Biocatalytic synthesis of (R)-(−)-mandelic acid from racemic mandelonitrile by cetyltrimethylammonium bromide-permeabilized cells of Alcaligenes faecalis ECU0401

Abstract

The nitrilase from Alcaligenes faecalis ECU0401 belongs to the category of arylacetonitrilase, which could hydrolyze 2-chloromandelonitrile, 3,4-dimethoxyphenylacetonitrile, mandelonitrile, and phenylacetonitrile into the corresponding arylacetic acids. To overcome the permeability barrier and prepare whole cell biocatalysts with high activities, permeabilization of Alcaligenes faecalis ECU0401 in relation to nitrilase activity was optimized by using cetyltrimethylammonium bromide (CTAB) as permeabilizing agent. The nitrilase activity from Alcaligenes faecalis ECU0401 increased 4.5-fold when the cells were permeabilized with 0.3% (w/v) CTAB for 20 min at 25 degrees C and pH 6.5. Consequently, almost all the mandelonitrile was consumed and converted to (R)-(-)-mandelic acid with greater than 99.9% enantiomeric excess (e.e.) by the CTAB-permeabilized cells. The permeability barrier has been significantly reduced in the hydrolysis of mandelonitrile by using CTAB-permeabilized cells and a dynamic resolution was successfully achieved, giving a 100% theoretical yield of (R)-(-)-mandelic acid. Efficient biocatalyst recycling was achieved as a result of cell immobilization in calcium alginate, with a product-to-biocatalyst ratio of 3.82 g (R)-(-)-mandelic acid g(-1) dry cell weight (dcw) cell after 20 cycles of repeated use.

Efficient production of (R)-o-chloromandelic acid by deracemization of o-chloromandelonitrile with a new nitrilase mined from Labrenzia aggregata

“…Screening for new enzymes with high selectivity and specific activity is an important prerequisite for their practical utilization. In the case of (R)-o-chloromandelic acid, a key precursor for the synthesis of Clopidogrel®, nitrilase-catalyzing deracemization of o-chloromandelonitrile has significant advantages over other enzymatic routes, but only very few biocatalysts were developed for this synthetic route, usually with low substrate loading (DeSantis et al 2002;He et al 2010). Therefore, lack of an effective biocatalyst fulfilling both the requirements for good ee and for high substrate loading was still the critical factor for practical production of (R)-o-chloromandelic acid via deracemization or dynamic kinetic resolution of o-chloromandelonitrile.…”

“…To the best of our knowledge, only a very few nitrilases as yet are available for the enantioselective hydrolysis of ochloromandelonitrile (DeSantis et al 2002;He et al 2010;Schreiner et al 2010). However, these nitrilases with good enantioselectivity towards o-chloromandelonitrile were often inhibited by the substrate even at a relatively low concentration (10-20 mM).…”

Reconstitution of the In Vitro Activity of the Cyclosporine-Specific P450 Hydroxylase from Sebekia benihana and Development of a Heterologous Whole-Cell Biotransformation System

“…For example, Bagherinejad et al reported that CTAB-treated cells increased the biotransformation of penicillin G by 9% in a penicillin G acylase-containing recombinant E. coli strain (38). The nitrilase activity of Alcaligenes faecalis ECU0401 was increased 4.5-fold when cells were permeabilized by using 0.3% (wt/vol) CTAB (43). A recombinant lipase was expressed in E. coli by using olive oil as the substrate, which gave a conversion rate of almost 100% when cells were treated with 0.3% (wt/vol) CTAB for 20 h, while this rate decreased to Ͻ50% when untreated cells were used (44).…”

Biotransformation of 1,3-Propanediol Cyclic Sulfate and Its Derivatives to Diols by Toluene-Permeabilized Cells of Bacillus sp. CCZU11-1

“…Cell walls of Bacillus sp. CCZU11-1 were disrupted with the ultrasonic cell disruptor (Ningbo Scientz Biotechnology, JY9Z-II, China) in a cold ice-water bath for 30 times at 400 W (working 3 s and intervals 7 s as one cycle [23]). After the treated samples were found to be disrupted entirely with microscope, the cell debris was removed by centrifugation (20,000×g) for 30 min at 4°C, and this supernatant obtained was designated as cell-free extract (CFE).…”