Biocatalysis Enzyme Toolkit Concept
The Challenge: Biocatalysis is as equally elegant as it is challenging, given that unlike most other related disciplines, more often than not, no enzyme has yet been characterised with the biochemical/biophysical characteristics desired, especially when considering the intricacies of requisite stereo-, regio- and chemo-selectivities. A process of biocatalyst screening is thus required, where panels of enzymes with closely related activities to the one desired are tested against the application, such as in the synthesis of a chiral API using a KRED kit.
"Pre-Genomics": In the earlier years of Biocatalysis, the greatest barrier to success was simply the absence of an effective enzyme toolbox, owing simply to the limited number and / or form of biocatalysts available. Many solutions were found by way of the use of either whole native microbial cells, such as Baker's Yeast, or crude / purified preparations from any microorganism identified as being capable of performing the biotransformation in question. Indeed, these approaches are still very effective today. Some genes encoding specific biocatalysis enzymes were cloned and expressed, but with limited genomic information (DNA sequences) being available, this route, although very effective, was unfortunately not scalable at that time.
"Post-Genomics" Era: The advent of whole genome sequencing projects enabled a new approach for biocatalyst development, based on the specific construction of panels of recombinant enzymes (enzymes produced using gene cloning technology). Recombinant enzymes offer notable advantages over their native counterparts, in that they can be made using standard methods, they offer generally higher activities per gram of powder, and most importantly, larger numbers can be accessed both rapidly and cost-effectively. Where a biocatalyst is identified against an application, production of the catalyst is also scalable using standard in-house or third-party facilities.
Current Routes to Success: The application of biocatalysis is growing rapidly, mirrored by an increase in availability of related products and services. For instance, a common route is now to screen a panel of biocatalysts, and then to evolve the best enzyme found, optimising it towards the "perfect" catalyst for the transformation in question. This route is certainly effective, but can be expensive and time-consuming. At the same time, the perpetual logarithmic increase of genomic data shows no signs of abating, and next-generation DNA sequencing technology already in place will ensure the pace is kept for at least the next five years, with no reason at all to doubt it will not continue to expand long after that. From its inception, this expanding, disruptive resource has lead to a series of step changes in many aspects of biological research, and the impact on biocatalyst development is proving to be no exception.
Enzyme Development Philosophy: While Prozomix acknowledges the utility of protein evolution techniques (such as "Error Prone PCR" and DNA Shuffling) when applied to biocatalysis, the sheer volume of current genomic data, coupled with its inevitable future growth, leads us to propose that the natural diversity of biocatalysts, i.e. that evolved over billions of years by Mother Nature, should be one of the first options considered when screening for novel activities (see here for a review of current commercial strategies). At the same time, it is acknowledged that panels of diverse unique enzymes (i.e. not containing site-directed mutants or otherwise artificially related enzymes) large enough to test this approach are not currently available, and that screening of ever larger numbers of commercial enzymes has its own cost implications. Thus the Prozomix Biocatalysis Enzyme Toolkit concept was devised to specifically address these issues.
The Prozomix Biocatalysis Enzyme Toolkit Concept: For a Biocatalysis Enzyme Toolkit to be as effective as possible, it must both contain a large number of relevant enzymes, yet be very cost-effective to access by the customer. To achieve the former, it was necessary to develop a proprietary high-throughput cloning approach, now termed GRASP™ (Genomics-based Related Activity Screening Protocol).
The GRASP™ protocol not only allows Prozomix to develop large numbers of related biocatalysts simultaneously, but additionally ensures those enzymes chosen represent as uniform a sampling of "visible primary sequence space", and thus also substrate specificities evolved, as possible. Moving beyond GRASP in 2014, Prozomix phased-in meta-GRASP technology, thus expanding the diversity of biocatalysts presented by the company, by taking advantage of in-house metagenome capabilities. With respect to being cost-effective enough to employ in today's highly competitive environment, Prozomix is offering its Biocatalysis Enzyme Toolkit via a new customer-focused commercial model, where under the terms and conditions of use:
1. ~100 mg of each enzyme as freeze-dried cell-free extract is supplied free-of-charge to the Toolkit Holder. Other advanced screening products can also be acquired free-of-charge, such as "kREDy-to-go™" colorimetric KRED screening plates, and 96-well plates containing 1 mg and / or 5 mg of each enzyme per well.
2. The customer uses the Toolkit components ad libitum for biocatalysis screening activities.
3. When a Toolkit enzyme is identified against an application of interest to the Tollkit Holder, larger quantities of the enzyme can be purchased at known list prices (see example here), or alternatively (for certain existing customers only), a permitted use fee (PUF) can be paid, and the expression construct acquired on non-exclusive basis.
4. A comprehensive range of hit optimisation, biocatalysts discovery / development and contract manufacturing services are also available from Prozomix.
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