Cross‐Linked Amorphous Nitrilase Aggregates for Enantioselective Nitrile Hydrolysis

Abstract

A recombinant Escherichia coli strain was constructed which efficiently expressed the enantioselective nitrilase from Alcaligenes faecalis DSMZ 30030 as a hisitidine-tagged enzyme variant under the control of a rhamnose inducible promoter. The enzyme was purified from cell extracts and used for the preparation of cross-linked enzyme aggregates (CLEAs). The conditions for the preparation of the CLEAs were optimized using various organic solvents and cross-linking agents and a procedure was developed which combined a precipitation with 85 % (v/v) isopropyl alcohol and a cross-linking with 30 mM glutaraldehyde. Thus, about 80 % of the initial nitrilase activity could be incorporated into the CLEAs. The hydrolysis of racemic mandelonitrile to (R)-mandelic acid was compared between the soluble nitrilase preparations and their CLEAs (nitCLEAs). The nitrilase activity in the CLEAs was at 30 8C and 608C about 5 times more stable than in the soluble preparations. The CLEAs could be reused 5 times with only about 10 % reduction in activity. The enantioselectivity of the nitrilase for the formation of (R)-mandelic acid from racemic mandelonitrile decreased for both preparations with increasing temperatures (10 8C to 508C), but this effect was significantly less pronounced for the CLEAs. A detailed analysis of solvent effects on nitrilase enantioselectivity allowed thermodynamic insights into contributions from free energy component (activation enthalpy and entropy) to chiral preference of nitrilase in such non conventional media.

Advances in enzyme immobilisation

“…A less-expensive method of enhancing enzyme proximity for cross-linking is by simply precipitating the protein and cross-linking the aggregates to form particles of about 50-100 lm diameter (López-Serrano et al 2002;Kaul et al 2007). These crosslinked enzyme aggregates (CLEA) were developed in Sheldon's laboratory (Cao et al 2001;Sheldon et al 2005; Fig.…”

“…4) and commercialised by CLEA Technologies (Netherlands). Examples of enzymes immobilised this way include nitrile hydratase (Kubáč et al 2008) lipase (López-Serrano et al 2002, nitrilase (Kaul et al 2007), penicillin acylase (Pchelintsev et al 2009), amino acylase (Bode et al 2003) and others (Sheldon 2007b).…”

“…Cross-linkers (such as glutaraldehyde, glutaraldehyde-ethylene diamine polymers, or dextran aldehyde) may be selected for optimal activity of a specific enzyme (Kaul et al 2007). Pchelintsev et al (2009) found that variation in the duration of the precipitation step prior to cross-linking influenced the activity and microstructure of penicillin acylase CLEA.…”

Identification of Amino Acid Residues Responsible for the Enantioselectivity and Amide Formation Capacity of the Arylacetonitrilase fromPseudomonas fluorescensEBC191

“…This trait has been used for the industrial production of (substituted) (R)-mandelic acid(s) from racemic (substituted) mandelonitrile(s) by dynamic kinetic resolution processes using different microorganisms (often strains of Alcaligenes faecalis) (19,34; M. Ress-Löschke, T. Friedrich, B. Hauer, and R. Mattes, 1998, DE19848129A1, German Patent Office). An enantioselective nitrilase from A. faecalis ATCC 8750 has been purified and characterized, and the encoding gene has been cloned (4,11,26,33).…”

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

“…1) [9][10][11]. Recently we explored solvent eVects on nitrilase enantioselectivity which allowed us to segregate contributions from activation enthalpy and entropy and elucidating the source of its high enantioselectivity [12]. (R)-(-)-mandelic acid is a key intermediate for the production of semi-synthetic cephalosporins [13] and penicillins [14].…”