Nitrilase Improvement

This method exhibits certain potential in exploring novel enzymes with required specificities from unannotated sequences. 

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Existing synthetic methods and commercial processes that employ nitrile hydratases (NHases) and nitrilases continue to be improved by directed evolution of existing enzymes, or by the discovery of new enzymes with improved properties, and new applications of these catalysts have recently been described. Numerous reviews have previously been published that describe applications of NHase [ 1-6] and nitrilase [ 1,4—11 ], and in this review we present examples of new applications of these nitrile-utilizing catalysts from journal articles, patent applications, and issued patents that have been published in the past 2-3 years. 

Recently, nitrilases have been applied to polymer modification, specifically to the modification of polyacrylonitrile (PAN). Nearly 3 x 106 tons of PAN are produced per annum and used in the textile industry. However, there is a great need to improve moisture uptake, dyeability with ionic dyes, and feel of this acrylic fiber. The cyano moieties of PAN have been successfully modified to carboxylates with the commercial Cyanovacta nitrilase, thus enhancing the aforementioned properties of PAN [98]. Nitrilase action on the acrylic fabric was improved... 

Recently, another interesting application of nitrilases has been demonstrated for the synthesis of pregabalin-the API of the neurophatic pain drug Lyrica. In this approach, the key step is the resolution of racemic isobutylsuccinonitrile (Scheme 10.8) [18], the process takes place with total regio- and stereoselectivity, and the (S)-acid is obtained and the (R)-substrate can be recycled under basic conditions. To improve the biocatalytic step, directed evolution was applied using the electronic polymerase chain reaction and in the first round of evolution a single C236S mutation led to a mutant with 3-fold increase in activity [19]. 

Chemical reactions enhanced by catalysts or enzymes are an integral part of the manufacturing processes for the majority of chemical products. The total market for catalysts and enzymes amounts to 11.5 billion (2005), of which catalysts account for about 80%. It consists of four main applications environment (e.g., automotive catalysts), 31% polymers (e.g., polyethylene and polypropylene), 24% petroleum processing (e.g., cracking and reforming), 23% and chemicals, 22%. Within the latter, particularly the catalysts and enzymes for chiral synthesis are noteworthy. Within catalysts, BINAPs [i.e., derivatives of 2,2 -bis(diphenylphosphino) -1, l -bis-l,l -binaphthyl) have made a great foray into chiral synthesis. Within enzymes, apart from bread-and-butter products, like lipases, nitrilases, acylases, lactamases, and esterases, there are products tailored for specific processes. These specialty enzymes improve the volumetric productivity 100-fold and more. Fine-chemical companies, which have an important captive use of enzymes, are offering them to third parties. Two examples are described here ... 

A method of efficient induction of fungal nitrilases in wild strains was established and seems to be of wide application throughout this enzyme group. However, heterologous expression of fungal nitrilases remains to be solved in order to facilitate the use of these enzymes in industrial biotechnology. To what extent the desirable properties (broad substrate specificity, thermostability, chemoselectivity and enantioselectivity) can be improved by mutagenesis of these enzymes should also be examined. 

The spectrum of known nitrilases and cyanide hydratases currently consists of a significant part of enzymes obtained by genome mining. These enzymes were, in many cases, helpful to the improvement of processes employing the hydrolysis of... 

A different method consisting of directed evolution was used with the nitrilases from P. fluorescens [26] and Alcaligenes faecalis [27]. The amide production by the former and the operational stability of the latter at a low pH were improved in this... 

Nitrilase production as well as its catalytic properties can be improved through culture optimization at the fermentation level and gene modification at the... 

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