Bioactive materials have been explored for a broad range ofapplications including biocatalysts, biosensors, antifouling membranes andother functional and smart materials. We report herein a unique method for preparation of bioactive materials through a spin coating process. Specifically, we investigatedthe preparation of protease Subtilisin Carlsberg-coated plastic films andexamined their activities for hydrolysis of chicken egg albumin (CEA). The process generated enzymic coatings with a typical loading of 13 g/cm2, retaining 46% ofthe enzyme activity for hydrolysis of CEA in aqueous solutions. Interestingly,the surface-coated protease thin film not only catalyzed the hydrolysis of CEAin aqueous solutions, but also showed good activity for solid-state CEA thatwas coated on top of the enzyme thin film.



Introduction


Enzymes have been widely used for food processing, chemicalsynthesis, pollutant degradation, clinical analysis, detergents, andtherapeutic agents. There is currently a growing interest in developing smartmaterials that can show desired functions in response to changes in theirsurrounding environments by conjugating enzymes with polymeric materials (1,2). Enzymesolid material incorporation has been mostly achieved via bothsurface attachment and adsorption or matrix entrapment. The incorporation ofenzymes into plastic materials is particularly challenging, as most industrialenzymes are not compatible with hydrophobic polymers. Nevertheless, advanceshave been made in manipulating the enzymes either chemically or physically to improvetheir hydrophobicity and thus allowing efficient incorporation with plasticmaterials (3, 4). For many applications, the functionalities or activities ofthe enzymes at the surface of materials are the most desired properties.Physical adsorption of enzymes to hydrophobic surfaces is generally inefficient(5), and the most effective surface attachment has been realized throughactivation of the materials followed by chemical tethering of the enzymes (6).In most cases, however, the activation of pre-prepared materials is not easy torealize. Strong chemical processing or radiation is usually required (7). Inthis paper, we report an alternative approach that does not require preactivationof plastic materials and is expected to be more suited for large-scalepreparation of smooth enzyme coatings on solid materials.


Spin coating is a process developed for preparation of materials of fine and controllable surface properties. The advantage of the spincoating process is the formation of uniform thin films (usually in the range ofmicrometers or even thinner) within a very short time period. Spin coating hasbeen applied for production of compact disks (DVD, CD ROM, etc.), flat displayscreens, and microcircuits (8, 9). The use of spin coatingtechnology for immobilizing enzymes onto biosensors, which usually involvedonly a small portion of the probe surfaces, has been reported previously in literature (10, 11). The current work examines the potential of spincoating enzymes for preparation of large-scale functional materials. Proteasesubtilisin Carlsberg was chosen as the model enzyme in this study. Among otherindustrial enzymes, proteases have the largest industrial production and account for about 60% of the total sale of enzymes worldwide for applicationssuch as food processing and detergent additives. We expect the development of protease coated materials may also extend the use of these enzymes to materials that havefunctions such as prohibiting surface accumulation of proteins, thus developinga unique class of materials for biomedical, bioprocessing, and personal careapplications.


Materials and Methods Materials


Chicken egg albumin (CEA, catalog no. A5378), casein, bovineserum albumin (BSA), protease subtilisin Carlsberg (protease SC), N-acryloxysuccinimide (NAS), 2-sulfoethyl methacrylate (2-SEM), and tyrosine werepurchased from Sigma-Aldrich (Missouri). Styrene was purchased from Arcos (New Jersey). Dimethyl sulfoxide (DMSO), tricholoroacetic acid (TCA), and4,4-azobis(4-cyanovaleric acid) were obtained from Wako Pure ChemicalsIndustries Ltd. (Osaka, Japan), and lutaraldehyde was from TIC America (Tokyo, Japan). Bradford G250 reagent was supplied by Biorad Laboratories (Hercules, Canada). All other reagents involved in the experiments were of analytical grade. Polystyrene Petridish disks 10 cm in diameter purchased from Fisher Scientific were applied asthe hydrophobic plastic plates for surface coating of enzyme.

 

Preparation of Polystyrene Modifier


The polystyrene with one end of the polymer chain functionalized for enzyme attachment was prepared by following a modified procedure as reported previously (6). In brief, a stock solution of a polymerizable surfactant, 2-sulfoethyl methacrylate (2-SEM), was prepared by dissolving 5 g of 2-SEM in 50 g of DI water and was then diluted to 100 g using DI water while the pH of the solution adjusted to 3.5 by using a solution of sodium hydroxide. (10 wt %). The polymerization reaction was conducted with 0.5 mL of 2-SEM stock solution, 392 mg of NAS, 1.2 mL of styrene, and 16 mL of DMSO. The reaction was initiated by adding 29.2 mg of 4,4-azobis(4-cyanovaleric acid), purging the solution with N2 for 1 min, and heating the system to 70 C in a water bath with stirring. The polymerization reaction was allowed to continue for 12 h, and the resulting polymer was collected after evaporating the solvent.


Preparation of Bioactive Thin Films of Protease SC via Spin Coating


A spin coating procedure was developed for preparation of protease SC thin film coatings on polystyrene plastic plates (Petri Dish disks of 10 cm in diameter). Typically, the one end-functionalized polystyrene was first applied to the surface of a plastic plate by spin coating 1 mL of its chloroform solution with a concentration of 50 mg/mL at 6000 rpm for 1 min under vacuum using a Laurell WS-400B-6NPP/LITE spin coater (Laurell Technologies corporation, North Wales, PA). Subsequently, 1 mL of protease SC solution (10 mg/mL) in a pH 7.0 and 0.05 M PBS buffer solution, 1 mL of 0.5% (V/V) of glutaraldehyde, and 1 mL of 10 mg/mL protease SC solution were coated sequentially layer-by-layer onto the surface of the plate. The spinning speed was controlled to be at 2000 rpm, and the spinning time for layer coating was allowed to last 1 min. The resultant coated plate was kept at 4 C for 12 h and was then washed with a Tris buffer solution of pH 8 and 0.05 M for 30 min to remove any unreacted free chemicals and enzyme. Finally, the coated plate was washed by DI water 3 times for 1 h for each wash, and the bioactive coated plate was then air-dried and cut into small pieces (1 cm _ 2 cm) for further uses. Plastic plate samples coated with a nonactive protein, BSA, were prepared by the same procedure and used for control analyses of enzyme activity and loading.


The amount of enzyme coated on the plate was determined by a previously reported method based on the Bradford protein assay (12). It was developed on the basis of the fact that the conjugation of Bradford dye with the enzyme and subsequent removal of the enzyme from the solution will lead to a reduced absorbance at 465 nm of the Bradford protein assay solution and that the change can be used to quantify the amount of enzyme immobilized on the solid materials. In detail, 1 mL of diluted Bradford reagent (1:10 by DI water) was incubated with the enzyme-coated plate samples (1 cm X 2 cm) under stirring for 5 min. The absorbance at 465 nm of the supernatant solution was measured with a UV-1601 spectrophotometer ( Shimadazu Corporation, Columbia, USA ). The amount of protease SC coated on the plate was calculated by applying a calibration curve developed with native protease SC.


In order to visualize the distribution of protease SC in the coating, the enzymes were labeled with Oregon green maleimide dye (Molecular Probes, USA). Briefly, protease SC coatings were incubated with a solution of the dye for 2 h. The excess of dye was washed out with DI water. After being dried with air, the samples were analyzed with a fluorescent microscope with 320_ magnification.


Standard Enzyme Activity Assay


Proteolytic activity of protease SC was determined by following the method of Folin and Ciocalteu (13). Typically, 0.65% (w/v) of casein in a PBS (0.05 M, pH 7.5) buffer solution was used as substrate, and a 100μL specimen solution of rotease SC was incubated with 500μL of substrate solution for 10 min at 37 C. The reaction was stopped by adding 500μL of 110 mM of TCA solution, and the equivalent of tyrosine in the TCA-soluble fraction was determined at 660 nm using Folin- iocalteau reagent. One unit of activity was defined as the amount of enzyme hydrolyzing casein to produce absorbance equivalent to 1.0 _ 10-3 mol of tyrosine per minute at 37 C. The activity of protease SC in the coating was determined in a similar manner except one piece of sample plate coated with protease SC (1 cm _ 2 cm) instead of an enzyme solution was applied.


 

Activity of Protease SC for Hydrolysis of CEA


The hydrolytic reaction of CEA was used for activity assay of protease SC. Briefly, 9 mL of a 1.8 mg/mL CEA solution was mixed with 1 mL of protease SC solution (0.5 mg/mL) at room temperature under shaking at 200 rpm. Aliquots of 100L of the sample were taken periodically for GPC analysis after dilution with 200L of DI water. GPC chromatography was performed with a computerized Shimazu LC system equipped with a PL-Aquagel OH mixed column (Polymer Laboratories, USA). DI water was used as mobile phase with a flow rate of 1.5 mL/min. CEA and its hydrolyzed products were detected at the UV wavelength of 280 nm. The hydrolysis reaction was done in triplicates, and the control reaction was conducted following exactly the same procedure but with 1 mL of DI water replacing the protein SC solution. The residual amount of CEA was determined from GPC chromatograms and applied for calculations of reaction velocity.


The same reaction as mentioned above was applied for activity assay of surface-coated protease SC. For activity in the liquid phase, 10 mL of 1.8 mg/mL CEA solution was incubated with 20 pieces of plastic plates that were coated with protease SC (1 cm X 2 cm) at room temperature. The hydrolytic reaction was monitored through the same procedure as that applied for native protease SC.


The activity of surface-coated protease SC against solid-state substrate was evaluated by spin coating a layer of CEA on top of the enzyme coating. Typically, 2 mL of 10 mg/mL CEA was spin coated onto the protease SC-coated plate at 2000 rpm for 1 min under vacuum. A thin film coating of CEA was quickly formed within the 1 min spin coating process. The plate was then cut into pieces of 1 _ 2 cm and was incubated at controlled reaction conditions with desired temperature and humidity. Samples were taken periodically and washed with 1 mL of DI water under shaking at 200 rpm for 15 min. The washing solution was analyzed for changes of content of CEA by using GPC.


Results and Discussions


Thin Film Formation of Protease SC via Spin Coating


Enzymes can be attached to the surface of solid supports through various strategies such as physical adsorption, covalent binding, and intermolecule cross-linking. Compared to other methods, spin coating has the potential to develop smooth coatings with a controllable amount of enzymes. We first examined several approaches for the preparation of surface-coated protease SC on the polystyrene plates, namely, physical adsorption, monolayer spin coating, and spin coating with glutaraldehyde (GA) cross-linking. Physical adsorption resulted in essentially no activity retention on the surface of the plastic after being washed with DI water, whereas spin coating with GA cross-linking achieved an enzyme loading and an activity density (per unit area of the plate) that were about 10 times that realized with monolayer spin coating. Accordingly, spin coating followed with GA cross-linking was the method chosen for preparation of the surface-coated enzyme in this study.



 

Enzyme loading on the surface of the plastics can be controlled by adjusting several processing parameters, including layers of coating, concentration of the enzyme and cross-linker (glutaraldehyde in this work) in the spinning solutions, and speed of spinning. Figure 1 A presents the effect of glutaraldehyde (GA) concentration on enzyme loading and residual activity of protease SC coated at a spinning speed of 2000 rpm. It was found that maximum activity of protease SC was achieved at a GA concentration of 0.5% (v/v), whereas enzyme loading generally increased with the concentration of GA. Apparently, GA with concentrations exceeding 0.5% significantly inactivated the protease. Figure 1B illustrates the influence of spinning speed with the GA concentration fixed at 0.5% (v/v). Interestingly, enzyme loading reached a maximum value at the spinning speed of 1200 rpm while the maximum enzyme activity was observed at the spinning speed of 2000 rpm. We tend to believe that best surface distribution of the enzyme may be realized when the spinning speed reached 2000 rpm. Therefore, the protease SC thin films were prepared for further studies under the optimized conditions with of 0.5% (v/v) GA and a spinning speed of 2000 rpm.


Typically a density of 13 g-enzyme/cm2 and a specific activity of 6 unit/cm2 of protease SC were achieved through the spin coating and cross-linking procedure. Since the spin coating procedure generally retains only a small portion of the spinning solution on the surface of the solid samples, the immobilization yield of the enzyme can be expected to be low. It was estimated that only 5% of the initially added protease SC had been attached on the polystyrene plates (Petri Dish disks). However, those spinning solutions are also expected to be reused, and much higher accumulated immobilization yield can be expected for practical applications. The distribution of protease SC in the thin film coating was visualized via fluorescent microscope by staining the enzyme with the Oregon green maleimide. Figure 2 illustrates the distribution of protease SC on a polystyrene plate prepared through the abovementioned procedure. It appeared that the enzyme was distributed on the surface in form of aggregates with dimensions in range of 2-20 m. The enzyme-covered area represented about 10% of the surface area of the plastic plate. That could be a result of uneven exposure of the active group of the functionalized polystyrene when it was coated on to the plastic surface. Nevertheless, the micrometer-scale spots of the enzyme distributed about uniformly throughout the whole surface of the plates.



Figure 2. Fluorescent image of spin coated protease SC on the surface

of poly(styrene) plates. The scale bar is 20 m.


The dimension of protease SC from Bacillus licheniformis is reported as 7.7 nm _ 5.5 nm _ 5.4 nm (14). Assuming each enzyme molecule occupies a surface area of 42.4 nm2, monolayer coverage of 1 cm2 would need _2.4 _ 1012 molecules of protease SC. When only 10% surface area was achieved (see Figure 2), it would require an enzyme loading of 0.011 g/cm2 of protease SC for monolayer coverage, and about 1,200 layers on average for enzyme-covered areas to achieve an enzyme loading of 13 g/cm2 on the plate. These calculations indicate that the average size of the enzyme aggregates should be in the order of _7 m, which is in agreement with the average size of the enzyme-covered area as shown in Figure 2.


 

Liquid-Phase Activities of Native and Surface-Coated Protease SC for Hydrolysis of CEA


The surface-coated protease SC was examined for the hydrolysis of CEA. Figure 3 shows typical GPC chromatograms demonstrating the progress of the hydrolysis of CEA. As shown in Figure 3, the CEA peak was found at a retention time of 6.2 min, while a product peak of the hydrolyzed polypeptide fragments appeared at 8.6 min (only one peak was observed as the column was not able to separate the fractions of the hydrolysis products). No hydrolysis of the protein substrate was observed when control samples of BSA coated and plain polystyrene plates were applied. Figure 4 presents the time courses of the hydrolysis reaction catalyzed by native and surface-coated SC. The reaction rate normalized with the amount of enzyme applied catalyzed by native protease SC was about 2.2-fold higher than that observed for the surfaced-coated enzyme, indicating the coated enzyme retained about 46% of the enzymes original aqueous activity. This is in fact a high retention of the activity for any immobilized enzyme, as in most cases immobilized enzymes show activities that are usually over 10 times lower than their native parent enzymes (15). The reduced activity of immobilized enzymes is generally resulted from the high mass transfer resistance associated with heterogeneous catalysis. However, the impact of mass transfer limitation is usually much less significant for slow reactions. The hydrolytic reaction examined in this work appeared to be a very slow reaction with a rate of 48 nmol/(h mg-enzyme) observed for native protease SC. Another apparent cause for high activity retention of the coated enzyme may be attributed to the fact that the coated enzyme was surface exposed.




 

Activity of Enzymic Thin Films for Solid-State Substrate


Many applications of materials may involve the exposure of the materials to substrates in the absence of a bulk liquid phase such as biocidal coating (16) and gas-phase biocatalysis (17). Although enzymes have been reported to be active in many unconventional reaction media, it appears to us that the literature hitherto lacks reports of enzyme-catalyzed reactions with solid substrates in the absence of bulk-phase water. Our tests demonstrated that the surface coated enzyme showed an interesting activity against solid-state protein substrate. Figure 5 shows the time course of the hydrolysis reaction of solid state CEA by surface coated protease SC. The solid-state CEA was prepared by spin coating the protein substrate onto the plastic plates which were coated with a thin film of protease SC. A CEA coating was formed within the 1 min spin coating process (under vacuum). According to the time course, the solid state hydrolysis was able to proceed with a reaction rate of 0.52 nmol/(hmg-enzyme), which is only about 2% of that observed for the surface-coated enzyme in liquid phase.


One may argue that the observation of the solid-state hydrolysis may be a result of reactions that took place during the spin coating process of the substrate and the washing step in preparation of samples for analysis. Reactions were certainly not inhibited during these preparations and sampling processes when there was a bulk phase of water. Considering the spin coating took only about 1 min to form a thin film and the washing step 15 min, it is unlikely that the reaction (which is also slow) taking place during those time windows contributed a significant portion to the observed hydrolysis. We further tested the activity of the coated enzyme under the washing conditions for 15 min with DI water and found no hydrolysis of CEA detectable following the same procedure. Moreover, as to be discussed in the following, the solid-state hydrolysis was found to be sensitive to changes in incubation conditions, indicating the reaction observed should have mainly taken place when the substrate was essentially dry.


Two operational parameters, namely, relative humidity (HR) and temperature, were varied to examine their impact on the solid-state hydrolysis reaction. Figure 6 A demonstrates the effect of relative humidity, which was regulated with a salt solution with desired water activity. The samples of solid-state reaction (plastic plates coated with protease SC and substrate CEA) and control samples were placed in a sealed chamber at room temperature along with DI water (for 100% RH) or a salted aqueous solution of lithium chloride (11.3 ( 0.3% RH), magnesium chloride (32.8 ( 0.2% RH), or potassium iodide (68.9 ( 0.2% RH). Conditioned air in the lab without additional humidity control was used as a condition of 49% RH. As shown in Figure 6A, the hydrolysis rate of CEA increased approximately linearly with increase in RH. This is similar to the observation of enzymatic reactions under other anhydrous conditions (18, 19). In addition to the generally expected effect of the hydration of enzymes on their activities, the RH effect observed in this work may also have included the contribution of water as a substrate for the hydrolysis reaction. Temperature of the incubation chamber also affected the solid-state hydrolysis significantly within the range of testing (Figure 6B). Generally speaking, increase in temperature generally accelerates the reaction rate. On the other hand, high temperature may also inactivate enzymes. As a result, enzymes generally have specific optimized temperatures to show the highest reaction rates. The optimized temperature of protease SC for the hydrolysis of protein was reported as 37 C (18). Surprisingly, the reaction rate tested for the solid-state hydrolysis continued to increase even after the temperature increased up to 80 C. The initial reaction rate at 80 C was almost 3 times higher than that at 25 C (1.9 vs 0.52 nmol/hmg-enzyme). It appeared that the spin coating and cross-linking of the enzyme has substantially improved the stability of the protease against thermal inactivation.

 




Stability of Surfaced-Coated Protease SC


Immobilization of enzymes generally stabilizes the biocatalysts. As indicated from the above activity tests, the surface-coated enzyme may also benefit from enzyme stabilization. Figure 7 depicts the experimental data on the stability of the surface-coated protease SC. As expected, cross-linking with GA improved the stability of the enzyme to a greater extent than the preparation without cross-linking (Figure 7A). The thermal stability tests were conducted by placing the immobilized enzymes into an incubation chamber set at 80 C. The half-lifetime of cross-linked enzyme was estimated to be 120 min, which was over 2-fold longer than that of the surface-coated enzyme without cross linking. We further examined the effect of water on the stability of the surface-coated enzyme by placing the immobilized enzyme (with GA cross-linking) in an aqueous solution at room temperature and comparing the stability results to that of the same enzyme placed in air (Figure 7B). It was found that about 50% of the initial activity of the enzyme was retained after incubating for 2 months in air, whereas only 8% retention was observed with the enzyme placed in aqueous solution. The results from these two stability tests strongly supported our observation of the activity of the coated enzyme at 80 C for solid-state catalysis. The reusability of surface-coated protease SC was also investigated. The same plastic plate coated with enzyme was reused after being washed with DI water for at least 3 times followed by drying in air before next activity test. The results indicated that the residual activity of protease SC on the plastic plate remains almost 85% after being reused for 5 times.


Conclusion


Spin coating was applied successfully to prepare plastic plates of smooth surfaces coated with evenly distributed enzyme. The surface-coated enzyme showed good activities and much improved thermal stability. In the absence of a bulk liquid phase, the surface-coated protease was able to catalyze the hydrolysis of solid-state protein substrate, indicating that enzyme-bound water was sufficient to enable the reaction at the interface between the solid-state enzyme coating and the solid-state protein substrate. The observed solid-solid-state catalysis responded to changes in humidity and temperature in a similar manner as observed for other nonaqueous enzymatic reactions. We expect the spin coating approach as reported in this work will provide a new methodology for preparation of a wide array of biofunctional materials.


Acknowledgment:


This work was supported by a research grant from Toyota Motor Engineering and Manufacturing North America, Inc. The authors thank Dr. J. Zhe for help with spin coating and Mr. Songtao Wu and Ramazan Komurcu for help with experimental work on enzyme characterization.


 

References and Notes


1)    Asuri, P.; Karajanagi, S. S.; Kane, R. S.; Dordick, J. S. Polymernanotube- enzyme composites as active antifouling films. Small 2007,3(1), 50-53.


2)    Roy, I.; Gupta, A.; Khare, S. K.; Bisaria, V. S.; Gupta, M. N. Immobilization of xylan-degrading enzymes from Melanocarpus albomyces IIS 68 on the smart polymer Eudragit L-100. Appl. Microbiol. Biotechnol. 2003, 61(4), 309-313.


3)    Wang, P.; Sergueeva, M. S.; Lim, L.; Dordick, J. S. Biocatalytic plastics as active and stable materials for biotransformations. Nat. Biotechnol. 1997, 15, 789-793.


4)    Novick, S. J.; Dordick, J. S. Protein-containing hydrophobic coatings and films. Biomaterials 2002, 23(2), 441-448. (5) Reis, P.; Holmberg, K.; Debeche, T.; Folmer, B.; Fauconnot, L.; Watzke, H. Lipase-catalyzed reactions at different surfaces. Langmuir 2006, 22(19), 8169-8177.


5)    Jia, H.; Zhu, G.; Wang, P. Catalytic behaviors of enzymes attached to nanoparticles: The effect of particle mobility. Biotechnol. Bioeng. 2003, 84(4), 406-414.


6)    Dagenais, P.; Desprez, B.; Albert, J.; Escher, E. Direct covalent attachment of small peptide antigens to enzyme-linked immunosorbent assay plates using radiation and carbodiimide activation. Anal. Biochem. 1994, 222(1), 149-155.


7)    Kucherenko, S. S.; Leaver, K. D. Modeling effects of surface tension on surface topology in spin coatings for integrated optics and micromechanics. J. Micromech. Microeng. 2000, 10(3), 299- 308.


8)    Schubert, D. W.; Dunkel, T. Spin coating from a molecular point of view: its concentration regimes, influence of molar mass and distribution. Mater. Res. InnoVations 2003, 7(5), 314-321.


9)    Kimura, J.; Saito, A.; Ito, N.; Nakamoto, S.; Kuriyama, T. Evaluation of an albumin-based, spin-coated, enzyme-immobilized membrane for an ISFET glucose sensor by computer simulation. J. Membr. Sci. 1989, 43(2-3), 91-305.


10)  Sant, W.; Pourciel-Gouzy, M. L.; Launay, J.; Do Conto, T.; Colin, R.; Martinez, A.; Temple-Boyer, P. Development of a creatininesensitive sensor for medical analysis. Sens. Actuators, B 2004, B103(1-2), 60-264.


11)  Bonde, M.; Ponoppidan, H.; Pepper, D. S. Direct dye binding-A quantitative assay for solid-phase immobilized protein. Anal. Biochem. 1992, 200, 95-98.


12)  Folin, O.; Ciocalteu, V. Tyrosine and tryptophan determinations proteins. J. Biol. Chem. 1927, 73, 627-650.


13)  Schmitke, J. L.; Stern, L. J.; Klibanov, A. M. The crystal structure of subtilisin Carlsberg in anhydrous dioxane and its comparison with those in water and acetonitrile. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 4250-4255.

14)  Szymanska, K.; Bryjak, J.; Mrowiec-Bialon, J.; Jarzebski, A. B. Application and properties of siliceous mesostructured cellular foams as enzymes carriers to obtain efficient biocatalysts. Microporous and Mesoporous Mater. 2007, 99(1-2), 167-175.


15)  Makal, U.; Wood, L.; Ohman, D. E.; Wynne, K. J. Polyurethane biocidal polymeric surface modifiers. Biomaterials 2006, 27, 1316-1326.


16)  Debeche, T.; Marmet, C.; Kiwi-Minsker, L.; Renken, A.; Juillerat, M. A. Structured fiber supports for gas phase biocatalysis. Enzyme Microb. Technol. 2005, 36(7), 911-916.


17)  Hartsough, D. S.; Merz, K. M. Protein dynamics, and salvation in aqueous, and nonaqueous environments. J. Am. Chem. Soc. 1993,

18)  115, 6529-6537.


19)  Zaks, A.; Klibanov, A. M. Enzymatic catalysis in nonaqueous solvents. J. Biol. Chem. 1988, 263 (7), 3194-3201.


 

About the Authors:


The authors are associated with Biotechnology Institute and Department of Bioproducts and Biosystems Engineering, University of Minnesota, St. Paul, Minnesota, Department of Chemical and Biomolecular Engineering, University of Akron, Akron, Ohio, and Materials Research Department, Toyota Motor Engineering and Manufacturing North America, Inc., Ann Arbor, Michigan, respectively.



To read more articles on Textile, Industry, Technical Textile, Dyes & Chemicals, Machinery, Fashion, Apparel, Technology, Retail, Leather, Footwear & Jewellery,  Software and General please visit https://articles.fibre2fashion.com


To promote your company, product and services via promotional article, follow this link: https://www.fibre2fashion.com/services/article-writing-service/content-promotion-services.asp