Based on the principles and metrics of green chemistry and sustainable development, biocatalysis is both a green and sustainable technology. This is largely a result of the spectacular advances in molecular biology and biotechnology achieved in the past two decades. Protein engineering has enabled the optimization of existing enzymes and the invention of entirely new biocatalytic reactions that were previously unknown in Nature. It is now eminently feasible to develop enzymatic transformations to fit predefined parameters, resulting in processes that are truly sustainable by design. This approach has successfully been applied, for example, in the industrial synthesis of active pharmaceutical ingredients. In addition to the use of protein engineering, other aspects of biocatalysis engineering, such as substrate, medium, and reactor engineering, can be utilized to improve the efficiency and cost-effectiveness and, hence, the sustainability of biocatalytic reactions. Furthermore, immobilization of an enzyme ...

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Role of Biocatalysis in Sustainable Chemistry

Enzymes as catalysts in synthetic organic chemistry gained importance in the latter half of the 20th century, but nevertheless suffered from two major limitations. First, many enzymes were not accessible in large enough quantities for practical applications. The advent of recombinant DNA technology changed this dramatically in the late 1970s. Second, many enzymes showed a narrow substrate scope, often poor stereo- and/or regioselectivity and/or insufficient stability under operating conditions. With the development of directed evolution beginning in the 1990s and continuing to the present day, all of these problems can be addressed and generally solved. The present Perspective focuses on these and other developments which have popularized enzymes as part of the toolkit of synthetic organic chemists and biotechnologists. Included is a discussion of the scope and limitation of cascade reactions using enzyme mixtures in vitro and of metabolic engineering of pathways in cells as factories for the production o...

Biocatalysis in Organic Chemistry and Biotechnology: Past, Present, and Future

Biocatalysis has found numerous applications in various fields as an alternative to chemical catalysis. The use of enzymes in organic synthesis, especially to make chiral compounds for pharmaceuticals as well for the flavors and fragrance industry, are the most prominent examples. In addition, biocatalysts are used on a large scale to make specialty and even bulk chemicals. This review intends to give illustrative examples in this field with a special focus on scalable chemical production using enzymes. It also discusses the opportunities and limitations of enzymatic syntheses using distinct examples and provides an outlook on emerging enzyme classes.

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Biocatalysis: Enzymatic Synthesis for Industrial Applications

Anatomy and physiology of the eye makes it a highly protected organ. Designing an effective therapy for ocular diseases, especially for the posterior segment, has been considered as a formidable task. Limitations of topical and intravitreal route of administration have challenged scientists to find alternative mode of administration like periocular routes. Transporter targeted drug delivery has generated a great deal of interest in the field because of its potential to overcome many barriers associated with current therapy. Application of nanotechnology has been very promising in the treatment of a gamut of diseases. In this review, we have briefly discussed several ocular drug delivery systems such as microemulsions, nanosuspensions, nanoparticles, liposomes, niosomes, dendrimers, implants, and hydrogels. Potential for ocular gene therapy has also been described in this article. In near future, a great deal of attention will be paid to develop non-invasive sustained drug release for both anterior and posterior segment eye disorders. A better understanding of nature of ocular diseases, barriers and factors affecting in vivo performance, would greatly drive the development of new delivery systems. Current momentum in the invention of new drug delivery systems hold a promise towards much improved therapies for the treatment of vision threatening disorders.

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Recent Perspectives in Ocular Drug Delivery

The review compiles artificial cascades involving enzymes with a focus on the last 10 years. A cascade is defined as the combination of at least two reaction steps in a single reaction vessel without isolation of the intermediates, whereby at least one step is catalyzed by an enzyme. Additionally, cascades performed in vivo and in vitro are discussed separately, whereby in vivo cascades are defined here as cascades relying on cofactor recycling by the metabolism or on a metabolite from the living organism. The review introduces a systematic classification of the cascades according to the number of enzymes in the linear sequence and differentiates between cascades involving exclusively enzymes and combinations of enzymes with non-natural catalysts or chemical steps. Since the number of examples involving two enzymes is predominant, the two enzyme cascades are further subdivided according to the number, order, and type of redox steps. Furthermore, this classification differentiates between cascades where al...

Artificial Biocatalytic Linear Cascades for Preparation of Organic Molecules

A leucine dehydrogenase has been successfully altered through several rounds of protein engineering to an enantioselective amine dehydrogenase. Instead of the wild-type α-keto acid, the new amine dehydrogenase now accepts the analogous ketone, methyl isobutyl ketone (MIBK), which corresponds to exchange of the carboxy group by a methyl group to produce chiral (R)-1,3-dimethylbutylamine.

Development of an amine dehydrogenase for synthesis of chiral amines

Status of protein engineering for biocatalysts: how to design an industrially useful biocatalyst

The reductive amination of ketones to produce chiral amines is an important transformation in the production of pharmaceutical intermediates. Therefore, industrially applicable enzymatic methods that enable the selective synthesis of chiral amines could be very useful. Using a phenylalanine dehydrogenase scaffold devoid of amine dehydrogenase activity, a robust amine dehydrogenase has been evolved with a single two-site library allowing for the direct production of (R)-1-(4-fluorophenyl)-propyl-2-amine from para-fluorophenylacetone with a kcat value of 6.85 s−1 and a KM value of 7.75 mM for the ketone substrate. This is the first example of a highly active amine dehydrogenase capable of accepting aliphatic and benzylic ketone substrates. The stereoselectivity of the evolved amine dehydrogenase was very high (>99.8% ee) showing that high selectivity of the wild-type phenylalanine dehydrogenase was conserved in the evolution process. When paired with glucose/glucose dehydrogenase, NADH cofactor can be effficiently regenerated and the reaction driven to over 93% conversion. The broad specificity, high selectivity, and near complete conversion render this amine dehydrogenase an attractive target for further evolution toward pharmaceutical compounds and subsequent application.

The Evolution of an Amine Dehydrogenase Biocatalyst for the Asymmetric Production of Chiral Amines

Approximately 90% of all ophthalmic drug formulations are now applied as eye-drops While eye-drops are convenient and well accepted by patients, about 95% of the drug contained in the drops is lost due to absorption through the conjunctiva or through the tear drainage A major fraction of the drug eventually enters the blood stream and may cause side effects [JC Lang, Adv Drug Delivery Rev 16 (1995) 39-43; C Bourlais, L Acar, H Zia, PA Sado, T Needham, R Leverge, Prog Retinal Eye Res 17 (1998) 33-58; MP Segal, FDA Consumer Mag (1991)] The drug loss and the side effects can be minimized by using microemulsion-laden soft contact lenses for ophthalmic drug delivery [D Gulsen, A Chauhan, Invest Ophthalmol Vis Sci 45 (2004) 2342-2347; D Gulsen, A Chauhan, Abstr Pap Am Chem Soc 227 (2004) U875] In order for microemulsion-laden gels to be effective, these should load sufficient quantities of drug and should release it a controlled manner The presence of a tightly packed surfactant at the oil-water interface of microemulsions may provide barrier to drug transport, and this could be used to control the drug delivery rates In this paper we focus on trapping ethyl butyrate in water microemulsions stabilized by Pluronic F127 surfactant in 2-hydroxyethyl methacrylate (HEMA) gels and measuring the transport rates of timolol, which is a beta-blocker drug that is used for treating a variety of diseases including glaucoma The results described here show that microemulsion-laden gels could have high drug loadings, particularly for drugs such as timolol base which can either be dissolved in the oil phase or form the oil phase of the microemulsions However, the surfactant covered interface of the Pluronic microemulsions does not provide sufficient barrier to impede the transport of timolol, perhaps due to the small size of this drug

Michael J. Abrahamson

Papers

Development of an amine dehydrogenase for synthesis of chiral amines

Status of protein engineering for biocatalysts: how to design an industrially useful biocatalyst

The Evolution of an Amine Dehydrogenase Biocatalyst for the Asymmetric Production of Chiral Amines

Timolol transport from microemulsions trapped in HEMA gels.

Entwicklung einer Amindehydrogenase zur Synthese von chiralen Aminen