Cross-Linked Enzyme Crystals Show Promise for Industrial, Clinical

Sep 28, 1992 - And for clinical uses, the enhanced enzymes might resist attack by proteases in order to serve as longer acting drugs that wouldn't be ...
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SCIENCE/TECHNOLOGY

Cross-Linked Enzyme Crystals Show Promise for Industrial, Clinical Uses • Cross-linking method makes enzymes more stable, resistant to proteases, compatible with mixed solvents Stu Borman, C&EN Washington f a protein engineer were to try to design near-perfect enzymes for industrial and clinical applications, what might their properties be? Such enzymes would retain substantially the same high activity as the natural enzymes they were patterned after, but would perhaps be more resistant to extremes of temperature and pH. Many St. Clair (left) and Navia discuss results of CLEC research in the cross-linked enzyme natural proteins have short shelf lives, so crystal laboratory at Vertex Pharmaceuticals it would be nice if the engineered enzymes could be freeze-dried, stored in- philized (freeze-dried) and stored in dry Despite the phenomenal efficiency of definitely at room temperature, and then form almost indefinitely at room tem- enzymes in biological systems, they are reconstituted easily when needed. For perature. They retain near-maximum not widely used in industrial processing process applications, compatibility with catalytic activity under harsh tempera- because of their instability and their aqueous-organic mixed solvents would ture and pH conditions, work in all strong tendency to denature in aqueousbe a desirable trait. And for clinical uses, kinds of solvents and solvent mixtures, organic solvent mixtures. Many industrithe enhanced enzymes might resist at- and resist proteolytic breakdown. al substrates and products are unstable tack by proteases in order to serve as Thus CLECs may have promise for or insoluble in aqueous media, where longer acting drugs that wouldn't be use as biocatalysts in the industrial pro- enzymes are most comfortable. The inbroken down quickly in the body. duction of fine chemicals and chiral compatibility of enzymes with many orAt Vertex Pharmaceuticals, a small pharmaceuticals. "If you're losing cata- ganic solvents also has precluded their structure-based drug design company lytic activity because your structure is use in processes in which solubility difin Cambridge, Mass., such enzymes are becoming unraveled," says Navia, "then ferences in aqueous and organic solvents not just a pipe dream. In a recent paper a cross-linked enzyme crystal will help are exploited to isolate products and imin the Journal of the American Chemical you." The crystals also may have poten- prove synthetic yields. CLEC technology Society [114, 7314 (1992)], Vertex bio- tial as reagents for use in biosensors, as addresses such stability and solvent physicists Nancy L. St. Clair and Man- long-acting therapeutic agents, and as compatibility concerns head-on. uel A. Navia report making cross- stabilized forms of catalytic antibodies. Biochemistry professor Frederic M. linked enzyme crystals (CLECs) that Whether or not CLECs provide an Richards and coworkers at Yale Unihave all of the above properties. economic advantage over existing tech- versity first cross-linked protein crysCLECs are made by reacting a crys- nology for such applications remains to tals with glutaraldehyde in the 1960s, tallized enzyme with the bifunctional be seen. However, says Navia, "We've as part of their effort to show that encompound glutaraldehyde, HCO(CH2)3- talked to about 60 companies, and about zymes were catalytically active in the CHO, whose aldehyde groups react 40 of them are actively evaluating our crystalline state—a contentious issue in with primary amino groups on proteins. materials to see if they make economic those days. Working primarily with CLECs also can be cross-linked using sense. The level of enthusiasm and the carboxypeptidase A, Richards and conumber of people working on this workers showed that cross-linked crysother bifunctional reagents. Cross-linking produces stable immo- would suggest that some of these 60 tals resist denaturation, don't dissolve bilized enzyme particles that can be lyo- companies are going to come on board." in salt solutions that would instantly

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dissolve non-cross-linked crystals, and "The thought occurred to us that are stable at high temperatures. How- if you could somehow stabilize a ever, they did not test the crystals for crystal sufficiently so that it wouldn't fall apart in solutions of compatibility with organic solvents. Now, in virtually the first major de- various types, so it would have velopment in protein cross-linking re- mechanical stability, then you'd search since the work at Yale 25 years have an immobilized enzyme ago, St. Clair and Navia have found particle that would be unique in that cross-linked enzyme crystals retain being extremely dense." near-maximum catalytic activity under Enzymes used in large-scale a range of harsh conditions, including biotechnology processing are ofexposure to aqueous organic solvents. ten immobilized on inert supThis is the basis for the filing of a broad port particles so they stay in patent on the technology by Vertex. place inside the reactor, enabling Richards admits: "We completely them to be used over and over missed the potential of this in the com- again. One disadvantage of such mercial sector. When Manuel called me immobilization is that the supup some time ago and told me what he port particles occupy much more had done in the lab and what he was space than the enzyme moleplanning to do, I was just overwhelmed. cules themselves, necessitating I thought it was terrific." In the 1960s, he much larger reactor vessels than Lyophilized thermolysin cross-linked enzyme says, biotechnology "wasn't a big deal. would otherwise be required. In this day and age, I don't think we The idea by St. Clair and Na- crystals could be useful for bioprocessing in would have missed the boat, and our via was to shrink the space re- aqueous-organic solvents patent would be sitting down in Wash- quired for equivalent catalytic ington." activity in such a bioreactor by eliminat- end of this year," says Navia. "The enRichards calls CLECs "a very interest- ing the inert support. "We felt this zymatic process seemed to be competiing development. If, in fact, the cross- would have economic value," says Na- tive, but not necessarily overpowering, linked crystals work at reasonable tem- via, "simply because the amount of real vis-à-vis the chemical process. So we peratures, are stable, and have a long shelf estate that your plant would be taking thought it would be fun to take the enzyme used in the enzymatic process and life, they could be amazingly useful, and up would be shrunk by that much." this could be a big step forward. I will The researchers first tested the cross- formulate it as a cross-linked enzyme watch with great interest to see whether linking technique on the enzyme ther- crystal—thinking that, if nothing else, this is pooh-poohed or picked up." ' molysin. Thermolysin is used industri- the compression of catalytic activity St. Clair and Navia's work on CLECs ally to make the sweetener aspartame, might give somebody an economic edge. stemmed from their x-ray crystallograph- although most aspartame is still manu- To our amazement, by doing this we ic studies on proteins. "A crystal is, in es- factured by a nonenzymatic (chemical) greatly enhanced the stability of the enzyme." Process studies have now shown sence, the densest possible immobilized process. enzyme particle you can get," says Navia. "Aspartame comes off patent at the that making a key aspartame precursor with thermolysin CLECs provides a 90fold increase in productivity, compared with using free thermolysin. In aqueous solution, cross-linked thermolysin crystals retain 80% of the activity of free thermolysin. Thermolysin CLECs are stable in solvents that denature the free enzyme, such as 50% aqueous solutions of acetonitrile, dioxane, acetone, and tetrahydrofuran (THF). The cross-linked crystals retain a high level of activity at pH 10 (the natural enzyme is inactive at pH 9) and after four days of continuous incubation at 65 °C (a temperature at which the natural enzyme lasts less than a day). The crystals also withstand four days' treatment with a mixture of proteases that digests most proteins (and that digests free thermolysin in 90 minutes). St. Clair and Navia Protein crystals suitable for x-ray crystallography (left) are about 0.5 mm long. CLECsspeculate that the "remarkable" temper(right) are only about 0.1 mm long, allowing relatively fast diffusion of substrates andature, pH, and solvent stability of CLECs may result from restricted access to key products into and out of the crystals SEPTEMBER 28,1992 C&EN 4l

SCIENCE/TECHNOLOGY conformations on the enzyme's denaturation pathway, owing to the rigidity of the covalently cross-linked crystal structure. Navia points out that, in cross-linking enzymes, "one of the things you have to be concerned about is diffusion. Diffusion will affect the apparent catalytic rate of the enzyme in crystalline form because substrate has to soak in, find an active site, and react, and then product has to soak back out." In crystals large enough for use in x-ray diffraction experiments (about 0.5 mm long), diffusion occurs too slowly for their use as practical catalysts. "I suspect that the beneficial properties of cross-linked enzyme crystals remained neglected and undiscovered all these years because of this effect," says Navia. "Our insight into this problem was the realization that diffusion was going to be manageable as crystals got smaller and their surface-to-volume ratio increased." The crystals used to make CLECs are about 0.1 mm long. From their x-ray crystallographic

work, St. Clair and Navia knew that a significant volume of most protein crystals is occupied by solvent—almost invariably water or an aqueous solution. The solvent generally forms welldefined channels through which smallmolecule substrates and products can migrate relatively freely, with diffusion times comparable to those in liquids. Use of CLECs "is restricted to smallmolecule reactions because the pores in the crystal lattice are of the order of 10 to 15 À in size," says Richards. "That means you're restricted, for any reasonable diffusion rate, to species that are the size of dyes, or perhaps trisaccharides— maybe a few hundred in molecular weight. But there are lots of molecules of that size that might be of interest commercially." In addition to thermolysin, Vertex researchers also have made CLECs of 10 other enzymes, including pig liver esterase (used to make chiral intermediates) and elastase. Organic chemist John W. Frost of Purdue University, who specializes in biotechnology ap-

Model of cross-linked thermolysin crystal unit cell shows considerable free space (occupied mostly by solvent), enabling substrates and products to diffuse in and out of the crystal. One thermolysin molecule in the crystal highlighted in blue, is shown bound to a key precursor (red) of the sweetener aspartame

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plied to chemical synthesis, suggests they might also want to try Œoss-linking isomerase, an enzyme used commercially for isomerizing glucose to fructose. "High-fructose corn syrup has to go through a purification step to increase the fructose concentration," says Frost. 'It's known that if you were to run the isomerization at higher temperature you would get that percentage of fructose without purification, but the problem has been that the catalyst doesn't last long enough. Any kind of technology that can potentially stabilize enzymes like isomerase has incredible ramifications." In previous research, some lyophilized enzymes have been found to be both stable and active when suspended in near-anhydrous organic solvents. However, in such systems, even a tiny amount of added water often leads to rapid loss of activity. What's unique about CLECs is that they're stable over the whole range of water concentrations in aqueous-organic mixed solvents. "If you read the literature on use of enzymes in organic solvents," says Navia, "it always refers to near-anhydrous organic solvents. If the water level goes up too high, it facilitates the breaking of bonds and the protein will unravel. We've demonstrated activity in 50% aqueous organic solvents, including 50% aqueous THF—a very harsh condition for an enzyme to be in." Chemistry professor Alexander M. Klibanov of Massachusetts Institute of Technology, who specializes in studies of biochemistry in nonaqueous media, comments that cross-linking of enzyme crystals "may turn out to be an improvement compared to other methods of putting enzymes in organic solvents, but if s really hard to say at this point because simply not enough is known. However, I would certainly view it as a significant advance and something that potentially may have very substantial implications." Because CLECs are self-supporting assemblies, they could be attractive for biosensor applications, where the largest possible signal per unit volume is often desired. The Vertex group has formulated jackbean urease CLECs for use in clinical biosensors to measure urea as an early indication of renal disease. The ability of CLECs to resist high temperatures and proteases also may make them valuable as therapeutic agents. Free enzymes used as drugs— such as streptokinase, used to treat myocardial infarctions—are often very short-

acting because they are attacked by proteases in the blood. CLECs, which are relatively stable to proteolysis, might be administered less frequently or in reduced amounts, and might even provide an alternative to gene therapy for enzyme replacement in patients with enzyme deficiency diseases. In addition, CLEC proteins could potentially be taken orally as drugs without being broken down by the digestive system. Vertex researchers are currently working on a proprietary strategy to get such drugs into the bloodstream once they have passed safely through the stomach. Still another potential application would be catalytic antibody CLECs. According to chemistry professor Stephen J. Benkovic of Pennsylvania State University, who studies catalytic antibodies, cross-linking of enzyme crystals "looks very promising. If it can be generalized to other enzymes—and catalytic antibodies fall into that category—it may very well prove to be quite useful." •

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