Synthesis of a Re-usable Cellobiase Enzyme Catalyst through In situ Encapsulation in Nonsurfactant Templated Sol Gel Mesoporous Silica

Transcription

1 Top Catal (2012) 55: DOI /s ORIGINAL PAPER Synthesis of a Re-usable Cellobiase Enzyme Catalyst through In situ Encapsulation in Nonsurfactant Templated Sol Gel Mesoporous Silica Yen Wei Sudipto Das David Berke-Schlessel Hai-Feng Ji John McDonough Lin Feng Xiang Zhang Wentao Zhai Yingze Cao Published online: 26 October 2012 Ó Springer Science+Business Media New York 2012 Abstract We present a re-usable enzyme catalyst system via direct encapsulation of cellobiase in nonsurfactant templated sol gel mesoporous silica host material with D-fructose as the template. The pore diameter and porosity of the silica host material, controlled by the fructose content, controlled the diffusion of substrate to the enzyme. This in situ immobilized cellobiase showed little or no leakage while could be repeatedly used as biocatalyst with little or no loss of activity after at least 9 cycles. Keywords Enzyme immobilization Cellobiase enzyme Mesoporous silica Nonsurfactant template Sol gel reactions 1 Introduction Biocatalysts have a very deep impact on the global drive towards green, environment friendly methodologies for chemical manufacturing [1 3]. The use of biocatalysts in chemical processes promotes relatively milder reaction conditions as well as high chemo-, regio- and stero-selectivities [2]. Enzymes are a class of biocatalysts that Y. Wei (&) D. Berke-Schlessel L. Feng X. Zhang W. Zhai Y. Cao Department of Chemistry, Tsinghua University, Beijing , China weiyen@tsinghua.edu.cn Y. Wei S. Das D. Berke-Schlessel H.-F. Ji Department of Chemistry, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104, USA J. McDonough Department of Materials Science and Engineering, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104, USA promote high activity, selectivity and specificity in complex chemical reactions under mild experimental and environmental conditions [4 7]. Cellobiase is an enzyme that hydrolyses variously b-linked diglucosides and aryl b-glucosides. The immense importance of this enzyme lies in its role in the enzymatic hydrolysis of cellulose [8, 9]. Cellulolytic enzymes act in conjunction with cellobiase in the degradation of crystalline cellulose to glucose, and cellobiase is the rate-limiting factor in this enzymatic hydrolysis of cellulose [9]. Enzyme immobilization is a key step for the application of these natural catalysts, as it enables easy separation and recovery of the enzyme catalyst from the reaction mixture. Utilization of solid supports has been the most explored approach for immobilization [10]. For enzyme immobilization in solid support material, the most widely used techniques include: adsorption of enzyme on the solid surface; covalent anchorage of the enzyme to the support material; electrostatic binding; and enzyme entrapment within inorganic inert matrices [11 17]. Physical adsorption of enzyme is an easy technique. However, the process involves a weak bonding of the enzyme to the support, which often leads to enzyme leaching [14, 18 20]. Covalent bonding of the enzyme to the solid support offers a very strong binding and leads to better stabilization of the enzyme on the support [13, 21]. But this technique often involves the chemical manipulation of the enzyme, altering the enzymatic activities [10, 22]. In this respect, the immobilization technique using enzyme-support electrostatic interactions provide high enzyme stability and also lead to easy regeneration of the support material [14, 23, 24]. But, a critical consideration in this technique is the operating ph conditions [11]. The operating environment should have a ph value, which must be compatible with the isoelectric point and activity of the enzyme.

2 1248 Top Catal (2012) 55: In this respect, enzyme immobilization by direct encapsulation in silica matrix via sol gel process has been widely explored in the recent years [16, 22, 25 33]. This method provides good compromise between the heterogeneous biocatalyst and the activity of the enzyme [11]. In this process, the enzyme is added to the hydrolyzed silica precursor sol (e.g. tetraethyl orthosilicate), and gelation is allowed to occur with the protein inside the silica matrix. This results in the formation of an amorphous silica matrix around the enzyme, thus entrapping the enzyme within the sol gel silica material. The sol gel synthesis of the silica material also generates interconnected open pore channels that allow accessibility to the encapsulated enzyme caged inside the host material. However, the conventional sol gel materials are microporous and the small pore diameter (\15 Å) of these materials restricts the diffusion of substrate molecules through the pores, to the enzyme inside the caged structure. This hinders the catalytic activity of the immobilized enzyme [27, 28]. The synthesis of nonsurfactant-templated mesoporous materials via sol gel chemistry has been reported by our group [34, 35]. We have developed a non-surfactant templated route to mesoporous sol gel silica, where organic sugar molecules like D-glucose and D-maltose has been used as the pore forming agent or template. After the synthesis, the extraction of the templates yielded mesoporous silica, which exhibited large pore volume, pore size and surface area [34, 35]. Unlike the surfactant templated route to mesoporous material synthesis, which usually requires high temperature calcination for template removal, the organic sugar templates could be very easily removed by simple extraction with water. Also, the pore size of the silica matrix could be controlled by varying the sugar (template) content during synthesis. Our group has reported direct encapsulation of acid phosphatase (ACP), alkaline phosphatsase (ALP) enzymes in D-glucose templated mesoporous sol gel silica [22, 36]. Here, we report a re-usable cellobiase catalyst system, developed by immobilization of the enzyme via direct encapsulation in sol gel silica via D-fructose templated route. In an earlier publication [37], we had reported the hydrolysis of pre-treated biomass by the application of this newly developed cellobiase catalyst, in conjunction with cellulase enzyme. Here, we report the synthesis and activity studies of the developed catalyst, using cellobiose as the substrate. It must be mentioned here that an easy and mild template extraction process is a very important consideration, as it makes the pores free and accessible by the substrate molecules. We have used D-fructose as template, since fructose is the most water-soluble sugar, thus leading to very easy extraction by water. The catalyst developed in presence of fructose showed significantly higher activity when compared to catalyst developed in absence of fructose. Also, the immobilized cellobiase catalyst enabled easy recovery, without significant loss of activity or enzyme leaching. 2 Experimental 2.1 Materials Tetraethyl orthosilicate (TEOS, 98 %), D-(-)-fructose (98 %) and sodium acetate- trihydrate (99 %) were obtained from Sigma Aldrich. The cellobiase enzyme (from Aspergillus niger) used was Novozym 188, a b-glucosidase obtained from Sigma Aldrich. The substrate cellobiose for the enzyme assay was also obtained from Sigma Aldrich. Spectrophotometric glucose assay reagents were obtained from Wako Autokit Glucose (cat # ) sold by Wako Pure Chemicals. All reagents were used as received, without any further purification. 2.2 Synthesis of Immobilized Cellobiase Sample Cellobiase was immobilized via the acid catalyzed sol gel reaction of TEOS with water (molar ratio of TEOS: H 2 O: HCl = 1:2:0.005). D-fructose was used as the template during synthesis. In a typical procedure for the preparation of 50 % fructose templated immobilized cellobiase sample, 9.0 g (0.5 mol) of distilled water was mixed with 0.63 g 2 M HCl in a two-necked 500-ml round-bottomed flask fitted with a thermometer and reflux apparatus. The mixture was stirred for 5 min at room temperature. To this mixture, 52 g of TEOS (0.25 mol) was added with slow stirring at room temperature. After the addition, the reaction was allowed to stir at room temperature under nitrogen. The solution homogenized and became transparent in 10 min, with the reaction mixture temperature rising up to 35 C. The mixture was heated to 60 C and briefly turned cloudy, at which point reflux began; the reaction mixture was refluxed at 60 C under nitrogen for 1 h. After the reflux, the reaction mixture was allowed to cool down to room temperature and high vacuum was applied to remove the ethanol produced as a byproduct during the hydrolysis of TEOS. The reaction mixture was subject to high vacuum until it reached 50 % of its original weight. After the vacuum extraction, 30 g of 50 % fructose solution (15 g of fructose dissolved in 15 g of distilled water) were added to the mixture with stirring. After a homogenous clear solution was obtained, the mixture was divided (by weight) equally into three 100-ml beakers. Each beaker contained 5 g of silica and 5 g of fructose. For each beaker, an enzyme solution of 625 ll of cellobiase in

3 Top Catal (2012) 55: ml 50 mm ph 5 sodium acetate buffers was prepared, and added to the sol gel solutions under heavy stirring at room temperature for 5 min. After the enzyme solutions were added, the reaction mixtures were sealed with parafilm and allowed to gel at 5 C. After gelling, holes were made in the parafilm, and kept at 5 C for solvent evaporation. After 2 days, the samples were kept at 0 C under high vacuum, until no further weight loss was observed. Each reaction beaker contained 5 g silica, 5 g fructose and 625 ll of cellobiase enzyme. After the drying process, the samples were ground to 40-mesh size using a mortar and pestle, and stored at 5 C for further analysis. Three different samples were made with three different template concentrations, with identical enzyme with respect to per gram silica in the sample. As control sample, cellobiase was immobilized under identical conditions without the addition of any fructose template. 2.3 Characterization In order to determine the total template content in each sample a Thermogravimetric Analysis (TGA) on a TA Q50 Thermogravimetric Analyzer (TA Instruments Inc., New Castle, DE) was performed. The samples were preheated to 80 C and kept isothermal at 80 C for 45 min in N 2 in order to drive out any remaining ethanol byproducts. During the actual thermal analysis, they were heated to 750 C at a heating rate of 10 C/min in air. Nitrogen adsorption desorption measurements were done on a Quadrasorb SI Automated Surface Area and Pore Size Analyzer (Quantachrome Instruments, Boynton Beach, FL) at -196 C. The template was extracted from the samples by washing in a large excess of distilled water. After extraction, the powder samples were dried at 40 C overnight, in a vacuum oven. The dried powders were degassed at 100 C and 0.2 torr overnight, before analysis. Quantachrome QuadraWin software (Version 5.02) was used for calculating the pore size and surface area of the samples. 2.4 Procedure for Assaying Enzymatic Activity The enzymatic activity of immobilized cellobiase samples were determined by assaying with cellobiose substrate, in which, the cellobiose was broken to glucose units by the enzyme. A 50 mm sodium acetate hexahydrate solution was prepared in distilled water, and the ph of the solution was adjusted to 5 using HCl. A 6.25 mg/ml solution of cellobiose was prepared in the sodium acetate buffer and used as the substrate. Prior to doing the assays, the immobilized samples were washed in aforementioned sodium acetate buffer in order to remove the template. Appropriate amounts of the immobilized samples were put into 15 ml falcon tubes, such that the amount of cellobiase enzyme present was calculated to be 12.5 ll; after washing, the liquid was carefully removed. The 0, 30, 50 and 70 % template content samples were each divided into 8 separate tubes (totaling 32 tubes), such that 0, 3.125, 6.25, 9.37, 12.5, 25, 50 and 62.5 mg/ml substrate concentrations could be added to each template content. The tubes were sealed, incubated at 37 C and gently agitated for 2 h. After 2 h, the tubes were taken out of the water bath and kept over ice, and the solid particles were allowed to settle down at the bottom of the tube. The glucose content of the liquid phase was determined by the procedure as described in the Wako Glucose Autokit (Wako Pure Chemicals), and expressed in terms of milligrams per milliliter of the liquid (mg/ml). The activity of the samples was expressed in terms of the glucose content (mg/ml). The washing solutions of the immobilized samples were also assayed to check the leaching of enzyme from the silica host material. 3 Results and Discussion 3.1 Thermogravimetric Analysis TGA was used to determine the amount of template in the as-synthesized samples. The weight loss obtained after heating the samples in air at 10 C/min to 750 C are reported as TGA weight loss in Table 1 [37]. This weight loss observed is due to the degradation of the fructose present in the samples. The TGA curves for all the as-synthesized samples are shown in Fig. 1 that is similar to our previous observation [37]. From Table 1, we can confirm that the original fructose contents in all the as-synthesized samples, calculated from the reaction stoichiometry, is in proportion to the TGA weight losses obtained. The weight loss in the 0 % template sample can be attributed to the presence of residual water molecules tightly bound to the silica matrix, and un-reacted ethoxy groups in the silica [1, 14, 20]. 3.2 Nitrogen Adsorption Desorption The Brunauer-Emmett-Teller (BET) surface area of the immobilized samples as well as their pore parameters listed in Table 1, were determined from the nitrogen adsorption desorption isotherms [21]. The nitrogen adsorption desorption isotherms of the samples at -196 C are shown in Fig. 2, similar to our previous report [37]. The control sample with 0 % fructose content, show a reversible type I isotherm [22], which are typically exhibited by xerogels having microporous structures [23]. In the 30, 50 and 70 %

4 1250 Top Catal (2012) 55: Table 1 Fructose template content, TGA weight loss, textural properties from nitrogen adsorption desorption, enzymatic activity and Michaelis Menten parameters of immobilized cellobiase samples Sample Weight % template added a TGA weight loss (%) BET surface area (m 2 /g) Pore volumev P (cm 3 /g) Pore diameter (Å) b Enzymatic activity (mg/ml) V max (mg/ml/h) K M (mg/ml) Free enzyme % template 0 \ % template % template % template a Weight % template added was calculated by assuming complete conversion of TEOS to SiO 2 b Obtained from maxima of the pore size distribution plots Fig. 1 Thermogravimetric analysis of the immobilized cellobiase samples. Partially reproduced from Das et al [37] with permission from Elsevier magnitude of the H2 hysteresis loop becomes bigger, shifting towards higher relative pressures (P/P 0 ). The pore size distributions and the pore volumes were calculated using the quenched solid density functional theory (QSDFT) method [26]. The pore size distribution is shown in Fig. 3 and the results are essentially the same as our earlier report [37]. The 0 % template sample shows peak maxima at 15 Å, which is microporous in nature. The pore size distribution peak maxima also indicates 30 % sample having average pore diameter in Å range, while 50 % and 70 % template samples have average pore diameters in Å range, which are mesoporous in nature. Increase in the template content from 0 to 70 % also led to a linear increase in the surface area of the immobilized samples, which suggests larger concentration of open pores in the silica microstructure, with increased Volume Adsorbed (cm 3 /g STP) % Template 50% Template 0% Template 30% Template Relative Pressure (P/P 0 ) dv/dd (cc.g-1.a o-1 ) % Template 70% Template 0% Template 30% Template Fig. 2 Nitrogen adsorption desorption isotherms of the immobilized samples, after extraction of the fructose template. Partially reproduced from Das et al [37] with permission from Elsevier template samples, as the fructose content increases; the isotherms go to type IV isotherms with H2 hysteresis loops [22], which is a characteristic of mesoporous molecular sieves [15, 24, 25]. With increasing template content, the Pore Diameter (A o A) Fig. 3 Pore size distribution of the immobilized samples after template extraction. Partially reproduced from Das et al [37] with permission from Elsevier

5 Top Catal (2012) 55: template content. Clearly, increased template contents led to larger pore sizes, increased surface area and pore volumes. Hence, from the nitrogen adsorption desorption, it is clear that the template (fructose) acted as a pore-forming agent. 3.3 Enzymatic Activity of Immobilized Cellobiase The enzymatic activities of the immobilized samples and free cellobiase enzyme were determined using cellobiase substrate. The activities were studied with varying substrate concentrations, and the highest values obtained are reported as enzymatic activity (mg/ml) in Table 1. The results demonstrate that the activities of the samples with higher template content (70 and 50 %) are significantly higher than the one prepared without any template addition (0 %). The 30 % sample exhibits activity lower than 50 and 70 %, but higher than the 0 % sample. It must be noted here that in an earlier study [37], we had applied our newly developed immobilized cellobiase enzyme system, in conjunction with cellulase enzyme, for the hydrolysis of biomass. In this work, for the activity studies, we have used pure cellobiase solution as the substrate. The enzymatic activity (mg/ml) of the immobilized cellobiase samples in this work (with pure cellobiose as the substrate), is observed to be considerably higher, when compared to the biomass hydrolysis [37]. The Michaelis Menten plots for the four samples are shown in Fig. 4 and the Michaelis Menten kinetics was studied according to Eq. (1) [7]: 1 ¼ K M 1 t 0 V Max ½SŠ þ 1 ð1þ V Max where [S] is the substrate concentration, t 0 (mg/ml/hr) is the rate of enzyme activity or the reaction rate of hydrolysis at any given substrate concentration, V max is the maximum reaction velocity or maximum rate of hydrolysis and K M is the substrate concentration at which the reaction velocity or the rate of hydrolysis is half of V max [27, 28]. The V max values of the immobilized samples and that of the free enzyme are listed in Table 1. It is clearly demonstrated that samples with higher template concentration have significantly higher V max than the sample with no template. This observed activity and V max enhancement could be attributed to the silica microstructure. It must be noted that the enzyme molecules are trapped inside the host matrix by gelation of the silica precursor sol around the enzyme molecules. The template, which was also inside the silica matrix prior to gelation, created an increased number of open pore channels, which allows accessibility to the enzyme inside the caged structure. Most importantly, the template also made the pore sizes bigger. The samples with high template concentration (70 and 50 %) are mesoporous material and have pore diameter in the range of Å. The sample with 30 % template is also mesoporous, but the pore diameter is in the range of Å. The 0 % sample (without any template) is microporous in nature, the pore diameter being *16 Å. The speed of the enzymatic reaction and the enzymatic activity depends on the accessibility of the enzyme by the substrate. The rate of diffusion of the substrate to the enzyme inside the caged silica structure is the limiting factor in the accessibility of the enzyme. With increase in the template content, the silica host material exhibited an increase in the pore diameters. Hence, the rate of diffusion of the substrate molecules through the pore channels to the caged enzyme inside the matrix is higher in samples with higher template content. With higher template content, we can also see an increase in the surface area of the silica host (Table 1). This is due to higher density of accessible pore channels and interconnected pores. This increased porosity and larger pore diameter also assist in the easier diffusion Fig. 4 Enzyme activity (Michaelis Menten) plots of the immobilized cellobiase samples. The x-axis represents the substrate concentration (mg/ml) and y-axis represents the reaction rate (mg/ml/h)

6 1252 Top Catal (2012) 55: Fig. 5 Activity of immobilized samples in recycled uses. All the tests were conducted in ph 5 sodium acetate buffer solution of the substrate molecules. With larger pore diameters and increased porosity, the rate of diffusion of the substrate through the pore channels to the enzyme is higher in the 70 and 50 % samples, compared to 30 and 0 % samples. In samples with small pore diameters, the substrate may not be able to even get inside the silica structure and access the enzyme inside. As a result, samples with higher template concentration exhibit significantly higher activities and V max. Also, as listed in Table 1, the pore volume of the silica host material increased with increase in the template content. As a result of this increased pore volume, the mobility of the substrate and also the accessibility of the enzyme to the substrate increased. The increased pore volumes, combined with increased porosity and pore diameters result in higher activities of samples with higher template content. When the immobilized cellobiase enzyme systems, in conjunction with cellulase enzyme, were used in hydrolysis of biomass [37], we had observed similar activity behavior. 3.4 Re-usability and Stability of the Immobilized Cellobiase Enzyme System The re-usability of the immobilized cellobiase samples was demonstrated by using the immobilized systems in multiple enzyme activity runs. After each run, the immobilized powdered samples were filtered out of the reaction mixture and washed with ph 5 sodium acetate buffer. To the washed samples, new substrate and buffer were added, and subsequently run for the next enzymatic activity assay. Figure 5 shows the enzymatic activity of the immobilized samples in multiple uses. The results exhibit that the enzymatic activity of the immobilized cellobiase samples are retained up to 9 uses with negligible loss in activity. The washing solutions (in between runs) were also assayed to check enzyme elution. All the solutions showed negligible enzymatic activity. Hence, it can be concluded that the enzyme is clearly immobilized inside the silica matrix, and not just adsorbed onto the silica surface, as it would have been washed away if the enzyme were just adsorbed onto the surface of the silica host material. The retained activity of the immobilized samples also demonstrates the fact that there is very little or no leaching of the enzyme from inside the pores to the reaction mixture, which signifies very tight and efficient encapsulation of the cellobiase inside the silica host material. The reusability studies were conducted over days. Going through 9 recycled uses, the encapsulated enzyme samples were in activity evaluation assays for days. The retained activities of the samples during the recycles effectively signify stability of the samples in use for 5 weeks. The as-synthesized fresh samples, stored under refrigeration (5 8 C) showed no loss in activity after weeks. 4 Conclusions In this study, cellobiase enzyme was directly immobilized in silica support via sol gel process, with tetraethyl orthosilicate (TEOS) as the silica precursor. In this encapsulation method, D-fructose was used as the template or pore-forming agent during the sol gel process, to obtain mesoporous silica host material. By varying the template content in the silica host material, different surface area, pore volume and pore diameters were obtained. These parameters defined the enzymatic activity of the immobilized cellobiase enzyme, because the immobilized enzyme

7 Top Catal (2012) 55: activity is restricted by the rate of diffusion of the substrate to the enzyme inside the solid support. Hence, higher template content silica support materials exhibited significantly higher enzymatic activity/reaction rate of the immobilized cellobiase enzyme. Also, when used in multiple batches, the immobilized samples showed negligible loss of the enzyme activity, up to 9 recycles. This clearly demonstrated the fact that the cellobiase enzyme was definitely caged within the silica matrix, and not just adsorbed on the silica surface. The retained activity also demonstrates very efficient encapsulation, with negligible leaking of the enzyme from the silica support. Acknowledgments We are indebted to Prof. Yury Gogotsi of the Department of Materials Science and Engineering, Drexel University, for letting us use his BET instrument. We are grateful to Prof. Jun Xi of the Chemistry Department, Drexel University, for discussions and valuable suggestions. We thank the Chinese Ministry of Science and Technology for the support via the National 973 Program (Grant No. 2011CB935700). References 1. Sheldon RA (2000) Pure Appl Chem 72:3 2. Sheldon RA (2007) Adv Synth Catal 349: Sheldon RA, Rantwijk FV (2004) Aust J Chem 57: Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, Fernandez-Lafuente R (2007) Enzyme Microb Technol 40: Koeller KM, Wong CH (2001) Nature 409: Iyer PV, Ananthanarayan L (2008) Process Biochem 43: Monier M, Wei Y, Sarhan AA (2010) J Mol Catal B Enzym 63:93 8. Abdel-Fattah AF, Osman MY, Abdel-Naby MA (1997) Chem Eng J 68: Calsavara L, De Moraes F, Zanin G (2001) Appl Biochem Biotechnol 91 93: Wang Y, Hsieh YL (2008) J Membr Sci 309: Macario A, Moliner M, Corma A, Giordano G (2009) Microporous Mesoporous Mater 118: Takahashi H, Li B, Sasaki T, Miyazaki C, Kajino T, Inagaki S (2001) Microporous and Mesoporous Mater 44 45: Palomo JM, Munoz G, Fernandez-Lorente G, Mateo C, Fernandez-Lafuente R, Guisan JM (2002) J Mol Catal B Enzym 19 20: Macario A, Giordano G, Frontera P, Crea F, Setti L (2008) Catal Lett 122: Grazú V, Abian O, Mateo C, Batista-Viera F, Fernandez-Lafuente R, Guisan JM (2005) Biotechnol Bioeng 90: OrÁaire O, Buisson P, Pierre AC (2006) J Mol Catal B Enzym 42: Noureddini H, Gao X (2007) J Sol-Gel Sci Technol 41: Macario A, Katovic A, Giordano G, Lucolano F, Caputo D (2005) Stud Surf Sci Catal 155: Salis A, Meloni D, Ligas S, Casula MF, Monduzzi M, Solinas V, Dumitriu E (2005) Langmuir 21: He J, Li X, Evans DG, Duan X, Li C (2000) J Mol Catal B Enzym 11: Palomo JM, Munoz G, Fernandez-Lorente G, Mateo C, Fuentes M, Guisan JM, Fernandez-Lafuente R (2003) J Mol Catal B Enzym 21: Wei Y, Xu J, Feng Q, Dong H, Lin M (2000) Mater Lett 44:6 23. MacArio A, Giordano G, Setti L, Parise A, Campelo JM, Marinas JM, Luna D (2007) Biocatal Biotransform 25: Corma A, Fornes V, Jorda JL, Rey F, Fernandez-Lafuente R, Guisan JM, Mateo C (2001) Chem Commun 5: Frings K, Koch M, Hartmeier W (1999) Enzyme Microb Technol 25: Ikeda Y, Kurokawa Y (2001) J Sol-Gel Sci Technol 21: Avnir D, Braun S, Lev O, Ottolenghi M (1994) Chem Mater 6: Dave BC, Dunn B, Valentine JS, Zink JI (1994) Anal Chem 66:1120A 29. Braun S, Rappoport S, Zusman R, Avnir D, Ottolenghi M (1990) Mater Lett 10:1 30. Reetz MT, Zonta A, Simpelkamp J (1995) Angew Chem Int 34: Noureddini H, Gao X, Philkana RS (2005) Bioresour Technol 96: Noureddini H, Gao X, Joshi S, Wagner PR (2002) J Am Oil Chem Soc 79: Noureddini H, Gao X, Joshi S (2003) J Am Oil Chem Soc 80: Wei Y, Jin D, Ding T, Shih W, Liu X, Cheng S, Fu Q (1998) Adv Mater 10: Wei Y, Xu J, Dong H, Dong JH, Qiu K, Jansen-Varnum SA (1999) Chem Mater 11: Wei Y, Xu J, Feng Q, Lin M, Dong H, Zhang W, Wang C (2001) J Nanosci Nanotechnol 1: Das S, Berke-Schlessel D, Ji H-F, McDonough J, Wei Y (2011) J Mol Catal B Enzym 70:49