octyl-b-Dxylotrioside, xylobioside and xyloside from xylan and n-octanol using acetone powder of Aureobasidium pullulans in supercritical fluids. Biotechnol Lett 2000;22:1183–9. [10] Sellek GA, Chaudhuri JB. Biocatalysis in organic media using enzymes from extremophiles. Enzyme Microb Technol 1999;25:471–82. [11] Ducret A, Trani M, Lortie R. Screening of various glycosidases for the synthesis of octyl glucoside. Biotechnol Bioeng 2002;77:752–7. [12] Greffe L, Bessueille L, Bulone V, Brumer H. Synthesis, preliminary characterization, and application of novel surfactants from highly branched xyloglucan oligosaccharides. Glycobiology 2004;15:437–45. [13] Matsumura S, Sakiyama K, Toshima K. One-pot synthesis of alkyl b-D-xylobioside from xylan and alcohol by acetone powder of Aureobasidium pullulans. Biotechnol Lett 1997;19:1249–55. [14] Kadi N, Belloy L, Chalier P, Crouzet JC. Enzymatic synthesis of aroma compound xylosides using transfer reaction by Trichoderma longibrachiatum xylanase. J Agric Food Chem 2002;50:5552–7. [15] Jiang Z, Zhu Y, Li L, Yu X, Kusakabe I, Kitaoka M, et al. Transglycosylation reaction of xylanase B from the hyperthermophilic Thermotoga maritima with the ability of synthesis of tertiary alkyl b-D-xylobiosides and xylosides. J Biotechnol 2004;114:125–34. [16] Tramice A, Pagnotta E, Romano I, Gambacorta A, Trincone A. Transglycosylation reactions using glycosyl hydrolases from Thermotoga neapolitana, a marine hydrogen-producing bacterium. J Mol Catal B Enzym 2007;47:21–7. [17] Zverlov V, Piotukh K, Dakhova O, Velikodvorskaya G, Borriss R. The multidomain xylanase A of the hyperthermophilic bacterium Thermotoga neapolitana is extremely thermoresistant. Appl Microbiol Biotechnol 1996;45:245–7. [18] Mamo G, Delgado O, Martinez A, Mattiasson B, Hatti-Kaul R. Cloning, sequence analysis and expression of a gene encoding an endoxylanase from Bacillus halodurans S7. Mol Biotechnol 2006;33:149–60. [19] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426–8. [20] Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD. Measurement of protein using bicinchonic acid. Anal Biochem 1985;150:76–85. [21] Matsumura S, Sakiyama K, Toshima K. Preparation of octyl b-D-xylobioside and xyloside by xylanase-catalyzed direct transglycosylation reaction of xylan and octanol. Biotechnol Lett 1999;21:17–22. [22] Prade RA. Xylanases: from biology to biotechnology. Biotechnol Genet Eng Rev 1996;13:101–31. [23] Subramaniyan S, Prema P. Biotechnology of microbial xylanases: enzymology, molecular biology, and application. Crit Rev Biotechnol 2002;22:33–64. [24] Biely P, Vrsanska´ M, Tenkanen M, Kluepfel D. Endo-beta-1,4-xylanase families: differences in catalytic properties. J Biotechnol 1997;57:151–66. [25] Lu¨ders H. Synthesis of alkyl glucosides and alkyl polyglucosides. In: Balzer D, Luders H, editors. Nonionic surfaces: alkyl polyglucosides (surfactant science series vol. 91). CRS Press; 2000. p. 20–75.
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Surfactants from xylan: Production of n-octyl xylosides using a highly thermostable xylanase from Thermotoga neapolitana Gashaw Mamo *, Sangita Kasture 1, Reza Faryar, Suhaila Hashim, Rajni Hatti-Kaul Department of Biotechnology, Lund University, Getingevagen 60, Box 124, SE-221 00 Lund, Sweden
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 September 2009 Received in revised form 5 January 2010 Accepted 6 January 2010
A highly thermostable recombinant xylanase from Thermotoga neapolitana was used as a catalyst for single-step synthesis of n-octyl xylobioside and n-octyl xylotrioside from xylan and n-octanol. Effect of xylan concentration, enzyme dose, reaction water content, reaction temperature and initial pH on the yield of these surfactants has been studied. The optimal conditions for n-octyl xylobioside and n-octyl xylotrioside synthesis are found to be different. The maximum n-octyl xylobioside and n-octyl xylotrioside yield were 120 and 38 mg/g of xylan, respectively. The n-octyl xylobioside yield achieved in this study was better than the yield achieved in all other enzymatic synthesis studies reported so far except what is achieved with the use of supercritical fluoroform under high pressure. The n-octyl xylotrioside yield is the highest ever achieved through enzymatic synthesis. An integrated system of production and recovery of n-octyl xylosides has improved the yield of n-octyl xylobioside and n-octyl xylotrioside by a factor of 1.7 and 2.4, respectively. ß 2010 Elsevier Ltd. All rights reserved.
Keywords: Octyl xylosides Nonionic surfactants Thermotoga neapolitana Xylan Family 10 xylanase Integrated product removal
1. Introduction Alkyl glycosides are a group of nonionic surfactants synthesized from naturally occurring renewable resources of fatty alcohols and sugars. Owing to their excellent surfactant properties, alkaline pH stability and biodegradability [1–4], alkyl glycosides have made their way into several applications, especially where the conventional petrochemical based surfactants are not desirable, such as in foods, hygiene, pharmaceuticals and cosmetics industries [3,5]. Their biocompatibility further makes them preferable in research with biological systems, e.g. for application in membrane protein extraction [6,7]. Production and applications of alkyl glycosides are expected to grow significantly mainly due to the demand for safe and eco-friendly products, and possibility of producing new surfactants with novel properties. Synthesis of alkyl glycosides from sugar and fatty alcohol as raw materials has been achieved by chemical and enzymatic routes. The latter has the advantage of providing a cleaner process involving fewer steps due to the high selectivity of enzymes, and is accomplished under mild reaction conditions without the need for toxic reagents [8,9]. Glycosyl hydrolases have been the main group
* Corresponding author. Tel.: +46 46 2224741; fax: +46 46 2224713. E-mail address: [email protected] (G. Mamo). 1 Current address: Biopharmaceutical Research Center, Panacea Biotech Ltd., B-1, E-12 Mohan Co-operative Industrial Estate, Mathura Road, New Delhi 110 044, India. 1359-5113/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.01.005
of enzymes used as catalysts for the synthesis of alkyl glycosides via reverse hydrolysis or transglycosylation mechanisms [8]. The enzymes are required to be active in the low water environment necessary for minimizing hydrolysis of the product, and high temperature for facilitating mixing of the hydrophilic and lipophilic components of the reaction. With these prerequisites, enzymes from (hyper-)thermophiles have proven to be the most effective biocatalysts, which besides their thermostability are known also to exhibit higher activity and stability at low water activity [10,11]. The majority of alkyl glycosides known are based on hexoses or their dimers as glycosyl moiety. As carbohydrates are structurally diverse, the use of different sugar groups as surfactant components would potentially provide molecules with novel properties. With increasing development in the production of second-generation bioethanol from glucose obtained from cellulose, the option of valorising other sugars from residual polysaccharides is attractive. Hemicelluloses constitute up to one-third of most plant materials and contain pentoses as primary building blocks. Xylose, the monomer of the dominant hemicellulose xylan, has been used in surfactant synthesis and the surfactants have indeed proven to have excellent properties [2,4,12]. Alkyl b-D-xylobiosides have shown interesting foaming properties and biodegradability [2]. Similarly, 1-O-alkenoyl-D-xylose and alkenyl-D-xylosides display a remarkable foaming ability at low concentrations and have demonstrated outstanding surface-active properties such as reduced surface tension, which is lower than that generally obtained for sugar esters [4]. Despite the obvious advantages,
G. Mamo et al. / Process Biochemistry 45 (2010) 700–705
enzymatic synthesis of pentose based surfactants is less explored. Most of the enzymatic transxylosylation studies reported so far have used b-xylosidases, while only a few reports have involved endo-xylanases [13–16]. Microbial species belonging to the order Thermotogales produce a variety of highly thermostable glycosyl hydrolases, which would have potential for synthetic applications. One of the members is the hyperthermophilic bacterium Thermotoga neapolitana with an optimum growth temperature of 80 8C. The biocatalytic potential of the organism was demonstrated by transglycosylation of 1hexanol, 9-fluorene methanol, 1,4-butanediol and geraniol using a crude cell homogenate [16]. T. neapolitana produces a thermostable family 10 endo-xylanase [17], which is used in this study for the synthesis of xylose based surfactants from xylan and n-octanol.
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for 10 min after which the reaction was stopped by adding DNS reagent. One unit of xylanase activity was defined as the amount of enzyme that releases 1 mmol of reducing sugar equivalent to xylose per minute under the experimental conditions. Protein concentration in the enzyme sample was determined by bicinchoninic acid method using bovine serum albumin as standard [20]. 2.4. Xylanase mediated synthesis of n-octyl xylosides from n-octanol and xylan A typical reaction was performed by mixing 0.6 mL of sodium acetate buffer, pH 5.5 containing 50 mg of xylan and T. neapolitana xylanase with 1.4 mL of n-octanol in a 4 mL reaction vial. The final concentration of the buffer in the reaction mixture was 20 mM. Unless otherwise mentioned, the enzyme amount added was 0.1 mL that corresponded to the xylanase activity of 0.5 U. The reaction mixture was incubated with shaking at 70 8C up to 24 h in a heating block (HLC Biotech, Bovenden, Germany). Samples (250 mL) were withdrawn at specific time intervals after briefly vortexing the vials. The samples were centrifuged at 6000 g for 10 min to separate the phases and pellet out insoluble material. The phases were then analysed to determine the concentration of n-octyl xylosides.
2. Materials and methods 2.1. Chemicals
2.5. Purification of alkyl xyloside products and preparation of standards
n-Octanol and birchwood xylan were purchased from Sigma–Aldrich, xylose was from Fluka (Switzerland), while silica gel 60 F254 precoated aluminum plates and acetonitrile were from Merck (Darmstadt, Germany). All the other materials used were of analytical grade
The xylanase catalysed reaction was performed in 200 mL reaction mixture in a 500 mL round bottomed flask at 70 8C in a thermostated oil bath with constant stirring at 700 rpm. The proportion of the reaction mixtures and the reaction conditions were similar to that described above. After running the reaction for 14 h, the aqueous and n-octanol phases were separated and alkyl xylosides were purified from the n-octanol phase. The alcohol was first removed under vacuum and the concentrated sample was loaded on an 80 mL silica column equilibrated with chloroform–methanol–water (65:25:2, v/v/v). The products were eluted using the same solvent system and the eluate was collected in 10 mL fractions that were analysed by thin layer chromatography (TLC). Fractions containing n-octyl monoxyloside, n-octyl xylobioside and n-octyl xylotrioside were pooled separately, dried and used as standards. The purity of these preparations was confirmed using HPLC and mass spectrometry.
2.2. Production and purification of recombinant T. neapolitana xylanase The recombinant E. coli BL21 (DE3) harboring the gene encoding T. neapolitana xylanase was grown in LB medium under conditions described earlier for recombinant Bacillus halodurans xylanase [18]. After cultivation, the cells were harvested by centrifugation for 10 min at 8000 g in a Sorvall refrigerated centrifuge. The cell pellet was resuspended in 10 mM Tris–HCl buffer pH 7.4 and treated with lysozyme (2 mg/mL) at room temperature for 1 h and subsequently subjected to freezing at 20 8C for 2 h and thawing prior to sonication using an ultrasonicator (Dr. Hielscher UP 400s, Germany) with 5 cycles of 1 min, 0.5 cycles/s, and 50% amplitude and 1 min interval between each cycle. The homogenate was centrifuged at 8000 g to remove the cell debris and the supernatant containing the enzyme was heated for 30 min at 80 8C to denature the contaminant host cell proteins. The clear supernatant obtained after centrifugation was used as the source of the enzyme. The specific activity of the enzyme preparation was 36 U/mg of protein. 2.3. Determination of enzyme activity and protein content The hydrolytic activity of T. neapolitana xylanase was determined based on the release of reducing sugars from xylan using the dinitrosalicylic acid (DNS) method [19]. A reaction mixture composed of 1% (w/v) birchwood xylan dissolved in 50 mM Na-acetate buffer, pH 5.5 and appropriately diluted enzyme was incubated at 80 8C
2.6. Semi-continuous process for synthesis of n-octyl xylosides A semi-continuous production and recovery of n-octyl xylosides was set up as shown in Fig. 1. The reaction mixture was composed of 5 g xylan, 350 mL n-octanol, 600 U xylanase, 10 mL of 1 M sodium acetate buffer (pH 5.5) and water to make up the volume to 500 mL. The reaction was performed at 70 8C and continuously stirred using a magnetic stirrer. During the course of the reaction, part of the reaction mixture was continuously pumped at a rate of 0.8 mL/min into a separating funnel to allow phase separation between the n-octanol and aqueous phases. After running the reaction for 3 h the n-octanol phase from the separation funnel was pumped over a Dowex1 50WX2-200(H) column (20 cm height, 1.8 cm internal diameter, 50 mL packed bed volume) pre-equilibrated with water saturated n-octanol. The water phase from the separation funnel as well as the
Fig. 1. Schematic presentation of the integrated system used for production and semi-continuous recovery of n-octyl xylosides. (1) Circulating water bath (70 8C), (2) magnetic plate stirrer, (3) reactor, (4) reaction mixture (5) magnetic stir bar, (6 and 7) pumps, (8) separation funnel, (9) adsorbent column, (10) elution solvent, and (11) product.
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column effluent, were circulated back to the reactor. This was continued for about 6 h after which the bound n-octyl xyloside products were eluted from the column using 50 mL of 99% (v/v) ethanol. The column was then re-equilibrated with noctanol (50 mL) and re-loaded for another 6 h. This was repeated for 3 cycles. The amount of products in the eluted samples was analysed by HPLC and compared to the amount achieved in the batch system (which had exactly the same set up but without the integrated Dowex column). 2.7. Determination of partition coefficient for alkyl xylosides from the reaction mixture The xylanase catalysed reaction between xylan and n-octanol was carried out in a 2 mL reaction mixture in 4 mL screw capped vials as described above. The phases were separated after centrifugation at 6000 g for 10 min and the amount of the alkyl xylosides was quantified by HPLC in both phases. The total concentration of each product in the reaction mixture was determined by diluting the biphasic reaction mixture with acetonitrile that resulted in a single homogenous phase from which samples were taken and analysed by HPLC. The partition coefficient, K of the different n-octyl xylosides was determined as the ratio of the concentration in the organic and aqueous phases at room temperature and pH 5.5. The amount of each product in the interphase was calculated by deducting the sum of its concentration in the top and bottom phase from its total concentration in the reaction mixture. 2.8. Anaylsis of n-octyl xylosides and reducing sugars Thin layer chromatography on silica gel 60 F254 precoated aluminum plates was used for qualitative analysis of the samples. The solvent system used was chloroform–methanol–water (65:25:2, v/v/v). After a single solvent ascent, spots corresponding to n-octyl xyloside products and reducing sugars were detected by spraying with 10% sulfuric acid in methanol and heating at 110 8C until spots were visible. n-Octyl-monoxyloside, -xylobioside and -xylotrioside had Rf values of about 0.73, 0.61 and 0.48, respectively. The concentration of n-octyl xylosides was quantitatively determined by HPLC (LaChrom system with an L-7100 pump, L-750 autosampler and a D-7000 interface system) using reverse phase RP-18 column (250 mm 4.6 mm, Kromasil1 1005C18, Akzo Nobel, Sweden) and a 500 ELSD evaporative light scattering detector (Alltech Associates, Deerfield, MI). The column was maintained at room temperature and the mobile phase used was acetonitrile:water (65:35) at a flow rate of 0.8 mL/min. The retention times of n-octyl-monoxyloside, -xylobioside and xylotrioside were 4.28, 3.25 and 2.55 min, respectively. The identity of the purified n-octyl xyloside products was confirmed by mass spectrometry using a quadrupole time of flight (Q-TOF) hybrid tandem mass spectrometer (API Qstar, MDS Sciex, Ontario, Canada) equipped with an electrospray ionization source (Turboion Spray1).
Table 1 Distribution behavior of the alkyl xylosides in n-octanol-buffer system. Product
Partition coefficient
Relative amount (%) Top phase
Interphase
Bottom phase
n-Octyl xyloside n-Octyl xylobioside n-Octyl xylotrioside
100 8.4 2.1
74 84 66
26 6 1
0 10 33
and in addition, improves xylan solubility. The experiments were carried out with n-octanol as the nucleophile, where n-octylmonoxyloside, -xylobioside and -xylotrioside were produced; their yields depending on the reaction conditions. 3.1. Partition coefficients of n-octyl xylosides in the reaction system The reaction between xylan (25 mg/mL) and n-octanol (70%) catalysed by the recombinant T. neapolitana xylanase in 2 mL volume was run for 9 h. The total amounts of n-octyl-monoxyloside, -xylobioside and -xylotrioside produced were about 0.34, 4.45 and 0.47 mg, respectively. Table 1 shows the distribution coefficient of n-octyl xyloside products in the biphasic system. nOctyl monoxyloside was distributed between the top phase and interphase, and was not detected in the aqueous phase. With increase in the number of sugar units, the relative amounts of the alkyl xylosides partitioned to the aqueous phase increased to 10% and 33% for n-octyl-xylobioside and -xylotrioside, respectively. According to an earlier report, only n-octyl xylobioside and noctyl xylotrioside (and not n-octyl xylomonoside) possess the desirable surfactant properties [2]. Hence, the major focus in this work has been to study the influence of various reaction parameters on the yield and optimize conditions for improved production of these products. 3.2. Influence of reaction parameters on production of n-octylxylobioside and -xylotrioside
3. Results and discussion Glycosyl hydrolase family 10 xylanases possess a retaining mode of reaction mechanism. The enzymes randomly cleave xylan and the covalent intermediate of enzyme-xylooligosaccharides formed reacts with the nucleophiles available to generate the products. Hydrolysis occurs when water is the nucleophile, while the presence of other nucleophiles results in the formation of transglycosylation products, e.g. alkyl xylosides are produced in the presence of an alcohol. The products formed are also subject to further attack by the same enzyme, resulting in other hydrolysis or transglycosylation products. Hence, knowledge of reaction conditions required for optimal production of the desired alkyl xylosides is important in order to control the degradation and maintain high yields of the product(s) of interest. Production of alkyl xylosides using xylan as raw material is attractive from technical as well as economic point of view as both the xylan hydrolysis and transglycosylation are combined in one step hence avoiding the need for prior isolation of hydrolysis products. Preliminary experiments on the reaction between xylan and different alcohols, catalysed by T. neapolitana xylanase, showed the occurrence of transglycosylation reaction up to an alcohol chain length of C12, however the reaction efficiency was found to decrease with increasing alkyl chain length. Investigation on the enzyme stability showed the xylanase to be fairly stable in the presence of n-octanol, retaining about 80% of its original activity after incubation for 8 h at 80 8C in 50 mM acetate buffer, pH 5.5 containing 50% (v/v) n-octanol. This property allows the use of high temperature when performing transglycosylation reaction
In the reaction medium with 70% (v/v) n-octanol, increasing xylan concentration from 5 to 50 mg/mL showed a decrease in the equivalent total product yield from 144 to 58 mg per gram of xylan; the decrease in n-octyl xylobioside concentration being more prominent (Table 2). At xylan concentration of 50 mg/mL, there was practically no water phase which resulted in unsuitable conditions for mixing and mass transfer, which in turn affected the product yield. On the other hand, lower xylan concentration results in a diluted product requiring larger reaction volumes, an undesirable effect for downstream processing of the products. In this study, a xylan concentration of 25 mg/mL that gave the total Table 2 Effect of xylan concentration on yields of n-octyl-xylobioside and -xylotrioside after 9 h of reaction with n-octanol catalysed by T. neapolitana xylanase. The reaction mixture was composed of xylan, 1.4 mL octanol, 0.6 mL buffer and 0.1 U xylanase. Product
Actual yield (mg/mL)
Equivalent yield (mg product/g xylan)
5
n-Octyl-xylobioside n-Octyl-xylotrioside
1.12 0.32
112 32
10
n-Octyl-xylobioside n-Octyl-xylotrioside
1.37 0.38
69 20
25
n-Octyl-xylobioside n-Octyl-xylotrioside
1.46 0.46
58 19
50
n-Octyl-xylobioside n-Octyl-xylotrioside
2.18 0.69
44 14
Xylan concentration (mg/mL)
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actual product yield of 1.92 mg/mL (the sum of n-octyl-xylobioside and -xylotrioside) was used for further studies. The hydrolysis of xylan (25 mg/mL) in the two-phase system was also followed. About 40% of the xylan was converted to reducing sugar during 9 h incubation, which according to TLC analysis contained a significant amount of xylose and xylobiose. Such a significant degree of hydrolysis is not surprising and is due to the high water content of the reaction. The possibility of producing n-octyl xylosides by reacting n-octanol with xylose and xylobiose was investigated but there was no product formation (data not shown). This seems to be in accordance with an earlier report by Matsumura et al. [21] showing that xylotetraose was the shortest xylooligosaccharide accepted by Aureobasidium pullulans xylanase for reaction with n-octanol. A low water activity would favor the reaction equilibrium towards product synthesis [8], however running transglycosylation reactions in water-free media suffer from drawbacks such as insolubility of sugar substrates, inactivation of glycosidases and/or unsuitable enzyme conformation. Accordingly, there was no detectable alkyl xyloside formation in a water-free reaction system. Besides acting as a competing nucleophile for the reaction, water serves as a medium for solubilizing the substrate xylan. Variation of the water content in the range of 10–50% (v/v) resulted in a low yield of alkyl xylosides both at low water content (10%, v/ v) where the xylan solubility is low and also at high water content (40–50%, v/v) that favors product hydrolysis. As shown in Fig. 2, the yield of n-octyl xylobioside increased gradually with time and was highest at a water content of 30% (v/v) while the highest level of n-octyl xylotrioside was reached at 20% (v/v) water and then decreased with further incubation due to hydrolysis to n-octyl xylobioside and xylose. The n-octyl xylobioside yield increased with increasing enzyme concentration up to 0.25 U/mL of the reaction volume. Further increase up to 1 U/mL did not make a significant difference except
Fig. 2. Time profile of the yields of (A) n-octyl xylobioside and (B) n-octyl xylotrioside at initial water content of 10% ( ), 20% (*), 30% (~), 40% (&), and 50%, v/v (^), respectively. Defined amounts of n-octanol were added to a mixture of 50 mg xylan, 0.1 U of T. neapolitana xylanase and sodium acetate buffer, pH 5.5 in a final reaction volume of 2 mL. The mixture was incubated with shaking at 70 8C.
*
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Fig. 3. Effect of recombinant T. neapolitana xylanase concentration on the yields of (A) n-octyl xylobioside and (B) n-octyl xylotrioside with time from xylan and noctanol at 70 8C. The enzyme was used at a concentration of 0.025 U (&), 0.05 U (*), 0.1 U (^), 0.25 U (&), 0.5 U (~) and 1 U/mL (*), respectively, in a reaction mix containing 50 mg of xylan, 1.4 mL of n-octanol and 0.6 mL of sodium acetate buffer, pH 5.5.
that the time required to achieve the maximum yield was shortened to 3–6 h (Fig. 3). At high enzyme dosage (0.5–1 U/mL) and extended incubation, n-octyl xylobioside concentration declined due to its hydrolysis. n-Octyl xylotrioside yield was higher when 0.05 and 0.1 U/mL enzyme was used, while at high enzyme dosage it was completely hydrolysed and hence not detected in the reaction mixture. In general, oligomers with higher degree of polymerization are cleaved more efficiently by xylanases and affinity constants decrease with sugar chain length [22]; higher affinity of a xylanase for n-octyl xylotrioside than n-octyl xylobioside has been demonstrated earlier [21]. This is related to the nature of the active site of the xylanases, which are known to have 3–5 subsites for binding the xylopyranose rings of xylan [23]. For a glycosidic bond to be broken the substrate should bind at least to one + subsite and one subsite. The longer the oligosaccharide, the higher the probability for the oligosaccharides to bind to the + and subsites, facilitating hydrolysis. Although there is no structural information available so far on the binding of alkyl glycosides to the xylanase active site, noctyl xylobioside is a large enough molecule for the sugar groups to bind to the subsites, with consequent hydrolysis of the glycosidic bond. Transglycosylation may also occur where a xylose unit is transferred to xylobiose (produced as the hydrolysis product) to form xylotrioside that in turn is hydrolysed to xylose and xylobiose [15]. Furthermore, family 10 endo-xylanases are known to have low xylosidase activities [24], and can slowly cleave smaller xylooligosaccharides. Fig. 4 shows the effect of temperature on the reaction yield. Higher yields of n-octyl-xylobioside and -xylotrioside were achieved in a temperature range of 70–80 8C. The initial reaction rate was nearly similar in this temperature range; however, a slightly higher yield of n-octyl xylobioside was obtained on prolonged incubation (up to 24 h) at 70 8C. This is probably due to
G. Mamo et al. / Process Biochemistry 45 (2010) 700–705
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Fig. 4. Synthesis of (A) n-octyl xylobioside and (B) n-octyl xylotrioside from xylan and n-octanol catalysed by recombinant T. neapolitana xylanase at 50 8C (^), 60 8C (&), 70 8C (~), 80 8C (*), and 90 8C ( ), respectively. The reaction mixture containing 1.4 mL n-octanol, 50 mg xylan and 0.6 mL sodium acetate buffer, pH 5.5 and 0.1 U xylanase was incubated at different temperatures in a shaking heating block and samples withdrawn at defined time intervals for analysis of the products.
*
higher stability of the enzyme. On the other hand, the maximum yield of n-octyl xylotrioside (over 35 mg/g xylan) was achieved within 9 h. With respect to the initial pH of the aqueous phase of the reaction, n-octyl xylobioside yield increased with time and was highest at pH 5 reaching 100 mg per gram xylan after 24 h of reaction (data not shown). On the other hand, the yield of n-octylxylotrioside increased up to 4 h and was highest at pH 7 (20 mg/g xylan). Subsequently, the n-octyl-xylotrioside underwent hydrolysis and was undetectable after 18 h of reaction at pH 5–6. The extent of hydrolysis was significantly lower at pH 7, which can be attributed to lower enzyme activity under these conditions. 3.3. Production of n-octyl xylooligosaccharides from xylan From the data obtained above it is clear that optimal conditions for synthesis of n-octyl-xylobioside and -xylotrioside
are slightly different. Production of n-octyl xylobioside is optimal in the reaction with initial water content of 30% (v/ v), enzyme dosage of 0.25 U/mL, pH 5 and 70 8C. On the other hand, n-octyl xylotrioside production is more favorable at a lower water content of 20%, enzyme dose of 0.1 U/mL, pH 7 and at 70–80 8C. The reaction was run under optimal conditions for n-octyl xylobioside production. About 100 mg n-octyl xylobioside/g xylan was produced during 18 h reaction and prolonged incubation up to 96 h resulted neither in formation of more surfactant nor product hydrolysis (data not shown). Further addition of equal amount of enzyme after 18 h did not improve the yield but instead led to product hydrolysis, which is in agreement with the effect of the increased enzyme dose shown in Fig. 3 and also indicates that the enzyme denaturation was not the cause for termination of the reaction. As shown in Table 3, comparison of the results obtained in this study with the earlier reports on xylosidation reactions using xylanase from other microbial sources shows that the productivity was higher than all the other systems except for the system using supercritical fluid (SCF) as the reaction medium where either comparable or relatively higher yields were reported depending on the SCF used. The maximum yield of n-octyl xylotrioside (38 mg/g xylan) is the highest ever achieved through enzymatic synthesis. Although the n-octyl xylobioside yield of about 10% (based on xylan amount) appears to be low, the enzymatic process provides several advantages. The endo-xylanase produces the more surface-active b-anomers [15,16], while the chemical synthesis results in anomerically mixed products dominated by a-anomers [2,25], from which the separation of b-anomer products is complex and results in poor yields. Moreover, the chemical methods often result in alkyl monosides [25], which have less desirable surfactant properties than alkyl-xylobioside and -xylotrioside produced by the xylanase [2]. 3.4. Integrating product removal with production Product inhibition is the major bottleneck in several biotechnology processes, limiting their productivity. In the system described above, it was observed that replacing 50% of the product-enriched n-octanol phase with fresh n-octanol after 6 and 12 h of reaction resulted in improvement of the n-octyl xylobioside yield by about 16%. Hence, a preliminary experiment on integrating product capture with the production system was set up as shown in Fig. 1. The reaction was first allowed to run under batch conditions and then the reaction mixture was pumped to a separating funnel for phase separation, after which the n-octanol phase was passed over a column of an ion exchange resin, Dowex 50WX2-200(H). This adsorbent was selected based on our screening studies on adsorption of n-octyl
Table 3 Maximum yield achieved per mL of reaction volume by different transxylosidation studies between 9 and 18 h of reaction. System
Water content (%v/v)
Total reaction volume (mL)
Xylan concentration (mg/mL)
Supercritical fluid
Pressure (kg/cm2)
T. neapolitanaa T. neapolitanab A. pullulans A. pullulans A. pullulans A. pullulans A. pullulansc
30 30 9.8 20 9.8 10 30
2 2 2.55 2.5 2.55 10 10
25 25 19.6 20 19.6 5 5
– – CO2 CHF3 – – –
– – 150 70 – – –
Productivity (mg/g xylan/h) n-Octyl xylobioside
n-Octyl xylotrioside
2.78 2.50 2.45 4.03 0.86 0.89 0.00
0.28 2.11 0.54 0.50 0.00 0.00 0.00
a and b denote systems with maximum production of n-octyl -xylobioside and -xylotrioside, respectively. a Reaction mixture containing 70% (v/v) n-octanol, 50 mg xylan, 0.5 U xylanase, and sodium acetate buffer, pH 5 was incubated at 70 8C for 18 h. b A mixture containing of 80% (v/v) n-octanol, 50 mg xylan, 0.1 U xylanase, and sodium acetate buffer, pH 5.5 was incubated at 70 8C for 9 h. c Only n-octyl-xyloside but no -xylobioside and - xylotrioside was produced.
Reference
This study This study [9] [9] [9] [9] [13]
G. Mamo et al. / Process Biochemistry 45 (2010) 700–705
705
Acknowledgment This work was performed within the framework of Greenchem, a research program supported by the Swedish Foundation for Strategic Environmental Research (Mistra). References
Fig. 5. Production of n-octyl xylosides in integrated (&) and batch reactor system ( ). The reaction mixture was composed of xylan (5 g), n-octanol (350 mL), xylanase (1.2 U/mL), 10 mL of 1 M sodium acetate buffer, pH 5.5 and water (to 500 mL). The total concentration for n-octyl monoxyloside (OX1), n-octyl xylobioside (OX2) and n-octyl xylotrioside (OX3) was determined after 24 h reaction time.
xylobioside to various resins (Sahoo et al., unpublished data). As shown in Fig. 5, the yield of the n-octyl xylosides was somewhat higher than that achieved in the batch system, the increase in noctyl xylotrioside yield being about 2.4 fold. Optimization of the integrated system parameters is expected to further increase the yields of n-octyl xylobioside and n-octyl xylotrioside per gram of xylan used. The integrated system has an added advantage of concentrating the n-octyl xylosides and easy separation from noctanol. Currently, recovery of alkyl glycosides from the respective alcohols used as substrates is carried out by distillation, which is a very energy intensive process. In the integrated system described here, the product is stripped from the bulk n-octanol phase by adsorption and is eluted from the adsorbent using ethanol, which is much easier to remove than the longer chain alcohols. 4. Conclusions This study clearly shows the advantages of using a thermostable xylanase for synthesis of interesting surfactant products, n-octyl-xylobioside and -xylotrioside directly from xylan. The use of xylan directly as substrate is attractive as it cuts down the costs for pre-hydrolysis to xylooligosaccharides for further transxylosidation to alkyl xylosides. At the same time, process technologies need to be developed that will overcome the limitations of the enzymatic processes, such as product inhibition and degradation so as to make the processes economically competitive. Pentose based surfactants are valuable products and their production can be integrated as a part of the lignocellulose biorefineries. Inspite of their relatively low yields, the reaction described in this paper results in other major side-products, i.e. xylooligosaccharides that can be recovered from the aqueous phase and used either directly or further valorized to other products thereby increasing the value addition to the raw material.
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