A Cut Above

enzymes by appealing to the obsession over purity Date: September 28, 1998 Specialty Enzymes Every once in a great while a discovery is made that not only sheds light on the true genius of nature but leaves one completely amazed that it could possibly work that way. The discovery of restriction enzymes in the mid 1970s and the explication of their role in nature was one of those discoveries. For biologists attempting to apprehend the way in which organic life reproduces itself, the finding ca

Sep 28, 1998
Brent Johnson

enzymes by appealing to the obsession over purity

Date: September 28, 1998 Specialty Enzymes

Every once in a great while a discovery is made that not only sheds light on the true genius of nature but leaves one completely amazed that it could possibly work that way.

The discovery of restriction enzymes in the mid 1970s and the explication of their role in nature was one of those discoveries. For biologists attempting to apprehend the way in which organic life reproduces itself, the finding came as a revelation, even though there doesn't seem to be any clear indication of how this mechanism could have begun in the first place. Never the ones to look a gift horse in the mouth, molecular biologists used this insight to develop techniques that mimic the genetic machinery of reproduction.

In what has amounted to the genetic equivalent of the Rosetta Stone, restriction enzymes are now the fundamental keys for extricating, deciphering, and replicating genetic matter. The economic implications of this technology have not gone completely unnoticed either. For individuals with the wit and ingenuity to take advantage of recombinant technology, the world has beaten a path to their door.

Companies such as New England Biolabs, Life Technologies, Promega, MBI Fermentas, Stratagene, Roche and others have developed extensive catalogs for many of the 220 restriction enzymes that are now commercially available. As these companies continue to grow and flourish with respect to the burgeoning science of molecular genetics, they also attempt to refine the process of enzyme production, enforcing new standards and raising expectations among consumers.

In 1976, New England BioLabs (NEB) was the first company to make restriction enzymes commercially available. From its initial offering of "Type II" Escherichia coli and Haemophilus influenza enzymes, NEB has dramatically expanded its repertoire to include nearly 200 enzymes, 30 percent of which were actually discovered by NEB. It is probably fair to say that NEB's products and services are the standard by which all others are compared. In fact, there are only about 10 restriction enzymes out of the approximately 220 currently available that NEB does not produce, and for those remaining 10, NEB has graciously listed the names of competing companies that do offer them in its catalog.

Atomic force microscope image of DNA bound with 6 EcoR I molecules. Image provided by Dave Allison, Oak Ridge National Laboratory, Oak Ridge, Tenn.
The REbase web site that NEB maintains is an electronic extension of its published catalog. It assists customers with one of the most all-encompassing databases of its kind. REbase is a vast warehouse of information, providing everything from product specifications to article abstracts. It is continually updated with new products and information, giving scientists up-to-the-minute news on available products and industry developments.

In response to the demand for restriction endonucleases, NEB has taken an aggressive approach in the current market. For the same price that used to buy 100 units of Pme I, you can now get 500 units at a savings of $220. For other restriction enzymes, such as BsoB I, the savings are even greater: 10,000 units of the enzyme for $50.00. The equivalent amount would have cost $500 previously. Such cost-cutting measures, though representing a boon to consumers, is no doubt being received with something less than enthusiasm by NEB's competition.

NEB isn't just in the business of making inexpensive products however. NEB has expanded its role as a provider of goods by taking an active interest in the training of research professionals. Through the creation of the REbase database and an ongoing educational series that gives researchers hands-on experience, more and more scientists are learning the techniques required for restriction enzyme digestion. The New England Biolabs Molecular Biology and PCR Summer Workshop is an intensive two-week course that attracts researchers from a wide pool of backgrounds and nationalities. Graduates of the program return home with a sophisticated understanding of the most advanced techniques used in experimental molecular biology.

Life Technologies was one of the first two companies to sell restriction enzymes, entering the market along with NEB in 1976. Its history of innovation has seen the cultivation of products such as the React Buffer System, which guarantees 100 percent activity, and a patented packaging system that enables the customer to use the vial container in screw-cap or flip-top mode.

Life Technologies' most recent addition to its stock is its room temperature stable enzymes (RTS). Rather than requiring packaging and shipping for overnight delivery, these dried enzymes survive indefinite exposure to room temperature conditions without significant loss of reactivity. When you want to use them, just reconstitute the enzymes with a glycerol solution and reaction buffer. The rehydrated enzyme works exactly like a typical "wet" enzyme. Any of the remaining unused portion can be stored in its liquid form at -20°C.

Life Technologies now has 10 such enzymes available for commercial use, including some of the most common, such as EcoR I and BamHI. The dried enzyme includes a reconstitution kit consisting of a reconstitution buffer, glycerol solution, and a reaction buffer. A special hotline assists those who are interested in using these enzymes for the first time.

Roche Molecular Biochemicals, formerly Boehringer-Mannheim, began as a pharmaceutical company at the turn of the century. When the market in biochemicals exploded in the 1980s, Boehringer followed suit by becoming a pipeline of raw materials for the revolution in molecular biology.

Today, the company is part of the largest diagnostics and health care company in the world, with a selection of 2,700 different products. From its original offering of EcoR I, Roche has expanded its selection of over 120 restriction enzymes to include even some rare cutters like Meganuclease Sce I which recognizes an extraordinary 18 base pair sequence.

According to product manager Brian Holaway, Roche has the reputation of being a provider of high quality, high activity restriction enzymes. Though these products may cost a little more, many researchers are willing to pay the extra price for advances such as the SuRE-Cut™ Buffer System, which, at least in theory, permits one to use any enzyme with every buffer. In conjunction with this buffer system, Roche has developed a detailed chart for picking the ideal buffer for a particular digest. Holaway claims the system is so effective that people using restriction enzymes from competitors will frequently use the Roche buffer system and its attending buffer chart in order to run their experiments. Testimony to the success of the SuRE-Cut™Buffer System and the quality of enzymes is the fact that these enzymes show 100 percent of initial activity through the entire control date period without the use of Bovine Serum Albumin (BSA). Holaway attributes these consistent results to a corporate philosophy attuned to getting the details right.

"The approach to quality control is almost obsessive-compulsive," he said. "The absence of specific exonuclease activity is due to tests that are done more rigorously, that challenge the enzyme to a greater degree."

Potential customers who are interested in Roche's line of restriction enzymes can also use its Restriction-MATE™ software, which, for around $30, looks for 339 different sequences and specific sites in addition to doing molecular weight conversions, stores melting temperatures for oligos up to 60 bases, and identifies methylation and amino acid codes.

MBI Fermentas is a bulk supplier of restriction enzymes whose products have, at one time or another, been private labeled by many of the large purveyors. Fermentas has earned its reputation as a guaranteed source for restriction enzymes because of its consistent procedures, evidenced by one of the first ISO 9000 certifications in the business. Achieving uniformity of these procedures is aided by the fact that all of Fermentas' restriction enzymes are produced at its own facilities, as opposed to farming its products out to other companies for manufacture. By keeping everything under one roof, so to speak, the company is better able to keep tabs on the production process, from enzyme digestion to packaging and shipping.

Fermentas' reputation as a dependable supplier reflects a production process that continually examines ways to enhance the performance of its products. The Tango™ Double Digest Buffer, which is effective in more than 95% of the company's restriction enzymes, is designed for use in double digests or as a universal buffer. This high performance buffer comes with each of Fermentas' enzymes and works well with most modifying enzymes, eliminating the need to change buffers after restriction enzyme digestion.

In the ongoing struggle for purity, Fermentas' Pure Extreme™ Restriction Endonucleases are some of its cleanest enzymes yet. The first weapon in their line of defense is the commonly used blue/white cloning assay, which tests the integrity of DNA ends within a margin of 3 percent. In this test, the lac Z gene on a plasmid is cut at a unique site with a restriction enzyme and is subsequently transformed into bacteria. The loss of even a single nucleotide at the ends of the cut plasmid will prevent the reconstitution of an active lac Z gene and leads to white colonies (When the Z gene is intact, colonies are blue) While the standards differ slightly for different kinds of cutters (staggered versus blunt ends, with staggered-end producers generally held to a higher standard) as well as from supplier to supplier, a few percent white colonies is considered to be acceptable by most suppliers.

The Labeled Oligonucleotide Assay (LO) is sensitive to the presence of nonspecific endonuclease, exonuclease, or phosphatase activities to an even greater degree. In this assay, 5¢-end-labeled oligos are overdigested with a restriction enzyme and the products analyzed on acrylamide gels for the presence of degraded oligos. Fermentas reports that enzyme preparations with as little as 0.2% white colonies (well within the standard accepted by most companies) nonetheless fail to pass the LO test, whereas every restriction enzyme in Fermentas' catalog must pass this test before it can be sold.

Promega, which has been in the business of making enzymes for the last 20 years, is banking on buffers as the "solution" to faster and cleaner enzyme digestion. In addition to its patented 4-Core Buffer system, which delivers optimal activity in the majority of enzymes, Promega supplies specific 10X buffers for restriction enzymes that don't respond to the normal mainline buffers. Each of Promega's buffers is continually reevaluated for effectiveness with respect to improved activity, specificity, heat stability, and reduced star activity.

As multiple digestion becomes more popular, better universal buffers are required that can obtain sustained levels of reactivity with more than one enzyme in digestion. Promega has been on the crest of this wave developing the Multi-Core buffer as an answer to persistent calls from the research community for improved all-purpose buffers. Although the Multi-Core is sensitive in the extreme to minor perturbations in pH and ionic concentration, Promega has achieved enzyme activities with Multi-Core up to 100 percent effectiveness.

Stratagene, which is the fifth largest producer of restriction enzymes in the United States (C. Wrotnowski. Genetic Engineering News, September 1, 1996, page 16) has produced a universal buffer that yields 80-100% activity for all but a few of its enzymes when used in one of four different dilutions (0.5X, 1.0X, 1.5X, and 2.0X). The universal buffer greatly assists scientists performing multiple digests. With each restriction enzyme purchase, customers receive an optimal buffer and/or 10X Universal buffer. To facilitate experimental planning, Stratagene has also developed a quick reference chart highlighting each enzyme's activity in the four concentrations of Universal Buffer.

In addition to its universal buffer, Stratagene has added two restriction enzymes to its catalog for 1997/98. Dpn I, which was actually discovered several years ago, has been included with some of its compatible neoschizomers MboI and Sau3AI. One of the benefits of Dpn I is that it can be used to produce specific cleavages at sequences 8-12 bp in length. The longer base hitters may prove more useful with complicated sequences such as mammalian or human genetic material. The other new enzyme, SanDI, which is truly unique, is derived from an unidentified strain of Streptomyces. Like Dpn I, it has no available isoschizomers but has the benefit of cutting methylated DNA.

Julie Robinson, product manager for Stratagene, is quite enthusiastic about her company's measure of quality control. "Our quality control procedures are very stringent and include precise unit activity determination, overdigestion of substrates, checking for exonuclease contamination, ligation and nicking assays, blue/white cloning assays, and continuous stability testing."

Quantum Biotechnologies, based in Montreal, Canada offers a line of over 80 enzymes, some manufactured in house and others that come from a variety of sources. The company prides itself on the quality control of each lot of enzyme--with respect to both the purity of the preparations (the absence of nucleases) and their stability. The company focuses on accessibility and affordability--being sure that enzymes in common use are available when needed and in quantities useful to its customers. Another aspect that Quantum has focused on is the development of a universal buffer system. Detailed information on each enzyme's activity in this "universal" buffer is available to enable researchers to adjust their reaction conditions accordingly. Robert Furic, product manager for Quantum's restriction enzymes, explained this is an interesting but difficult product line to manage. "It's tough keeping inventory on a number of different enzymes" in such a competitive market.

One solution to the problem of maintaining an inventory of restriction enzymes has been to share customers. There is a high degree of interdigitation among companies that produce restriction enzymes. What they tend to do is concentrate in a specific area, providing enzymes for a targeted application.

"In our case, we actually purchase enzymes from virtually all the manufacturers, "said Warren Shore, president of United States Biological (USB).

Like USB, many of the companies that are in this business as manufacturers are actually distributors of their competitors' products, which makes for some unusual alliances. Shore explained that such proprietary research agreements are very common and that they are usually conducted in advance of sales. The intention is to pool the combined resources of two or more companies that have strengths in disparate areas of research. The result is a network of companies that complement each other in the variety and quality of restriction enzymes that they can offer.

Perhaps this fluid arrangement, which makes rivals virtual subcontractors, shouldn't be too surprising. What many big businesses are discovering is that unorganized competition is wasteful. Why battle it out over customers and resources when there are enough to go around for everyone?

Pvu II crystals (Ira Schildkraut and Joseph Bonventre, New England Biolabs, Inc.)
Amersham Pharmacia Biotech is the merger of two companies formerly known as Amersham Life Sciences and Pharmacia Biotech. This powerful confederation has given Amersham a strong presence in the market with an extensive line of over 100 enzymes. Some of Amersham's restriction enzymes are identified with the Enforcement Cloning Tested logo. The logo indicates the suitability of the enzyme for cloning applications, which describes over 30 of Amersham's cataloged enzymes.

In addition to its private-labeled enzymes, Amersham is one of the few companies that offers Takara Shuzo's Fse I for distribution. The international scope of Amersham's dealings has given the company a significant interest in Japan as well as Europe.

One piece of technology that Amersham won't be trading with its competition anytime soon, however, is its new analysis software. Designed by John Antoniw, this software permits users to identify restriction sites and enzyme supplies and allows manipulation of DNA sequences in virtual space. Think of it, no more poring through catalogs to find the right binding site. The program will even predict the size and number of fragments generated. It would seem that the future of biochemicals relies as much on finding them as it does in making them.

Sigma-Aldrich is yet another huge conglomerate that has extended its tendrils around the entire planet by diversifying its products and services through mergers and leveraged buy-outs. Storming its way into the market for restriction enzymes, Sigma promises to be a contender for domestic sales of restriction enzymes.

By acquiring Fluka BioChemika in Buchs, Switzerland, Sigma has well positioned itself to expand its production of biochemical reagents, which earned the company a hefty portion of its over one billion dollars in sales last year. Sigma currently has 69 restriction enzymes in its bank with even more in development. Its strategy for competing in this volatile market is to rely on the strength of their worldwide shipping and distribution infrastructure that provides next day service to almost any point on the globe.

Even though PanVera is not an enzyme manufacturer per se, as the exclusive distributor of restriction enzymes for Takara Shuzo in North and South America, it is positioned to take on a significant share of the market. Beginning September 1, 1998 PanVera will offer Takara Shuzo's complete line of enzymes and thereby become one of the biggest purveyors of restriction enzymes in the country. Amidst this veritable windfall of enzymes will be some unusual enzymes such as the rare cutters Fse I and Sse 8387 I.

PanVera is most proud of the length to which Takara Shuzo has gone to challenge the integrity of its enzymes. The pKF3 Cloning Test Takara Shuzo uses on all of its restriction enzymes is applied by several companies including Amersham. The Enforcement Cloning System is similar in principle to the blue/white colony selection. A vector, pKF3 containing several key enzymes in determining antibiotic resistance, is cut within a gene (rpsL) whose expression confers strep sensitivity to a strep-resistant host bacterium. While transformations with the intact plasmid confers ("enforces") strep sensitivity, plasmid that has been cut and re-ligated will fail to confer strep sensitivity when contaminating nucleases are present. While similar in principle to the blue/white assay, this assay is less subjective in that it does not require making judgement-calls based on the amount of color in a colony. By exploiting the property of streptomycin resistance in the rpsL gene when exposed to exonuclease contamination, the pKF3 Cloning Test can be used to screen for very small amounts of nuclease, thus providing a high degree of purity in the treated samples.

There is a subcategory of enzyme manufacturers that offer limited selections of enzymes for the one-stop shopper. Although they may not have the great variety of NEB or Life Technologies, they do offer a convenient service for researchers who like to get all of their products from one source. This can be especially helpful if purchase orders are funneled through state agencies and require a substantial amount of time to process. That is one of the reasons why companies such as Advanced Biotechnologies, Sibenzyme, Chimerx, American Allied Biochemical, and Shinko American have extended their services to include restriction enzymes. These companies focus on the most popular enzymes available, targeting the broad midrange of restriction enzyme applicability.

Advanced Biotechnologies has a selection of thermophilic isoschizomers that can be found in the inventory of few other companies. Rather than lock horns with the larger manufacturers, the British-owned Advanced Biotech has carved out a niche market for enzyme duplicates.

Sibenzyme is a major producer of restriction enzymes in Russia. Founded by a former researcher at NEB, Sibenzyme is now the only foreign manufacturer that makes enzymes that are private labeled by NEB. Although Sibenzyme has a catalog that is nearly as comprehensive as some of its American competitors,' NEB relies on its production of rare cutters to complement its prodigious offering.

Chimerx, a division of Molecular Biology Resources (which produces restriction enzymes for private-label and commercial use) produces over 200 reagents for life science research, 50 of which include a selection of restriction enzymes. The latest release of CviJ I, which is customized for the creation of cosmid sublibraries, generates random DNA fragments for use in large-scale DNA sequencing projects. Benefits of the enzyme include higher cloning efficiencies and the elimination of the need for preligation, end repair, chemical extraction, and agarose gel electrophoresis and elution.

American Allied Biochemical (AAB) is a small manufacturer that focuses exclusively on the production of restriction enzymes. Ninety-five to ninety-eight percent of its customers are universities with applications as varied as bacterial testing and ribosomal typing. Jennifer Blakely, a representative for AAB, attributes the company's position in the marketplace as owing to low prices and customer service. AAB claims to keep its production costs down by maintaining a small facility and by producing native enzymes rather than clones, and it passes the savings along to consumers.

Toyobo Biochemicals, which began its existence as the biggest spinning mill in Japan, is now spinning strands of genetic material as one of the leading foreign producers of restriction enzymes. Distributed exclusively by Shinko American in the U.S., Toyobo's battery of 120 enzymes represents more than a casual interest in gaining a share of the American market.

The author can be reached at bjohnson@the-scientist.com

To list all the available restriction enzymes would be an onerous task, and, as it happens unnecessary, given the data bases that exist both on the web and in print. Instead, we have chosen to highlight some of the more unusual enzymes that have been discovered, which have applications in genetic engineering. Some of these are:

Rare Cutters: While the majority of restriction enzymes in common use require between four and six bases in the recognition sequence, a few enzymes have been found that have longer recognition sites, as many as eighteen bases. These rare cutters can be useful for generating long fragments for preparing libraries of complex genomes.

Two Base Cutters: At the other extreme, a novel two base cutter has been isolated and used in the generation of random fragments, as an alternative to shearing or sonication. Uses include the generation of random sublibraries for large-scale human shotgun sequencing.

Sequence Specific Intron Encoded Endonucnucleases: are a collection of proteins that are encoded by genes with mobile, self-splicing introns. They promote the movement of their own sequence to intronless targets by making a site-specific double-strand break at a target (intronless) site. The recognition sequences for these enzymes tend to be long, between 14 and 44 bases.

Class IIS Restriction Enzymes: (hapoxoterministic enzymes) are characterized by cleaving outside the recognition site, or within an interrupted palindrome, at bases not specified. They produce asymetric staggered ends which are unique and hence cannot join with other fragments generated by the same enzyme. They can only hybridize in the original configuration from which they were derived. These enzymes find uses in site- specific mutagenesis or the isolation of large intact DNA fragments. The table provides information on where you can purchase some of these rare and unusual restriction endonucleases.

Specialty Enzymes