Correction to: Release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126: a comparative study.

In the original version of our paper entitled "Release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126: a comparative study" (2005) 27:415-424, some references to already published articles were inadvertently left out.

Strategies for discovery and improvement of enzyme function: state of the art and opportunities.

Developments in biocatalysis have been largely fuelled by consumer demands for new products, industrial attempts to improving existing process and minimizing waste, coupled with governmental measures to regulate consumer safety along with scientific advancements. One of the major hurdles to application of biocatalysis to chemical synthesis is unavailability of the desired enzyme to catalyse the reaction to allow for a viable process development. Even when the desired enzyme is available it often forces the process engineers to alter process parameters due to inadequacies of the enzyme, such as instability, inhibition, low yield or selectivity, etc. Developments in the field of enzyme or reaction engineering have allowed access to means to achieve the ends, such as directed evolution, de novo protein design, use of non-conventional media, using new substrates for old enzymes, active-site imprinting, altering temperature, etc. Utilization of enzyme discovery and improvement tools therefore provides a feasible means to overcome this problem. Judicious employment of these tools has resulted in significant advancements that have leveraged the research from laboratory to market thus impacting economic growth; however, there are further opportunities that have not yet been explored. The present review attempts to highlight some of these achievements and potential opportunities.

Enantioselective nitrilase from Pseudomonas putida: cloning, heterologous expression, and bioreactor studies.

Nitrilases have attracted tremendous attention for the preparation of optically pure carboxylic acids. This article aims to address the production and utilization of a highly enantioselective nitrilase from Pseudomonas putida MTCC 5110 for the hydrolysis of racemic mandelonitrile to (R)-mandelic acid. The nitrilase gene from P. putida was cloned in pET 21b(+) and over-expressed as histidine-tagged protein in Escherichia coli. The histidine-tagged enzyme was purified from crude cell extracts of IPTG-induced cells of E. coli BL21 (DE3). Inducer replacement studies led to the identification of lactose as a suitable and cheap alternative to the costly IPTG. Effects of medium components, various physico-chemical, and process parameters (pH, temperature, aeration, and agitation) for the production of nitrilase by engineered E. coli were optimized and scaled up to a laboratory scale bioreactor (6.6 l). Finally, the recombinant E. coli whole-cells were utilized for the production of (R)-(-)-mandelic acid.

Predicting enzyme behavior in nonconventional media: correlating nitrilase function with solvent properties.

The insolubility of nitrile substrates in aqueous reaction mixture decreases the enzymatic reaction rate. We studied the interaction of fourteen water miscible organic solvents with immobilized nitrile hydrolyzing biocatalyst. Correlation of nitrilase function with physico-chemical properties of the solvents has allowed us to predict the enzyme behavior in such non-conventional media. Addition of organic solvent up to a critical concentration leads to an enhancement in reaction rate, however, any further increase beyond the critical concentration in the latter leads to the decrease in catalytic efficiency of the enzyme, probably due to protein denaturation. The solvent dielectric constant (epsilon) showed a linear correlation with the critical concentration of the solvent used and the extent of nitrile hydrolysis. Unlike alcohols, the reaction rate in case of aprotic solvents could be linearly correlated to solvent log P. Further, kinetic analysis confirmed that the affinity of the enzyme for its substrate (K (m)) was highly dependent upon the aprotic solvent used. Finally, the prospect of solvent engineering also permitted the control of enzyme enantioselectivity by regulating enantiomer traffic at the active site.

Studies on the production of enantioselective nitrilase in a stirred tank bioreactor by Pseudomonas putida MTCC 5110.

Nitrilases constitute an important class of hydrolases, having numerous industrial applications. The present work aims to address the production of nitrile hydrolyzing enzymes from Pseudomonas putida MTCC 5110 in a 6l bioreactor. Effect of various physico-chemical conditions and process parameters like pH, temperature, aeration and agitation rates and inducer concentration was studied. Further, the enzyme activity was enhanced by adopting the inducer feeding strategy. Various biochemical engineering parameters pertaining to the cultivation of P. putida in different physico-chemical conditions were reported. Finally, segregation of growth phase from the enzyme production phase allowed significant reduction in total fermentation time.

Stereoselective nitrile hydrolysis by immobilized whole-cell biocatalyst.

The present work attempts to deal with the stability and reusability aspect of nitrilase from Alcaligenes faecalis for the production of (R)-(-)-mandelic acid. Four entrapment matrixes were screened to search for a suitable support, and alginate was found to have significant process advantages over its other counterparts. Thermodynamic analysis allowed us to account for decreased enantioselectivity (E) as a result of immobilization. The system was also characterized based on the Thiele modulus (phi). Efficient reusability of the biocatalyst up to 35 batches was achieved by immobilization as compared to 9 batches for free cells, and cross-linking extended it further to 40 batches. Finally, synthetic utility of the immobilized biocatalyst was demonstrated on a preparative scale to produce 640 g of (R)-(-)-mandelic acid with 97% enantiomeric excess (ee).

Enhancing the catalytic potential of nitrilase from Pseudomonas putida for stereoselective nitrile hydrolysis.

(R)-mandelic acid was produced from racemic mandelonitrile using free and immobilized cells of Pseudomonas putida MTCC 5110 harbouring a stereoselective nitrilase. In addition to the optimization of culture conditions and medium components, an inducer feeding approach is suggested to achieve enhanced enzyme production and therefore higher degree of conversion of mandelonitrile. The relationship between cell growth periodicity and enzyme accumulation was also studied, and the addition of the inducer was delayed by 6 h to achieve maximum nitrilase activity. The nitrilase expression was also authenticated by the sodium dodecyl phosphate-polyacrylamide gel electrophoresis analysis. P. putida MTCC 5110 cells were further immobilized in calcium alginate, and the immobilized biocatalyst preparation was used for the enantioselective hydrolysis of mandelonitrile. The immobilized system was characterized based on the Thiele modulus (phi). Efficient biocatalyst recycling was achieved as a result of immobilization with immobilized cells exhibiting 88% conversion even after 20 batch recycles. Finally, a fed batch reaction was set up on a preparative scale to produce 1.95 g of (R)-(-)-mandelic acid with an enantiomeric excess of 98.8%.

Purification and characterization of an enantioselective arylacetonitrilase from Pseudomonas putida.

The highly enantioselective arylacetonitrilase of Pseudomonas putida was purified to homogeneity using a combination of (NH4)2SO4 fractionation and different chromatographic techniques. The enzyme has a molecular weight of 412 kDa and consisted of approximately nine to ten identical subunits (43 kDa). The purified enzyme exhibited a pH optimum of 7.0 and temperature optimum of 40 degrees C. The nitrilase was highly susceptible to thiol-specific reagents and metal ions and also required a reducing environment for its activity. These reflected the presence of a catalytically essential thiol group for enzyme activity which is in accordance with the proposed mechanism for nitrilase-catalyzed reaction. The enzyme was highly specific for arylacetonitriles with phenylacetonitrile and its derivatives being the most preferred substrates. Higher specificity constant (kcat/K(m)) values for phenylacetonitrile compared to mandelonitrile also revealed the same. Faster reaction rate achieved with this nitrilase for mandelonitrile hydrolysis was possibly due to the low activation energy required by the protein. Incorporation of low concentration (<5%) of organic solvent increased the enzyme activity by increasing the availability of the substrate. Higher stability of the enzyme at slightly alkaline pH and ambient temperature provides an excellent opportunity to establish a dynamic kinetic resolution process for the production of (R)-(-)-mandelic acid from readily available mandelonitrile.

Release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126: a comparative study.

Nitrilases constitute an important class of hydrolases, however, cheap and ready availability of enzyme sources limit their practical synthetic applications. The present investigation was directed to compare the applicability of various physical cell disintegration methods namely, solid shear, liquid shear and sonication, for the release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126. Different parameters associated with each method were optimized in order to ensure maximal release of active nitrilase. The methods were also compared under optimal conditions for their efficiency of nitrilase release and extent of cell disruption, and enzyme release were visualized under a differential interference contrast microscope (DIC) and SDS-PAGE, respectively. Maximum release of the enzyme protein from the cells was observed in case of liquid shear method employing high-pressure homogenization, however, the specific activity of nitrilase was highest in cell-free extract (CFE) generated by sonication. Both the solid shear and liquid shear proved to be equally effective for maximum release of intracellular enzymes, however, from the specific activity point of view, sonication was found to be a better one compared to other two methodologies. The generated cell-free extract can be further employed for the production of enantiopure chiral carboxylic acids, which are important chiral building blocks.

A high-throughput amenable colorimetric assay for enantioselective screening of nitrilase-producing microorganisms using pH sensitive indicators.

Based on the color change of an indicator due to the release of hydrogen ion from a nitrilase-catalyzed reaction, a rapid colorimetric method was established for the enantioselective screening of nitrilase-producing microorganisms. The formation of acids due to the nitrilase-mediated hydrolysis of nitriles causes a drop in the pH, which in turn results in a change of color of the solution (containing indicator) that can be observed visually. The buffer (0.01 M phosphate, pH 7.2) and indicator (Bromothymol blue, 0.01%) were selected in such a way that both have the same affinity for the released protons. The enantioselectivity of nitrilases was estimated by comparing the hydrolysis of (R)-mandelonitrile with that of racemate under the same conditions. The method was used to screen a library of nitrilase-producing microorganisms, isolated in the authors' laboratory for their ability to enantioselectively hydrolyze mandelonitrile to mandelic acid, an important chiral building block.