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Industrial enzyme technology

A recent two-day meeting* was designed to bring together academics and industrialists in the field of enzyme technology. According to the advertising brochures, the con- ference was targeted at ‘Cutting- edge technologies for a competitive advantage through novel, environ- mentally safe, cost-effective appli- cations’. Inasmuch as industry is prepared to reveal its latest develop- ments, this objective was satis- factorily achieved. However, as a means of technology transfer be- tween academia and industry, the conference was less successful: the relatively few academic research pres- entations may indicate that costs of registration have deterred aca- demic participants. Support from the major European and US biotechnol- ogy companies active in this area was substantial, with more than 80% of the 100 participants being industrial representatives, including researchers and senior management personnel. The atmosphere of serious science (or at least the serious prospect of making money from enzyme appli- cations) was not noticeably affected by the proximity of the venue to the DisneyWorld complex, nor by the prevalence of Mickey Mouse imagery (a telephone in the form of a foot- high Mickey Mouse can be a dis- turbing sight at 6 a.m.)!

The current state of the enzyme industry

The introductory presentation on the current status of industrial enzymes by Elisabeth Hefti (Biotech Business Link, East Brunswick, NJ, USA) effectively set the scene. With cautious optimism, she predicted that the current global enzyme market of US$1.4 billion will rise by over lo”/0 per annum in sales volume, with a value in- crease of 4-j% per annum. The apparent discrepancy between these figures is attributed to the tendency of successful industrial enzymes to acquire commodity status rapidly, where prices and profit margins shrink as a result of increasing competition.

*The meeting ‘Exploiting Enzyme Tech-

nology for Industrial Applicationr’ was organ- ized by the IBC, and was held at Lake Buena

Vista, FL, USA, 26-27 February 1996.

While the detergent, food and starch processing industries still ac- count for -75% of industrial enzyme usage (mostly hydrolases such as pro- teases, amylases, lipases and cellu- lases), it is a sign of the developing sophistication of the industry that over 60% of such enzymes now used are recombinant products. A signifi- cant proportion of these have been modified by site-directed or random mutagenesis to enhance one or more functional properties.

Hiding among these global figures is the growing industrial application of non-hydrolytic (often high-speci- ficity), ‘speciality’ enzymes. Such enzymes now account for 10% ofthe market, and are finding increasing application in the development of diagnostics and analytical technol- ogies, as well as in the pharmaceuti- cal industry. ‘Added value’ is possible through an enzyme’s specificity in the synthesis and resolution of ra- cemic intermediates, and the conse- quent cost-savings compared with conventional chemical routes of production (Jay Shetty; Solvay Enzymes, Elkhart, IN, USA). Illus- trating these points with diverse examples, including the use of oxido-reductases for the synthesis of gluconic acid, fumarase for the syn- thesis of L-malic acid, and fi-uctosyl transferase for the synthesis of fi-ucto- syl polymer, Shetty focused on the use ofglucosyl transferases for produc- ing a new class of sugars - isomalto- oligosaccharides (IMOs). IMOs have potential commercial applications in the food and health industries as non- fermentable carbohydrate (bulking agent), anti-cariogenic agent, and antistaling agent (in baking).

Specialist enzymes in industrial enzymology

Some of the most interesting pres- entations dealt with developments in the industrial use of high-specificity enzymes. John Sih (Pharmacia- UpJohn, Kalamazoo, MI, USA) re- viewed the steps in the scale-up of a lipase-catalysed chiral resolution of an intermediate in the synthesis of the anticancer drug Camptosar. Surpris- ingly, the key to high yields of pure chiral product lay in the immobiliz- ation technology, providing a reminder of the empiricism that is inherent in many areas of applied biocatalysis.

The issue of biocatalyst stability (or lack of it) was addressed by Alexey Margolin (Altus Biologics, Cambridge, MA, USA), who pre- sented a comprehensive review of the functional advantages of Cross- Linked Enzyme Crystals (CLECs), whereby enzyme microcrystals grown from aqueous solution are cross- linked with a bifunctional reagent such as glutaraldehyde. The advan- tages of CLECs (extremely high specific-activities, enhanced enzyme solvent and temperature stability) would seem to be counter-balanced by the complexity of developing reli- able large-scale crystallization tech- nology. However, success with a number of different enzymes was reported.

The development of enzymatic methods for the production of gly- oxylic acid and glyphosate (David Anton; E. I. DuPont de Nemours, Wilmington, DE, USA) was an excellent example of the power- ful combination of good enzymol- ogy, chemistry and genetics. The successful process, which utilizes a recombinant glycolate oxidase

hyper-expressed in Pichia pnstorir

together with some simple Schiff’s base chemistry, ingeniously over- comes serious problems of yield, unwanted side reactions and catalyst cost.

Recent developments in the traditional uses of industrial enzymes

Nevertheless, a high proportion of the two-day programme dealt with the traditional applications of enzymes in industry. The unequal balance between the traditional uses of hydrolases and more developed specialized enzyme processes was emphasized in a series of presen- tations from representatives of some of the major biotechnology compa- nies. Kathleen Clarkson (Genencor International, South San Francisco, CA, USA) reported on the current and developing uses of cellulase- complex enzymes in the textile industry - modern stone-washing of jeans is biological, not geological! Despite the apparent simplicity of this enzyme technology, the cloning of the individual components of the Ttichoderma cellulase complex (endo- glucanases, cellobiohydrolases and glucosidases) has led to an improved understanding of cellulase activity relative to the molecular structures of different cellulosic polymers, and the development of customized

Meeting report

Copyright 0 1996, Elsevier Science Ltd. All rights reserved. 0167 - 7799/96/$15.00 PII: 50167.7799(96130009-7 TIBTECH JUNE 1996 IVOL 14)

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cellulases for specific ‘stone-washing’ processes.

A recurring theme in the presen- tations on traditional enzyme apph- cations was the modification of the functional properties of enzymes, generally by site-directed mutagen- esis. Recombinant approaches to enhancing protein thermostability, reducing oxidation sensitivity, and increasing resistance to solvents and proteolytic degradation were all reported. Anders Pedersen (Novo Nor&k A/S, Bagsvaerd, Denmark) reviewed experiments on the inser- tion of prolines into various sites in subtilisin (the bacterial proteinase that is a major component of the enzyme complement in ‘biologi- cal’ washing powders). Mutations based on apparently favourable psi/phi angles generated both positive and negative changes in the protein melting temperature (T,). The observation that positive changes in T, were never greater than about 3°C reflects results from the work on site-directed muta- genesis stabilization of proteins by several academic laboratories. This report emphasized the disturbing fact that, even with access to high- resolution three-dimensional struc- tural data for proteins, intelligently selected site-directed mutations may produce unexpected (and unwanted) results.

Subtilisin has also been the target of engineering attempts to enhance stability (Donn Rubingh; Proctor & Gamble, Cincinnati, OH, USA). Calcium-chelating compounds (‘builders’) are required for good per- formance of detergents, but can result in autolytic inactivation of the protease through complexation with enzyme-bound calcium ions. How- ever, simple deletion of the loop that forms the tetra-coordinate ‘tight’ calcium-binding site in subtilisin resulted in a highly unstable (although builder-insensitive) struc- ture, and further site-specific engi- neering was required to stabilize the conformation.

An approach that combines the screening of natural sources of enzymes with structure-based enzyme engineering and random mutagenesis linked with robotic screening of pooled mutants is being adopted by Novo Nordisk to iden- tify and optimize enzymes for in- dustrial use. The stabilization of Copri- nus cineveus peroxidase (used in dye decolouration and wastewater treat- ment) by random mutagenesis using

TIBTECH JUNE 1996 (VOL 14)

‘Mn2+-poisoned’-PCR, together with robotic screening of recombi- nants (Joel Cherry; Nova Nordisk Biotech, Davis, CA, USA), was an excellent example of the strength of this approach. Of the many mu- tations isolated by either site-specific or random mutagenesis that were assessed, a single change at Gln239 repeatedly showed enhanced stabil- ity. Robotic screening of subse- quently prepared random pools of Gln239Xaa yielded an active mutant with enhanced stability-this approach can also rapidly identify any synergy resulting from double mutations.

The pulp and paper industry dorn- nated much of the latter part of the second day, with technical presen- tations by Pirkko Suominen (Primalco Biotec, Rajamaki, Finland), Dave Senior (ICI Forest Products, Canada) and Thomas Jet&es (Forest Products Laboratories, Madison, WI, USA). Predicted throughout the late 1980s to be the next major field of bulk industrial enzymology, xylanase and mannanase technology appears to have struggled to cross that barrier from ‘potential’ to ‘real’ application. It was heartening to hear that enzyme bleaching is routinely used in Finnish pulp mills. Tom Jeffries demonstrated that xylanase treatment is reliably capable of re- ducing the usage of chlorine in pulp bleaching. However, his emphasis on the competition between chlo- rine, chlorine dioxide, hydrogen peroxide and enzyme bleaching technologies was a sobering reminder of the constraints affecting the de- velopment of enzyme technology - cost-effectiveness being not the least significant.

Enzymes from extremophiles A recurring theme of the meeting

was the potential of enzymes from ‘extremophiles’ - organisms that grow under extremes of, for example, temperature, salinity, or pH. Both Greg Zeikus (Michigan Biotech- nology Institute, Lansing, MI, USA) and Don Cowan (Helix Biotech- nology, University College, London, UK) reviewed the role of extremo- philes in recent discoveries on the fundamental mechanisms of protein stability. The innate thermostability and thermophilicity that permit higher reaction rates and increased product formation at higher tempera- tures is due to the enzymes’ rigidity - these characteristics are not lost if the enzymes are cloned and expressed in industrial hosts. Greg Zeikus

explained that chemical stability at high temperatures results from possession of fewer amino acids that are susceptible to deamidation, destruction of sulphide bonds, or peptide-bond hydrolysis. He pre- sented the view that, in order to design an application-specific thermo- stable enzyme, one should start with a representative thermozyme, then engineer enzyme kinetics by site- directed mutagenesis.

The use of extremophile enzymes as valuable resources, both for pro- viding design rules for the engineer- ing of stability into other proteins and as novel biocatalysts, was stressed by both speakers. The relevance of this theme was highlighted by the presence throughout the meeting of representatives of one of California’s newest biotechnology companies, Recombinant Biocataly- sis (La Jolla, CA, USA). The use of novel and exciting approaches to the screening and selection of new biocatalysts (many of which are derived from hyperthermophilic organisms isolated by the world’s leading thermophile microbiologist, Karl Stetter) generated enormous interest amongst the industrial representatives.

Concluding remarks Although the two presentations on

the regulation of new and genetically engineered enzymes in food and related products were well-presented and addressed an important topic, they were perhaps the least relevant to the target audience as they focused specifically on Canadian regulations. While the Canadian regulatory en- vironment is a model that could be beneficially emulated in other coun- tries, the opportunity to transfer information on regulatory mecha- nisms between the European and US biotechnology sectors was sadly wasted.

Possibly the best reflection of the quality and interest of the meeting was that attendance at the final pres- entation showed little evidence of audience dissipation. The organizers can be assured that their selection of topics and speakers was sufficiently inspired to compete effectively against the adjacent attractions of DisneyWorld!

Don Cowan