181

New applications for biocatalysts

Sheldon W May
Biocatalysis biotechnology biocatalysts Significant production biochemical has always been a key focus area in and new approaches have continued progress for the utilization of over the past year. to emerge

has been made in the biocatalytic using genetic and of new and

of both synthetic and natural polymers, in of novel biocatalysts approaches or through identification

order to facilitate biocatalytic and biomedical applications. Finally, new approaches are being pursued co enable the modulation of the stereochemistry of enzymatic reactions, thus harnessing one of the key defining features of biological catalysis.

the generation

Polymer synthesis and modification

Materials science and engineering is widely recognized as a key scientific and technological frontier area in which major advances are rapidly being made. In my opinion, a premier initial interface between biotechnology and materials science is the utilization of biocatalysis for the production of polymeric materials with specialized properties. For example, the high scereoselectivity exhibited by many enzymes opens up the attractive possibility of producing chiral polymeric materials from racemic starting reagents. A recent example is the stereoselective lipase-catalyzed ring-opening polymerization of racemic a-methyl-P-propiolactone reported by Svirkin et a/. [2*], who achieved the production of optically active S-enriched poly(a-methyl-P-propiolactone). Polymerizations conducted in toluene and heptane proceeded more rapidly than those carried out in dioxane, and the enantiomeric ratios in toluene, heptane and dioxane were 4.1, 0.9 and 2.0, respectively. Thus, from the point of view of reaction rates and enantioselectivity, toluene is the preferred solvent. Polymer products prepared in toluene using lipase from Pseudomonas jhmescens had number average relative molecular mass values in the range 2600 to 2900gmol-1 and [a]D values from +12.2 to +lY. Analysis of the polymer chain end structure by IH and 13C NMR showed that these products possess hydroxyl and carboxylic acid termini. On the basis of the analysis of chain stereosequence distributions by 13C NMR, it was concluded that stereoselectivity during propagation results from catalyst enantiomorphic-site control. Lipase-catalyzed polyester synthesis in anhydrous ether and fluoroform was investigated by Chaudhary et a/. [3**] with the goal of producing polymer with low dispersity and controlled molecular weight. Enzymatically synthesized polyesters were characterized using laser desorption mass spectrometry, which enables accurate structural and size characterization of polymers with relative molecular masses (iVrs) up to 10000, as well as by the more conventional techniques of gel permeation chromatography and NMR and IR spectroscopy. In supercritical fluoroform, as the pressure is raised, the molecular weight of both soluble and precipitated polyester increases; however, the precipitated polyester retains low dispersity after precipitation, because it is subject to a very low rate of enzymatic chain extension. At a pressure of 14OOpsi, a 6-8% overall yield of monodisperse polyester was obtained. In a related work, Matsumura and co-workers [4] reported

biological sources, their modification modulation

in the immobilization with amphiphilic

of biocatalysts

polymers, and in the of enzymatic reactions.

of the stereochemistry

Address School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA Current Opinion in Biotechnology 1997, 8:181-l 86

Electronic identifier: 0956-l 669-008-00181 0 Current Biology Ltd ISSN 0956-l 669 Abbreviations GDH glucose dehydrogenase relative molecular mass 4 PEG poly(ethylene glycol) racemic temperature Tr

Introduction
Biocatalysis has long been a key focus area in biotechnology [l]. Enzymes catalyze a rich variety of metabolic transformations, and do so with very high catalytic rates under mild conditions with high reaction selectivity and stereospecificity. These characteristics are obviously highly attractive from an industrial perspective. Moreover, within the past few years, the horizons of biocatalysis have expanded greatly to encompass both catalytic RNA molecules (ribozymes) and catalytic antibodies (abzymes). Thus, the potential of biocatalysis extends well beyond the particular chemical reactions that normally occur within living cells.

This review will describe a number of significant new developments in the application of biocatalysis made during the past year. Important progress has been made in the utilization of biocatalysis for the production of specialized synthetic polymeric materials, as well as for the in vitro production of peptides containing unnatural amino acids, thus significantly enhancing the rapidly developing interface between biotechnology and materials science. The generation of novel biocatalysts has been accomplished using genetic and biochemical approaches, and progress has continued on the systematic identification of extremophiles or other sources for enzymes with unusual properties. Novel approaches have been reported for the immobilization of biocatalysts and for the covalent attachment of amphiphilic polymers to soluble enzymes in

182

Biochemical engineering

using the lipase-catalyzed ring-opening polymerization of B-propiolactone in bulk to produce the corresponding polyester with Mr>SOOOO. The molecular weight of the polyester produced was inversely dependent on the concentration of lipase, and both molecular weight and polymerization rate increased with increasing temperature. Similarly, benzyl B-malolactonate was readily polymerized by porcine pancreatic lipase or Novozyme 435 lipase at 60C to yield poly(benzy1 B-malate) with M,>7000 [5]. The biocatalytic synthesis of novel fluorinated polyesters has also been recently reported [6]. The enzymatic synthesis of a fluorescent polymer of Z-naphthol was reported by Premachandran et al. [7*], using the oxidative enzyme horseradish peroxidase encapsulated in AOT/isooctane reversed micelles. In this 2-naphthol monomer reaction system, the amphiphilic partitions on the oil-water interface with its hydroxyl moieties directed toward the microaqueous core in which the enzyme is encapsulated. The precipitated poly(Z-naphthol) polymer produced had the morphology of single and interconnected microspheres and was found to be soluble in a range of polar and nonpolar organic solvents. The poly(Z-naphthol) exhibited the fluorescence characteristics of the naphthol chromophore plus an additional well-resolved fluorescence that was attributed to an extended quinonoid structure attached to the polymer backbone. Further evidence for this quinonoid structure was obtained using UV, IR and NMR spectroscopy. Biocatalysis can also enable the in vitro production of naturally occurring polymeric materials, thus obviating the need for the isolation and purification of these substances from natural sources. A step in this direction is described in the recent report [8] of the successful in vitro enzymatic synthesis of cellulose. This was achieved through the cellulase-catalyzed polycondensation of B-cellobiosyl fluoride monomer in a mixed solvent of acetonitrile and acetate buffer. The key to this success was the use of partially purified cellulase and an appropriate mixture of acetonitrile/buffer as the reaction solvent. Depending on polymerization conditions, either native cellulose I, a metastable allomorph, or the crystalline allomorph cellulose II, a thermodynamically more stable form, was produced. The authors contend that the formation of two cellulose allomorphs implies that the polarity of the glucan chain ordering can be controlled, and they propose the term choroselectivity for this spatial control of the ordering of the macromolecular chain. In addition, the cellulose analogs 6-0-methylated cellulose and xylan were successfully synthesized with regio- and stereoselectivity using this enzymatic polymerization technique. Although the technology for the chemical synthesis of peptides is now very well developed and has been reliably automated, the incorporation of unnatural amino acids or peptidomimetics into synthetic peptides is often quite difficult to accomplish. In principle, biocatalysis can make

a significant contribution in this area through the use of proteases or lyases operating in the reverse (synthetic) direction. In this respect, Fernandez et a/. [9] examined several proteases as potential catalysts for the enzymatic synthesis of oligopeptides containing the unnatural amino acid allylglycine (Ag), the overall objective being the synthesis of a reactive tetrapeptide that could be chemically polymerized into a potentially biocompatible or biodegradable material. Commercially available proteases were screened for esterase activity toward the methyl ester of allylglycine (DLAgOMe) to identify potential catalysts for dipeptide synthesis. Proteases from Aspergillus oryzae and Aspe7gillu.r sojae, pronase E and protease Nagarse synthesized the protected dipeptide Cbz-L-Ag-L-PheNHz from Cbz-DL-AgOMe and L-PheNH2 but were unable to catalyze the synthesis of Cbz-L-Phe-L-AgOEt. Thus, although these enzymes could use allylglycine as the acyl donor, they could not employ it as the acyl acceptor in peptide synthesis. In contrast, chymotrypsin was able to use allylglycine ethyl ester as the acyl acceptor in the synthesis of Cbz-L-Phe-L-AgOEt, but was unable to synthesize Cbz-L-Ag-I,-PheNHZ. The two dipeptides Cbz-I,-Ag-L-Phe and L-Phe-L-AgOEt were then used in the thermolysin-catalyzed synthesis of the desired tetrapeptide, Cbz-L-Ag-L-Phe-L-Phe-L-AgOEt. Polysaccharides are also attractive targets for enzymatic synthesis [lo-121. The novel enzymatic synthesis of cyclodextrins from maltose using cyclomaltodextrin glucanotransferase in two-phase water-organic solvent systems was recently reported [lo]. B-cyclodextrin was obtained when the reaction was carried out in water/cyclohexane at 7C whereas both the ~1and B forms were obtained in various aqueous/alkanol solvent mixtures. Enzymatic syntheses of polymers containing nicotinamide mononucleotide [13], of mosaic nucleic acids composed of 50% RNA and 50% DNA [14], and of uniformly 13ClsN-labeled DNA oligonucleotides in milligram quantities for NMR studies [15] have also been described recently.

Generation and identification biocatalysts

of novel

The generation of novel biocatalysts using genetic or biochemical methods, as well as the identification of extremophiles or other sources for enzymes with unusual properties, represent important objectives in the biotechnological development of biocatalysis. An example of the genetic approach is provided by the recent work of Moore and Arnold [16**], who report the directed evolution of an esterase to be used for antibiotic deprotection in aqueous-organic solvents through sequential generations of random mutagenesis and screening. One esterase variant was found to perform as well in 30% dimethylformamide as the wild-type enzyme does in water, reflecting a 16-fold increase in esterase activity. The random pairwise gene recombination of two positive variants led to a further twofold improvement in activity. Considering also the increased expression level achieved

New applications for biocatalysts May

183

during these experiments, the net result of four sequential generations of random mutagenesis and one recombination step was a SO-60-fold increase in total activity. The authors point out that although the contributions of individual effective amino acid substitutions to enhanced activity are small (<twofold increases), the accumulation of multiple mutations by directed evolution allows significant improvement of the biocatalyst for reactions on substrates and conditions not already optimized in nature. The positions of the effective amino acid substitutions were identified using an esterase structural mode1 based on homology to acetylcholinesterase and triacylglycerol lipase. None of the substituted amino acid residues appear to interact directly with the antibiotic substrate, which underscores the difficulty of predicting mutational effects in a rational design effort. A more molecular approach to the generation of novel biocatalysts is illustrated by the work of Kim et a/. [17**] on the construction of hybrid restriction enzymes. Two novel site-specific endonucleases were successfully produced by linking two different zinc-finger proteins to the cleavage domain of F&I endonuclease. Both fusion proteins are active, and under optima1 conditions each cleaves DNA in a sequence-specific manner. Thus, these results demonstrate the feasibility of constructing a variety of artificial nucleases that will cut DNA near predetermined sites, by linking the FoAI endonuclease module to a specificity module such as a zinc-finger motif. Turning to the approach of identifying novel sources for biocatalysts, Rasskazov and co-workers [ 181 recently reported data on the distribution among marine invertebrates of 1,3-B-glucanases, proteinases and deoxyribonucleases. These data were obtained in a systematic study carried out by the Russian Academy of Sciences [IS], and the results provide evidence that hydrolytic enzymes from marine invertebrates exhibit some unique properties and specificities. Similarly, Sode and co-workers [ 191 report the isolation of a novel glucose dehydrogenase (GDH) from the marine bacterium Cytop/zaga marino&zva. This enzyme catalyzes the oxidation of a hydroxyl group in glucose, but does not react with substituents at the Cl position of the substrate. This novel GDH was then utilized for the enzymatic determination of 1,.5-anhydro-D-glucitol, using 2,6-dichlorophenolindophenol and phenazine methosulfate as electron mediators. An extremely thermostable B-glucosidase has recently been described [ZO] that served as a biocatalyst for the synthesis of new glucoconjugates at elevated temperatures. The molecular basis for the enhanced stability exhibited by a number of enzymes isolated from thermophiles is a subject of considerable biotechnological significance. In this respect, Sode et a/. [Zl] report that a novel thermostable GDH isolated from a soil bacterium near a hot spring varies its temperature optimum by altering its quarternary structure. On the basis of native elec-

trophoresis and SDS-PAGE, the authors conclude that at moderate temperatures the enzyme is a hetero-oligomeric complex constructed from two distinct peptides with M,s of 67000 and 43 000, with an optimum temperature for reaction of 45C. Incubation of this hetero-oligomer at 7oC dissociates the subunits, resulting in a single peptide enzyme ofMr 67 000, which exhibits optimal GDH activity at 75C as well as markedly enhanced thermal stability.

Advances in immobilization attachment

and polymer

A clever new method for enzyme immobilization has been described [ZZ] that entails the generation of a fusion protein of yeast a-glucosidase with a polycationic hexaarginine fusion peptide at its carboxyl terminus. This fusion protein can then be directly adsorbed from crude cell extracts onto polyanionic matrices. After ionic adsorption onto the support, the fusion protein is stable toward thermal, pH or urea denaturation, and the immobilized enzyme exhibits normal kinetic and thermodynamic properties. Because the authors find that the operational stability of the enzyme immobilized by this method is significantly increased compared to that of the soluble form, such fusion proteins containing polyionic peptide sequences may represent a versatile approach to biocatalyst immobilization. The covalent attachment of water-soluble polymers to enzymes for the purpose of facilitating biocatalytic and biomedical applications has attracted a good deal of attention in recent years. Morpurgo et a/. [23*] report on the covalent attachment of two poly(ethy1ene glycol) (PEG) derivatives (linear, M, 5000; branched, M, 10000) and of a new polymer (poly[acryloylmorpholine], M, 5500) to the oxygenative enzyme tyrosinase. These polymers are amphiphilic, and thus the polymer-tyrosinase conjugates were examined for possible use in the bioconvertion of phenolic substrates in organic solvents, as well as for potential pharmaceutical applications. The polymer-tyrosinase conjugates were characterized as to kinetic parameters, thermostability, stability toward inactivation by quinone oxidation products, half-life in blood circulation and catalytic behaviour in organic solvents. Polymer attachment was found to have increased both enzyme stability in aqueous solution and enzyme solubility in organic solvents; however, circular dichroism measurements indicated that organic solvent solubilization had altered the conformation of the enzyme, resulting in irreversible loss of catalytic activity. It is important to note that tyrosinase is an oxidoreductase, a class of enzymes that are recognized to have outstanding biotechnological potential [24-311. Indeed, the ability of tyrosinase to oxidize phenolic moieties suggests a number of potential applications in the polymer modification area. A recent example is provided by the work of Payne et al. [32], who reported that upon treatment of thin chitosan films with tyrosinase and a phenolic reactant, the spectra

184

Biochemical engineering

and the acid-base properties of the films were markedly altered as compared to those of untreated films. Many current and proposed biotechnological applications of biocatalysis entail the utilization of lipases, because these enzymes are obtained from many sources, are relatively stable, and can accommodate a variety of organic substrates. Obviously, simple and efficient methods for lipase immobilization are highly desirable for such applications. Reetz et al. [33] report lipase immobilization by entrapment in chemically inert hydrophobic silica gels that are prepared by the hydrolysis of alkyl-substituted silanes in the presence of the enzyme using sodium fluoride as a catalyst. The authors find that this method yields immobilized lipases with esteri~~ation activities enhanced by a factor of up to 88 compared to commercial enzyme powders. Moreover, studies on the stability of immobilized lipases under reaction conditions or storage (dry or in aqueous or organic media) revealed excellent retention of enzymatic activity. Rose11 eta!. [34*] report the use of packed-bed hollow-fiber membrane reactors to carry out the lipase-catalyzed esterification of dodecanol and decanoic acid in hexane at constant water activity. The lipase was immobilized on microporous polypropylene and packed in the shell space of the reactor. Water activity control was accomplished by pumping saturated salr solutions through the microporous hollow-fiber polypropylene membranes. Water generated by reaction in the organic phase, pumped continuously through the shell of the reactor, was transferred into the bulk of the aqueous phase under the water activity gradient. The performance of the reactor was found to be strongly influenced by water activity, and, with careful control, complete esterification was achieved. The reactor could be operated continuously for 100 h with no degradation in its performance. The immobilization of acid phosphatase into a bovine serum albumin-PEG hydrogel was reported by DUrso and Fortier 135.1. The operational stability of the enzyme at 37C was increased markedly after immobilization, and the diffusional restrictions caused by the hydrogel decreased when the size of the microbeads was decreased or when the chain length of PEG was increased, resulting in an increase in the porosity of the hydrogel. New designs of macroporous polymers and supports that may find application in immobilized enzyme reactors have also been recently described [36].

been recognized that altered enzyme enantioselectivity can occur at unusual reaction temperatures and Phillips and co-workers propose a simple theoretical framework for this phenomenon. The enantiomeric ratio of products formed in an enzymatic reaction proceeding by a simple mechanism is determined by the differential free energy of activation (MGf) for the formation of the enantiomers. Since MG* =MHi-TM@, consideration of reaction rate theory allows one to define a racemic temperature, T,, at which there will be no stereochemical discrimination. At temperatures below T,, the stereochemical course of the reaction is dominated by the activation enthalpy difference (MHZ) and the stereochemical purity of the products will decrease with increasing temperature. At temperatures above T,, the stereochemistry is mainly determined by TMSS, and the stereochemical purity of the products should increase with increasing temperature. Moreover, the major products obtained at T<T, and T>T, should have opposite chirality; thus, a temperature-dependent reversal of stereochemistry is predicted. The authors find an example of just such an enantiospecificity reversal in the reaction of secondary alcohol dehydrogenase from Thermoanaerobacter ethanoficus with Z-butanol: S-Z-butanol is the preferred substrate below the T, of 26X, whereas K-Z-butanol reacts more rapidly above 26C. In contrast, because the T, for Z-pentanol is 70C the S-enantiomer is preferred at all temperatures below this value and enantiospeci~city increases as the temperature is lowered. As expected, the effect of temperature on stereochemistry is a function of both the reactivity and the binding characteristics of the particular enzyme and substrate in question. In a recent example, Kasche et a/. [40*] found that the stereoselectivity of penicillin amidase decreases by almost an order of magnitude when the temperature is increased from 5C to 45C, while stereoselectivicy increases with substrate reactivity (R,,,/Khl). An example of complex behavior is provided by the lipase-catalyzed transesterification of 3,7-dimethyl-6-octanol, where enantiospecificity increases between 15C and ZYC, decreases at 38X2, and then increases at 60C 1411. (It should also be noted that the stereochemistry of enzymatic reactions can be markedly influenced by solvent [42].) From a biotechnological perspective, Phillips [37] predicts that temperature modulation of stereochemistry will be most pronounced for esterases, dehydrogenases and lipases. These enzymes generally operate on lipophilic substrates where the MHi and TM% for enzyme-substrate interaction are likely to be closely balanced, thus giving rise to substantial variation in MG$ with temperature.

Modulation of biocatalyst stereospecificity

From the viewpoint of enzyme technology, the ability to modulate the stereochemistry of enzymatic reactions and thus produce products of desired chirality would be highly desirable. The work of Phillips and co-workers [37**,38,39] on the effect of temperature on the stereochemistry of enzymatic reactions has drawn attention to an important aspect of such stereochemical modulation. It has long

Conclusions
As biotechnology enters its third decade, it is quite evident that renewed emphasis on biocatalysis has emerged. This interest has undoubtedly been sparked by remarkable new insights into protein structure and function that have resulted from the emergence of powerful techniques for probing the dynamics and folding of macromolecules,

New applications for biocatalysts May

185

and from advances in genetic technology. During the past year, important progress has been made toward the application of biocatalysis to increasingly complex biotechnological needs. Particularly significant in this respect are the utilization of biocatalysts for the production of new or modified polymeric materials, the generation of novel biocatalysts using genetic and biochemical approaches, and the refinement of immobilization and modification technology in order to facilitate biocatalytic and biomedical applications. Future efforts will certainly focus on enhancing these achievements through the use of more sophisticated approaches for rational design and optimization, in order to produce new or improved biocatalysts for a variety of applications.

9. ..

Fernandez NM, Margot AO, Falender CA, Blanch HW, Clark DS: Enzymatic synthesis of peptides containing unnatural amino acids. Enzyme Microb Technol 1995, 17:964-971. The incorporation of modified amino acids or peptidomimetics into synthetic peptides cannot be accomplished using standard molecular biological approaches and may also be incompatible with the chemistry used in automated peptide synthesizers. Biocatalysis can make a significant contribution in this area because many proteases (as well as other lytic enzymes) are quite tolerant of secondary structural variations in substrate analogs. 10. Morita T, Yoshida N, Karube I: A novel synthesis method for cyclodextrins from maltose in water-organic solvent systems. Appl Biochem Biotechnoll996, 56:31 l-324. Monsan P, Paul F: Enzymatic synthesis of oligosaccharides. FEMS Microbial Rev 1995, 16:187-l 92. Bonnin E, Thibault JF: Galactooligosaccharide production by transfer reaction of an exogalactanase. Enzyme Microb Technol 1996, 19:99-l 06. Liu R, Orgel LE: Enzymatic synthesis of polymers containing nicotinamide mononucleotide. Nucleic Acids Res 1995, 23:3742-3749. Conrad F, Hanne A, Gaur RK, Krupp G: Enzymatic synthesis of 2-modified nucleic acids: identiication of important phosphate and ribose moieties in RNase P substrates. Nucleic Acids Res 1995, 23:1845-l 853. Zimmer DP, Crothers DM: NMR of enzymatically synthesized uniformly 1sClsN-labeled DNA. Proc Nat/ Acad Sci USA 1995, 92:3091-3095.

11.

12.

References

and recommended

reading

13.

Papers of particular interest, published within the annual period of review, have been highlighted as: 14. .
l

of special interest of outstanding interest May SW: Biocatalysis in the 90s: a perspective. Enzyme Microb Technol 1992, 14:80-84.

1.

15.

Svirkin YY, Xu J, Gross RA, Kaplan DL, Swift G: Enzymecatalyzed stereoselective ring-opening polymerization of amethyl-P-propiolactone. Macromolecules 1998, 29:4591-4597. What is noteworthy about this work is the clear evidence that optically active s-enriched product is obtained from the lipase-catalyzed polymerization of racemic substrate. Although enantioselectivity varied with reaction conditions, the results constitute a very good example of the benefits of biocatalysis in polymer synthesis. 2. . Chaudhary AK, Beckman EJ, Russell AJ: Rational control of polymer molecular weight and dispersity during enzymecatalyzed polyester synthesis in supercritical fluids. J Am Chem Sot 1995, 117~3728-3733. This is an excellent, clear-cut account of the use of supercritical fluids to control enzyme-catalyzed polymerization. Of particular note is the fact that the characterization of the molecular weight and dispersity of the polyester products was carried out very rigorously. These issues are often inadequately addressed by other investigators. 3. .. 4. Matsumura S, Beppu H, Tsukada K, Toshima K: Enzyme-catalyzed ring-opening polymerization of P-propiolactone. Biotechnol Lett 1998, 18:1041-1046. Matsumura S, Beppu H, Nakamura K, Osanai S, Toshima K: Preparation of poly(beta-malic acid) by enzymatic ring-opening polymerization of benxyl P_malolactonate. Chem Lett 1996, 795-796. Kim 0, Gross RA, Hammar WJ, Newmark RA: Microbial synthesis of poly(beta-hydroxyalkanoates) containing fluorinated sidechain substituents. Macromolecules 1996, 29:4572-4581.

Moore JC, Arnold FH: Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents. Nat Biotechnol 1998, 14~458-467. See annotation [171. 16. .. Kim YG, Cha J, Chandrasegaran S: Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Nat/ Acsd SC; USA 1996, 93:1156-l 160. These two papers [16,17**] are excellent illustrations of the potential of genetic and biochemical approaches for the generation of biocatalysts with enhanced catalytic activity or altered specificity characteristics. The authors also point out some of the challenges and unresolved problems inherent in the rational design approach. 1 7. .. 18. Rasskazov VA, Elyakova LA, Kozlovskaya EP, Sova VV, Artukov AA: Hydrolytic enzymes of marine invertebrates and prospects for their utilization. Mar Technol Sot J 1996, 30:29-34. Tsugawa W, Horuichi S, Tanaka M, Wake H, Sode K: Purification of a marine bacterial glucose dehydrogenase from Qtophaga merit~oflava and its application for measurement of 1,5anhydro-D-glucitol. Appl Biochem Biotechnoll996, 56:301-310. Fischer L, Bromann R, Kengen SWM, De Vos WM, Wagner F: Catalytical potency of f%glucosidase from the extremophile Pyrococcus furiosus in glucoconjugate synthesis. Nat Biotechnol 1996, 14:88-91.

19.

5.

20.

6.

Premachandran RS, Banerjee S, Wu XK, John Vr, McPherson GL, Akkara J. Ayyagari M, Kaplan D: Enzymatic-synthesis of fluorescent naphthol-based polymers. Macromolecules 1996, 29:6452-6460. This paper reports fluorescent polymer synthesis using horseradish peroxidase encapsulated in AOTlisooctane reversed micelles. . Kobayashi S, Okamoto E, Wen X, Shoda S: Chemical synthesis of native-type cellulose and its analogs via enzymatic polymerization. J Macromol Sci - Pure Appl Chem 1996, 33:1375-1384. In this pioneering work, synthesis of the native form of cellulose was achieved for the first time using the cellulose-hydrolyzing enzyme, cellulase, in a mixed solvent system. 8. ..

7.

Sode K, Tsugawa W, Yamazaki T, Watanabe M, Ogasawara N, Tanaka M: Novel thermostable glucose dehydrogenase varying temperature properties by altering its quaternary structures. Enzyme Microb Technol 1996, 19:82-85. This thermostable dehydrogenase isolated from a soil bacterium near a hot spring varies its temperature optimum by altering its quarternary structure. 21. . Stempfer G, Hoell-Neugebauer B, Kopetzki E, Rudolph R: A fusion protein designed for noncovalent immobilization: stability, enxymatic activity, and use in an enzyme reactor. Nat Biotechnol 1996, 14:481-484. This approach to immobilization is certainly much more laborious than the standard techniques for covalent attachment for which activated supports are commercially available; however, the advantage here is that the locus of attachment to the support is presumably confined to the ionic fusion peptide. This should help preserve the structure and activity of the immobilized biocatalyst. 22. ..

186

Biochemical

engineering

Morpurgo M, Schiavon 0, Caticeti P, Veronese FM: Covalent modification of mushroom tyrosinase with different amphiphilic polymers for pharmaceutical and biocatalysis applications. Appl Biochem Biotechnol 1996, 55:59-72. Polymer-tyrosinase conjugates, which were evaluated for possible use in the bioconversion of phenolic substrates and for potential pharmaceutical applications, are described. 23. . 24. May SW: The potential of oxidoreductase enzymes biotechnology. Nat Biotechnol 1963, 1~677-666. in

Rosell CM, Vsidya AM, Halling PJ: Continuous in situ water activity control for organic phase biocatalysis in a packed bed hollow fiber reactor. Biotechnol Bioengin 1996, 49:284-269. This is an example of how biocatalysis in solvents can be markedly influenced by water activity in an actual working reactor. DUrso EM, Fortier G: Albumin-polyfethytene glycol) hydrogel as matrix for enzyme immobilization: biochemical charactedzation of crosslinked acid phosphatase. Enzyme Microb Technoll996, l&462-400. Immobilization was found to markedly increase the operational stability of this enzyme. Svec F, Frechet JM: New designs of macroporous polymers and supports: from separation to biocatalysis. Science 1996, 273:205-211. This paper describes how polymer monoliths containing intricate pore networks can be obtained in any desired shape by a simple molding process. These supports provide advantages such as fast kinetics, high reactivity, and high throughput. Applications ranging from immobilized enzyme reactors to fast media for the separation of synthetic or biopolymers are discussed. Phillips RS: Temperature modulation of the stereochemistry of enzymatic catalysis: prospects for exploitation. Trends Biotechnoll996, 14:13-l 6. This article summarizes how temperature can influence the stereochemistry of enzymatic reactions, presents a simple theoretical framework for this phenomenon, and discusses the prospects for future applications of temperature modulation of the stereochemical course of enzymatic reactions. 36. Phillips RS, Zheng C, Pham VT, Andrade FA, Andrade MA: Effects of temperature on stereochemistry of enzymatic reactions. Biocatalysis 1994, 10:77-06. Phillips RS: Temperature effects on stereochemistry of enzymatic reactions. Enzyme Microb Technol 1992, 14:417-419. 37. .. 36. .. 35. .

34. .

25.

Katopodis AG, Smith HS, May SW: New oxyfunctionalization capabilities for o-hydroxylases: asymmetric sulfoxidation and branched ether demethylation. J Am Chem Sot 1966, 110:697-699. Sirimanne SR, May SW: Facile stereoselective hydroxylatlon by dopamine-P-monooxygenase. 1966, 110:7560-7561. allylic J Am Chem Sot

26.

27.

May SW, Katopodis AG: Hydrocarbon monooxygenase system of Pseudomonas olaovorans. Methods Enzymol 1990, 188:3-g. Bes MT, Villa R, Roberts SM, Wan PWH, Willetts A: Oxidative biotransformattons by microorganisms: production of chiral synthons by cyclopentanone monooxygenase from Pseudomonas sp. NCIMB 9672. J Mel Catel B - Enzym 1996, 1:127-134. Kato Y, Yamada H, Asano T: Stereoselective synthesis of opinetype secondary amine carboxylic acids by a new enzyme opine dehydrogenase: use of recombinant enzymes. J MO/ Catal B - Enzym 1996, 1:151-160. Stachyra T, Guillochon D, Pulvin S, Thomas D: Hemoglobin, horseradish peroxidase, and heme-bovine serum albumin as biocatalysts for the oxidation of dibenzothiophene. Appl Biochem Biotechnol 1996, 59:231-244. Genet R, Benetti PH, Hammadi A, Menez A: t-tryptophan 2,3-oxidase from Chromobactarium violeceum. Substrate specificity and mechanistic implications. J Biol Chem 1995, 270:23540-23545. Payne GF, Chaubal MV, Barbari TA: Enzyme-catalyzed polymer modification reaction of phenolic compounds with chitosan films. Polymer 1996, 37:4643-4646. Reetz MT, Zonta A, Simpelkamp J: Efficient immobilization of lipases by entrapment in hyrophobic sol-gel materials. Biotechnol Bioengin 1996, 49:527-534.

26.

29.

39.

30.

31.

Kasche V, Galunsky B, Murk A, Piotraschke E, Rieks A: The dependency of the stereoselectivity of penicillin amidase enzymes with R-specific S-l -subsite and S-specific 5 (I)subsite on temperature and primary structure. Biotechnol Lett 1996, l&455-460. This paper reports that the stereoselectivity of penicillin amidase decreases by almost an order of magnitude as the temperature is increased from 5C to 45C, whereas stereoselectivity increases with substrate reactivity. 41. Parmar VS, Prssad AK, Singh PK, Gupta S: Lipase-catalyzed transesterifications using 2,2,2+rifluoroethyl butyrate, effect of temperature on rate of reaction and enanttoselectivity. Tetrahedron - Asymmetry 1992, 3:1395-l 396. Terradas F, Testonhenry M, Fitzpatrick PA, Klibanov AM: Marked dependence of enzyme prochiral selectivity on the solvent J Am Chem Sot 1993,115:390-396.

40. .

32.

33.

42.