Biocatalysis

Tyler JohannesMichael R. SimurdiakHuimin ZhaoDepartment of Chemical and Biomolecular Engineering, University of Illinois,Urbana, Illinois, U.S.A.

INTRODUCTION

Biocatalysis may be broadly dened as the use ofenzymes or whole cells as biocatalysts for industrialsynthetic chemistry. They have been used for hundredsof years in the production of alcohol via fermentation,and cheese via enzymatic breakdown of milk proteins.Over the past few decades, major advances in ourunderstanding of the protein structurefunction relation-ship have increased the range of available biocatalyticapplications. In particular, new developments in proteindesign tools such as rational design and directedevolution have enabled scientists to rapidly tailorthe properties of biocatalysts for particular chemicalprocesses. Rational design involves rational alterationsof selected residues in a protein to cause predictedchanges in function, whereas directed evolution, some-times called irrational design, mimics the naturalevolution process in the laboratory and involvesrepeated cycles of generating a library of different pro-tein variants and selecting the variants with the desiredfunctions (see the entry Protein Design). Enzymeproperties such as stability, activity, selectivity, andsubstrate specicity can now be routinely engineered inthe laboratory. Presently, approximately 100 differentbiocatalytic processes are implemented in pharma-ceutical, chemical, agricultural, and food industries.[1]

The products range from research chemicals to com-modity chemicals and the number of applications con-tinue to grow very rapidly. In spite of these successes,however, the vast potential of biocatalysis has yet tobe fully realized.

In this entry, we briey outline the scope of biocata-lysis and discuss its advantages and disadvantages ascompared to chemical catalysis. We then review suchtopics as enzyme and whole-cell based biocatalysis,biocatalysts used in nonaqueous media, biocatalystimmobilization, discovery and engineering of novelenzymes, and hybrid approaches combining chemicaland biological synthesis. An overview of the six generalclassications of enzymes along with their relative usein industry is discussed. Selected industrial applicationsof whole-cell based biocatalysis including the produc-tion of lactic acid and 1,3-propanediol are also studied.

THE SCOPE OF BIOCATALYSIS

Advantages and Disadvantages of Biocatalysisvs. Chemical Catalysis

Similar to other catalysts, biocatalysts increase thespeed in which a reaction takes place but do not affectthe thermodynamics of the reaction. However, theyoffer some unique characteristics over conventionalcatalysts (Table 1). The most important advantage ofa biocatalyst is its high selectivity. This selectivityis often chiral (i.e., stereo-selectivity), positional(i.e., regio-selectivity), and functional group specic(i.e., chemo-selectivity). Such high selectivity is verydesirable in chemical synthesis as it may offer severalbenets such as reduced or no use of protectinggroups, minimized side reactions, easier separation,and fewer environmental problems. Other advantages,like high catalytic efciency and mild operationalconditions, are also very attractive in commercialapplications.

The characteristics of limited operating regions,substrate or product inhibition, and reactions inaqueous solutions have often been considered as themost serious drawbacks of biocatalysts. Many of thesedrawbacks, however, turn out to be misconceptionsand prejudices.[2,3] For example, many commerciallyused enzymes show excellent stability with half-livesof months or even years under process conditions. Inaddition, there is an enzyme-catalyzed reaction equiva-lent to almost every type of known organic reaction.Many enzymes can accept non-natural substrates andconvert them into desired products. More importantly,almost all of the biocatalyst characteristics can betailored with protein engineering and metabolicengineering methods (refer to the section BiocatalystEngineering and see also the entry Protein Design)to meet the desired process conditions.

Biocatalytic processes are similar to conventionalchemical processes in many ways. However, whenconsidering a biocatalytic process one must accountfor enzyme reaction kinetics and enzyme stabilityfor single-step reactions, or metabolic pathways formultiple-step reactions.[4] Fig. 1 shows the key steps

Encyclopedia of Chemical Processing DOI: 10.1081/E-ECHP-120017565Copyright # 2006 by Taylor & Francis. All rights reserved. 101

in the development of a biocatalytic process. Itusually starts with the identication of a targetreaction, followed by biocatalyst discovery, character-ization, engineering, and process modeling. In manycases, biocatalyst engineering is the most time-consuming step, often involving two major ap-proaches: rational design and directed evolution. Inaddition to biocatalyst development, product isolationis an important step. The overall process economicsdepends on all these factors, which needs to bedemonstrated in a pilot-scale plant before scale-up.Biocatalysts can constitute a signicant portion ofthe operating budget; however, their cost can bereduced by reusing them when immobilized (refer tothe section Biocatalyst Immobilization).

Enzyme Based Biocatalysis vs. Whole-CellBiocatalysis

Both isolated enzymes and whole cells can be used asbiocatalysts. Compared to whole cells, isolatedenzymes offer several benets, including simpler reac-tion apparatus, higher productivity owing to highercatalyst concentration, and simpler product purica-tion.[2] Until recently, only enzymes that were abun-dantly produced by cells could be used in industrialapplications. Now it is possible to produce largeamounts of an enzyme through the use of recombinantDNA technology. In brief, the DNA sequenceencoding a given enzyme is cloned into an expressionvector and transferred into a production host such as

Table 1 Advantages and disadvantages of biocatalysis in comparison with chemical catalysis

Advantages Disadvantages

Generally more efcient (lower concentration of enzyme needed) Susceptible to substrate or product inhibition

Can be modied to increase selectivity, stability, and activity Solvent usually water (high boiling point and heatof vaporization)

More selective (types of selectivity: chemo-selectivity,regio-selectivity, diastereo-selectivity, and enantio-selectivity)

Enzymes found in nature in only one enantiomeric form

Milder reaction conditions (typically in a pH range of 58 and

temperature range of 2040C)Limiting operating region (enzymes typically denatured

at high temperature and pH)

Environment friendly (completely degraded in the environment) Enzymes can cause allergic reactions

(From Ref.[2].)

Fig. 1 Flowchart of the develop-ment of a biocatalytic process.

102 Biocatalysis

Escherichia coli or Saccharomyces cerevisiae for geneexpression. The overexpressed enzymes are puriedfrom the cell extracts based on their chemical andphysical properties. The most commonly used enzymepurication techniques include electrophoresis, centri-fugation, and chromatography. Centrifugation sepa-rates enzymes based on their differences in mass orshape, whereas electrophoresis separates enzymesbased on their differences in charge. Liquid chromatog-raphy separates enzymes based on their differencesin charge (ion-exchange chromatography), in mass(gel ltration chromatography), or in ligand-bindingproperty (afnity chromatography).

The whole-cell biocatalysis approach is typicallyused when a specic biotransformation requires multi-ple enzymes or when it is difcult to isolate the enzyme.A whole-cell system has an advantage over isolatedenzymes in that it is not necessary to recycle the cofac-tors (nonprotein components involved in enzyme catal-ysis). In addition, it can carry out selective synthesisusing cheap and abundant raw materials such ascornstarches. However, whole-cell systems requireexpensive equipment and tedious work-up becauseof large volumes, and have low productivity. Moreimportantly, uncontrolled metabolic processes mayresult in undesirable side reactions during cell growth.The accumulation of these undesirable products as wellas desirable products may be toxic to the cell, and theseproducts can be difcult to separate from the rest ofthe cell culture. Another drawback to whole-cellsystems is that the cell membrane may act as a masstransport barrier between the substrates and theenzymes.

Nonaqueous Biocatalysis

Historically, enzymes have been used extensively inaqueous media. Enzymes are well suited to theirnatural aqueous environment; however, biotransfor-mations in industrial synthesis often involve organicmolecules insoluble in water. More importantly,because of its high boiling point and high heat ofvaporization, water is usually the least desired solventof choice for most organic reactions. Thus, shiftingenzymatic reactions from an aqueous to an organicmedium is highly desired.

Over the past 15 yr, studies have shown thatenzymes can work in organic solvents.[5] However,the enzymatic activity is quite low in an organic solventcompared to that in water. Recent advances in proteinengineering and directed evolution have aided in thedevelopment of enzymes that show improved activityin organic solvents. Progress has also been made indeveloping simple, scalable, and low-cost techniques toproduce highly active biocatalyst preparations for use

in organic solvents.[6] One such method improvesenzyme activity in organic solvents by lyophilizing(freeze-drying) an aqueous biocatalyst solution in thepresence of organic and inorganic molecules called excip-ients. These excipients include nonbuffer salts, crownethers, cyclodextrins, and solid-state buffers.[7] Someremarkable results have also been achieved by usingionic liquids as solvents in biocatalytic reactions.[8]

Biocatalyst Immobilization

Immobilization is the process of adhering biocatalysts(isolated enzymes or whole cells) to a solid support.The solid support can be an organic or inorganic mate-rial, such as derivatized cellulose or glass, ceramics,metallic oxides, and a membrane. Immobilized bioca-talysts offer several potential advantages over solublebiocatalysts, such as easier separation of the biocata-lysts from the products, higher stability of the biocata-lyst, and more exible reactor congurations. Inaddition, there is no need for continuous replacementof the biocatalysts. As a result, immobilized biocata-lysts are now employed in many biocatalytic processes.

More than one hundred techniques for immobiliz-ing enzymes have been developed which can be dividedinto ve major groups summarized in Table 2.[9]

Adsorption of the enzyme onto a surface is the easiestand the oldest method of immobilization. Entrapmentand cross-linking tend to be more laborious enzymexation methods, but they do not require altering theenzyme as much as other techniques. The formationof the covalent linkage often requires harsh conditions,which can result in a loss of activity because of confor-mational changes of the enzyme. It is important to notethat most of these techniques can also be used toimmobilize whole cells. In addition, although thesetypes of immobilization are considered to be relativelyold and well established, the emerging eld of nano-tube biotechnology has created another possible meansof immobilizing biocatalysts.[10]

Biocatalyst Discovery: Sourcesand Techniques

Traditionally, potentially commercial enzymes areidentied by screening micro-organisms, which arefrequently isolated from extreme environments, forbiocatalytic activity. Commercial enzymes are selectedby probing libraries of related enzymes for a range ofproperties, including activity, substrate specicity, sta-bility over a temperature range, enantio-selectivity, orcompatibility under various physical and chemicalconditions. Unfortunately, most of the commerciallyviable enzymes have been isolated in only a few micro-bial species such as Bacillus and Pseudomonas because

Biocatalysis 103

of the limitations in micro-organism cultivation tech-niques.[11] It has been widely acknowledged that themajority of microbial species (up to 99%) have neverbeen cultivated and thus have never been investigated.To access this vast untapped microbial diversity,several companies such as Diversa Corporation(San Diego, California, U.S.A.) and TerraGen Dis-covery (Vancouver, British Columbia, Canada) havesuccessfully developed modern bioprospecting tech-niques such as multiple metagenome cloning to isolatenovel industrial enzymes.[12]

New methods for exploring natural biodiversityhave been greatly facilitated by high-throughputscreening technologies and robust expression in recom-binant organisms. Recombinant DNA technologymakes it possible to produce enzymes at levels 100-foldgreater than native expression and allows expressionof genes from organisms that cannot be cultured.Although some problems may be resolved by screeninglarger libraries of DNA, this may not be the mostefcient or expedient method of obtaining a viablebiocatalyst. A more efcient means of obtaining agood biocatalyst may involve engineering the catalystitself using various protein engineering and metabolicengineering techniques (refer to the section BiocatalystEngineering and see also the entry Protein Design).

Biocatalyst Engineering

Nature has supplied us with a vast array of biocatalystscapable of catalyzing numerous biological reactions.Unfortunately, naturally occurring biocatalysts are oftennot optimal for many specic industrial applications,

such as low stability and activity. Moreover, naturallyoccurring biocatalysts may not catalyze the reactionwith the desired non-natural substrates or producethe desired products. To address these limitations,molecular techniques have been developed to createimproved or novel biocatalysts with altered industrialoperating parameters. It should be noted that, forenzyme based biocatalysts, many molecular techniqueshave been developed for engineering enzymes withnovel or improved characteristics. Readers are referredto the entry Protein Design. In this section, wemainly discuss the molecular techniques used forwhole-cell based biocatalyst engineering, or metabolicengineering.

Metabolic engineering is a rapidly growing areawith great potential to impact biocatalysis.[13] It hasbeen broadly dened as the directed improvementof product formation or cellular properties throughmodications of specic biochemical reaction(s) orthe introduction of new one(s) with the use of recombi-nant DNA technology.[14] In an industrial context,the ultimate goal of metabolic engineering is the devel-opment of optimal biocatalysts. In the past twodecades, metabolic engineering has been successfullyused to engineer micro-organisms to produce awide variety of products, including polymers, aro-matics, carbohydrates, organic solvents, proteins, anti-biotics, amino acids, and organic acids. According tothe approach taken or the aim, these applicationscan be classied into seven groups: 1) expression ofheterologous genes for protein production; 2) exten-sion of the range of substrate for cell growth andproduct formation; 3) design and construction of path-ways for the production of new products; 4) design and

Table 2 Methods of enzyme immobilization

A. Covalent attachmentIsolated enzymes usually attached through amino or carboxyl groups to a solid supportVariety of supports such as porous glass, cellulose, ceramics, metallic oxides

B. Adsorption

Ion-exchangers frequently used in industry because of simplicityIndustrial applications include anion-exchangers diethylaminoethyl cellulose (DEAE-cellulose) and the cation-exchangercarboxymethyl cellulose (CM-cellulose)

C. Entrapment in polymeric gels

Enzyme becomes trapped in gel volume by changing temperature or adding gel-inducing chemicalEnzymes may be covalently bound to gel (for instance, polyacrylamide cross-linked with N,N 0-methylenebisacrylamide)or noncovalently linked (calcium alginate)

D. Intermolecular cross-linkingEnzyme cross-linked with bifunctional reagents

Popular cross-linkers are glutaraldehyde, dimethyl adipimidate, dimethyl suberimidate, and aliphatic diamines

E. EncapsulationEnzymes enveloped in semipermeable membrane, which allows low molecular weight substrates and products to passthrough the membrane

Enclosed in a variety of devices: hollow bers, cloth bers, microcapsules, lm

(From Ref.[9].)

104 Biocatalysis

construction of pathways for degradation of xenobio-tics; 5) engineering of cellular physiology for processimprovement; 6) elimination or reduction of by-product formation; and 7) improvement of yield orproductivity.[15] Several of these applications have beenimplemented at industrial-production scale (refer tothe section Industrial Applications of Whole-CellBased Biocatalysis) and the number of applicationsshould continue to grow. In particular, with the recentadvances in genomics, proteomics, and bioinformatics,many new genes and pathways will be discovered andthe regulation of metabolic network will also be betterunderstood, all of which will accelerate the develop-ment of more commercially viable bioprocessesthrough metabolic engineering.

Hybrid Approaches Combining ChemicalSynthesis and Biocatalysis

Biocatalysts exhibit exquisite catalytic efciency that isoften unmatched by conventional catalysis. Nonethe-less, conventional organic synthesis will likely remainthe staple of the chemical and pharmaceutical indus-tries. In the future, the integration of these twoapproaches will probably offer the optimal route forindustrial synthesis. An illustration of this principlecan be found in the selective deprotection of reactivefunctional groups. Enzymes are unique deprotectingtools for combinatorial synthesis because of theirremarkable selectivity and ability to operate undermild reaction conditions. A recent example is thesynthesis of long multiply lipidated peptides containingvarious side-chain functional groups.[16] In this study,penicillin acylase was used for selective N-deprotectionof a highly labile S-palmitoylated oligopeptide. Afterremoval of the protecting group, the S-palmitoylatedoligopeptide was used as a building block in furthersynthetic steps.

INDUSTRIAL APPLICATIONS OF ENZYMEBASED BIOCATALYSIS

With the rapid technical developments in gene discov-ery, optimization, and characterization, enzymes havebeen increasingly used as biocatalysts. According tothe International Union of Biochemistry and Mole-cular Biology (IUBMB) nomenclature system, allenzymes are classied into six classes on the basis ofthe general type of reactions that they catalyze(Table 3). Within each class are subclasses and theenzymes themselves. The result is an ordered systemof enzymes and the reaction(s) that each catalyzes. Itis important to note that, in biological processes, everyclass of enzyme is utilized in the cell to a large extent.However, this is not the same in industrial processes,where certain classes of enzymes are used more oftenthan others. As shown in Fig. 2, most of the enzymesthat have been used as biocatalysts in industry arehydrolases (65%), even though oxidoreductasesare typically much more useful than hydrolases ascatalysts. The utility of an enzyme class depends onthe relative commercial importance of the productsthat each enzyme produces, the accessibility of theenzymes, and the specic characteristics of the enzymes(e.g., stability, activity, and selectivity).

Oxidoreductases

Oxidoreductases catalyze oxidation and reductionreactions that occur within the cell. They are veryappealing for industrial uses because of the reactionsthat they are able to catalyze. However, they oftenneed expensive cofactors such as nicotinamide adeninedinucleotides (e.g., NAD=NADH) and avines (e.g.,FAD=FADH2) in the reactions. In fact, nicotinamideadenine dinucleotides are required by about 80%of oxidoreductases. Fortunately, several NAD(H)

Table 3 Classication of enzymes

Enzymes Type of reactions Representative subclasses

Oxidoreductases Catalyze the transfer of hydrogen or oxygenatoms or electrons from one

substrate to another

Oxidases, oxygenases, peroxidase, dehydrogenases

Transferases Catalyze the group transfer reactions Glycosyltransferases, transketolases, methyltransferases,transaldolases, acyltransferases, transaminases

Hydrolases Catalyze hydrolytic reactions Esterases, lipases, proteases, glycosidases, phosphatases

Lyases Catalyze the nonhydrolytic removalof groups

Decarboxylases, aldolases, ketolases, hydratases,dehydratases

Isomerases Catalyze isomerization reactions Racemases, epimerases, isomerases

Ligases Catalyze the synthesis of various types

of bonds with the aid of energy-containingmolecules

Synthetases, carboxylases

Biocatalysis 105

regeneration systems have been developed, the mostwidely used being the formate=formate dehydrogenase(FDH) system.[17]

An example of a pharmaceutical synthesis reactioninvolving an oxidoreductase is the synthesis of3,4-dihydroxylphenyl alanine (DOPA).[2] 3,4-Dihy-droxylphenyl alanine is a chemical used in the treat-ment of Parkinsons disease. The industrial processthat synthesizes DOPA utilizes the oxidoreductasepolyphenol oxidase. As shown in Fig. 3, the monohy-droxy compound is oxidized by the regio-specicaddition of a hydroxyl group. It is worth mentioningthat epinephrine (adrenaline) can also be synthesizedby a similar reaction path using the same enzyme.[2]

Another example is the use of leucine dehydrogen-ase coupled with FDH for the reductive amination oftrimethylpyruvate to L-tert-leucine (Fig. 4). The whole

process is carried out in a membrane reactor in whichthe cofactor NAD is regenerated by FDH. Thisprocess has now reached ton-scale production atDegussa (Germany).[18]

Transferases

Transferases catalyze the transfer of functional groupssuch as methyl, hydroxymethyl, formal, glycosyl, acyl,alkyl, phosphate, and sulfate groups by means of anucleophilic substitution reaction. They are not widelyused in industrial processes; however, there are a fewexamples of industrial processes that utilize transferases.

A classical example of industrial application oftransferases is the use of various glycosyltransferasesfor the synthesis of oligosaccharides. Oligosaccharidesand polysaccharides are important classes of naturallyoccurring compounds, which play vital roles in cellularrecognition and communication processes.[19] Becauseof the required use of many protection and deprotec-tion groups, chemical synthesis of complex oligosac-charides represents a daunting challenge in syntheticorganic chemistry. By contrast, enzymatic synthesisof oligosaccharides by glycosyltransferases requiresvery few protection and deprotection steps because ofthe high regio- and stereoselectivity of glycosyltrans-ferases, thus offering an attractive alternative.[2]

Another example is the use of a glucokinase (a trans-ferase) in combination with an acetate kinase forthe production of glucose-6-phosphate (Fig. 5). Thisprocess is carried out in multikilogram scale by theJapanese company Unitika.[1]

Fig. 2 The relative use of enzyme classes in industry. (FromRef.[2].)

Fig. 3 Enzymatic synthesis of 3,4-dihydroxylphenyl alanine(DOPA).

Fig. 4 Enzymatic synthesis of L-tert-leucine.

Fig. 5 Enzymatic synthesis of glucose-6-phosphate.

106 Biocatalysis

Hydrolases

Hydrolases catalyze the addition of water to a sub-strate by means of a nucleophilic substitution reaction.Hydrolases (hydrolytic enzymes) are the biocatalystsmost commonly used in organic synthesis. They havebeen used to produce intermediates for pharmaceuti-cals and pesticides, and chiral synthons for asymmetricsynthesis. Of particular interest among hydrolasesare amidases, proteases, esterases, and lipases. Theseenzymes catalyze the hydrolysis and formation of esterand amide bonds.

Lipases can hydrolyze triglycerides into fatty acidsand glycerol. They have been used extensively toproduce optically active alcohols, acids, esters, andlactones by kinetic resolution. Lipases are unique, inthat they are usually used in two-phase systems. Aclassic example is the use of a lipase for the productionof (S,R)-2,3-p-methoxyphenylglycyclic acid, an inter-mediate for diltiazem. In this process, methyl-p-methoxyphenylglycidate is stereospecically hydro-lyzed by a lipase immobilized in a hollow bermembrane reactor. The enzyme is located at the inter-facial layer between an organic and an aqueousphase.[1]

Proteases such as a-chymotrypsin, papain, andsubtilisin are also useful biocatalysts for regio-selectiveor stereoselective hydrolytic biotransformations. Forexample, dibenzyl esters of aspartic and glutamic acidcan be selectively deprotected at the 1-position bysubtilisin-catalyzed hydrolysis (Fig. 6).[2] In addition,a-chymotrypsin is used in the kinetic resolution ofa-nitro-a-methyl carboxylates, which results in L-congured enantiomers of the unhydrolyzed esterswith high optical purity (>95% e.e.).[2]

Lyases

Lyases are the enzymes responsible for catalyzingaddition and elimination reactions. Lyase-catalyzedreactions involve the breaking of a bond between acarbon atom and another atom such as oxygen, sulfur,or another carbon atom. They are found in cellularprocesses, such as the citric acid cycle, and in organicsynthesis, such as in the production of cyanohydrins.[2]

Several industrial processes using lyases as catalystshave been reported. Perhaps the most prominent lyase-catalyzed process is the production of acrylamide fromacrylnitrile. This process is carried out by the NittoChemical Company of Japan at a scale of more than40,000 tons per year.[20] Another example is the use ofa fumarase for the production of (S)-malic acid fromfumaric acid. As shown in Fig. 7, a water molecule isadded to the double bond in fumarate by means ofan addition reaction. The result is a cleavage of thecarboncarbon double bond, and a formation of anew carbonoxygen bond. A third example is bio-catalytic production of a cyanohydrin from a ketone.This reaction is catalyzed by a lyase called oxynitrilase.It consists of the cleavage of one carbonoxygen bond,and the addition of a HCN molecule. The chirality ofthe product is based on the form of the enzyme used(R-oxynitrilase or S-oxynitrilase).[2]

Isomerases

Isomerases catalyze isomerization reactions such asracemization and epimerization. They have not beenused in many industrial applications. However, oneof the most successful enzyme based biocatalytic pro-cesses involves an isomerase: the use of glucose isomer-ase for the production of high-fructose corn syrup(HFCS) (Fig. 8). High-fructose corn syrup is used asan alternative sweetener to sucrose in the food andbeverage industry. The isomerization of glucose toHFCS on an industrial scale is carried out in continu-ous xed-bed reactors using immobilized glucoseisomerases. The total amount of HFCS produced byglucose isomerase exceeds a million tons per year.[21]

Ligases

Ligases catalyze reactions that involve the creation ofchemical bonds with nucleotide triphosphates. They

Fig. 6 Regio-selective ester-hydrolysis catalyzed by subtilisin.

Fig. 7 Enzymatic synthesis of (S)-malic acid.

Fig. 8 Enzymatic synthesis of high-fructose corn syrup.

Biocatalysis 107

are important in certain cellular processes, such as con-necting nucleotides in DNA replication. However,similar to isomerases, ligases have very few industrialapplications.[2] It is important to note that DNAligases are essential tools in recombinant DNA tech-nology and are used almost in every biology-relatedlaboratory.

INDUSTRIAL APPLICATIONS OF WHOLE-CELLBASED BIOCATALYSIS

Whole-cell based biocatalysis utilizes an entire micro-organism for the production of the desired product.One of the oldest examples for industrial applicationsof whole-cell biocatalysis is the production of aceticacid from ethanol with an immobilized Acetobacterstrain, which was developed nearly 200 yr ago.[1] Thekey advantage of whole-cell biocatalysis is the abilityto use cheap and abundant raw materials and catalyzemultistep reactions. Recent advances in metabolicengineering have brought a renaissance to whole-cellbiocatalysis. In the following sections, two novel indus-trial processes that utilize whole-cell biocatalysis arediscussed with emphasis on the important role playedby metabolic engineering.

Lactic Acid

L-lactic acid has long been used as a food additive andhas recently received great attention because it can beused as an important feedstock for the production ofother chemicals such as polylactic acid (PLA), acetal-dehyde, polypropylene glycol, acrylic acid, and penta-dione.[22] Among them, PLA is the most importantproduct as it can be used to manufacture thermo-formed containers, packaging, nonwovens, paper-coated articles, and lm products.[23] Lactic acid canbe produced from sucrose, whey (lactose), and maltoseor dextrose from hydrolyzed starch using Lactoba-cillus strains.

Compared to other polymeric materials such aspolyethylenes, polylactic acid has several advantagesincluding an increased hydrophilicity, resistance toultraviolet light, ability to be dyed with dispersiondyes, a range of melting temperatures between 120Cand 170C, and low ammability and smoke genera-tion. Most importantly, polylactic acid is biodegrad-able and is derived from renewable resources,utilizing energy from the sun and lowering the fossilfuel dependence for production.[23]

In recognition of the superior properties and thehuge potential market of polylactic acid, Cargill Inc.and The Dow Chemical Company started a jointventure Cargill Dow LLC to produce lactic acid using

fermentation (whole-cell biocatalysis) several yearsago. A production plant was built in Blair, NE, in2001 and it now produces 140,000 metric tons of poly-lactic acid per year. It is predicted that the eventualcost of polylactic acid will be between $0.50 and$0.75 per pound (http:==www.cargilldow.com).

One of the drawbacks in the current commercialfermentation process is that the predominant form ofthe product is the deprotonated lactate rather thanlactic acid, requiring more expensive and wastefulproduct purication steps. This is because the Lacto-bacillus fermentation operates at a minimum pH of5.05.5 which is above the pKa of lactic acid (3.87).To overcome this limitation, a powerful strainimprovement method, genome shufing, was used toimprove the acid tolerance of a poorly characterizedindustrial strain of Lactobacillus.[24] A population ofstrains with subtle improvement in pH tolerance wasisolated using classical strain improvement methodssuch as chemostats, and were then shufed by recursivepool-wise protoplast fusion to createmutant strains thatgrow at substantially lower pH than does the wild-typestrain.

1,3-Propanediol

1,3-Propanediol is an intermediate that is widely usedin the synthesis of polyesters and polyurethanes. Poly-mers based on 1,3-propanediol are very useful in thecarpet and textile industry because of their good lightstability and biodegradability.[25] The conventionalmethods for producing 1,3-propanediol rely on petro-leum derivatives and are quite capital intensive and=orgenerate waste streams containing environmental pol-lutants. Thus, the use of micro-organisms to produce1,3-propanediol from glucose represents an attractivealternative.

Both the biological production of 1,3-propanediolfrom glycerol and that of glycerol from glucose havebeen known for many years.[13] However, there is nosingle micro-organism that could convert basic carbonsources such as glucose to the desired 1,3-propanediolend-product. Such a micro-organism is highly desiredin the process as it requires less energy input and usesan inexpensive starting material.

A team of researchers from DuPont and Genencorhas successfully used metabolic engineering techniquesto engineer such a micro-organism. The conversion ofglucose to 1,3-propanediol requires the combination oftwo natural pathways: glucose to glycerol and glycerolto 1,3-propanediol. The best natural pathway for theproduction of glycerol from glucose was found in theyeast Saccharomyces cerevisiae, which consists of twoenzymes: dihydroxyacetone-3-phosphate dehydrogenaseand glycerol-3-phosphate phosphatase. The best natural

108 Biocatalysis

pathway for production of 1,3-propanediol fromglycerol was found in Klebsiella pneumoniae, whichconsists of glycerol dehydratase and 1,3-propanedioldehydrogenase. The genes encoding these two naturalpathways were cloned and expressed in E. coli. E. coliwas chosen as the production strain because it has beenused in large-scale production on an industrial level, ithas many genetic tools, and its metabolism and physiol-ogy are well characterized. This engineered E. coli wasfound to produce over 120g=L of 1,3-propanediol in40hr fed-batch fermentation.[13]

CONCLUSIONS

Biocatalysis has become an important tool for indus-trial chemical synthesis and is on the verge of signi-cant growth. In the past several decades, manybiocatalytic processes have been implemented to pro-duce a wide variety of products in various industries.Most of them use naturally occurring enzymes ormicro-organisms as catalysts. With the help of innova-tive biocatalyst discovery methods and advances inprotein engineering and metabolic engineering, thetime and cost of developing new biocatalysts can bereduced signicantly. Most importantly, the bio-catalysts can be readily tailored to their specic appli-cations and process conditions through proteinengineering and metabolic engineering. It is possiblethat in the future they can be rationally designed toact specically in any chemical reaction of interest,fullling the holy grail of catalysis: catalysis by design.In addition, the use of biocatalysts in organic solventsin combination with the integration of biocatalysis andchemical catalysis will continue to broaden the scopeof the applications of biocatalysts. New advances ingenomics, proteomics, and bioinformatics will fuelthe development of biocatalysis as an integral part ofindustrial catalysis.

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