Journal of Molecular Catalysis B: Enzymatic 63 (2010) 109–115 Contents lists available at ScienceDirect Journal of Molecular Catalysis B: Enzymatic journal homepage: www.elsevier.com/locate/molcatb Immobilization of Geobacillus pallidus RAPc8 nitrile hydratase (NHase) reduces substrate inhibition and enhances thermostability Idan Chiyanzu a , Don A. Cowan b , Stephanie G. Burton c,∗ a Department of Chemical Engineering, University of Cape Town, Rondebosch 7701, Cape Town, South Africa b Institute for Microbial Biotechnology and Metagenomics, University of Western Cape, Bellville 7535, Cape Town, South Africa c Biocatalysis and Technical Biology Group, Cape Peninsula University of Technology, Bellville 7535, Cape Town, South Africa article info Article history: Received 26 September 2009 Received in revised form 9 December 2009 Accepted 10 December 2009 Available online 21 December 2009 Keywords: Nitrile hydratase Geobacillus pallidus RAPc8 Immobilization Eupergit Thermostable enzyme Thermophilic abstract The nitrile hydratase (NHase) from the thermophilic strain Geobacillus pallidus RAPc8 was investigated for its potential application in the biocatalytic production of amides from nitriles. The recombinant NHase was immobilized to a range of insoluble matrices using various cross-linking agents. The immobilized preparation using Eupergit ® C with 1-ethyl-3-(dimethylamino-propyl) carbodiimide (EDAC) cross-linking exhibited the highest immobilization efficiency (93%). The pH range and optimal tempera- ture for activity were unchanged by immobilization but the thermostability of the Eupergit ® C-NHase was improved compared with the soluble enzyme; at 60 ◦ C the half-life of the immobilized NHase was 330 min as compared with 54.5 min for the soluble enzyme. Kinetic parameters V max (4.5 mol mL -1 min -1 ), K m (17.3 mM) and k cat (3543.3 min -1 ) were obtained for the immobilized NHase at 50 ◦ C, as compared with 48.8 mol mL -1 min -1 , 10.2 mM and 37777.1 min -1 respectively for the soluble enzyme. The operational stability was improved significantly by immobilization, with 85.7% of initial activity maintained after reuse for eight cycles. Most notably, the Eupergit ® C-immobilized NHase showed substantially lower substrate inhibition (K i = 194.7 mM) than the soluble enzyme (K i = 101.0 mM). In the presence of various co-organic solvents, Eupergit ® C-EDAC NHase showed statistically higher retention of activity than the non-immobilized control. © 2009 Elsevier B.V. All rights reserved. 1. Introduction The nitrile hydratases (NHases: EC3.5.5.1) catalyze the hydra- tion of nitriles to their corresponding amides [1]. The versatile nature of these enzymes, associated with their very broad substrate specificity, has resulted in a number of commercial applications, including manufacture of the commodity chemicals acrylamide [2], nicotinamide [3] and 5-cyanovaleramide [3], and in treatment of organocyanide industrial effluents [4]. However, the instability of many NHases currently limits successful application at com- mercial scale, particularly in the production of acrylamide and nicotinamide [5–7]. Consequently, whole-cells are often employed, in processes which are operated at relatively low temperatures [3], resulting in reduced reactivity due to diffusion limitation and low specific activity [8]. Further, many NHases are inhibited at high concentrations of substrate and product [9]. Attempts to overcome substrate and product inhibition have been made by keeping these concentrations low [10], but this compromises industrial capacity for bulk amide production. ∗ Corresponding author. E-mail address: Burtons@cput.ac.za (S.G. Burton). More recently, isolated enzymes with high selectivity have been used effectively to catalyse industrial reactions and high volumet- ric productivity has been achieved by increased biocatalyst loading [11]. However, in view of the noted instability of NHases, if isolated and purified NHases are to be applied in industry, enhancing their stability is seen as one prerequisite for their effective use [12]. The use of thermostable enzymes isolated from thermophilic micro- bial sources presents one opportunity to achieve higher stability. Further, immobilized biocatalysts are often regarded as the appro- priate form for application of enzymes in industry as they can be reused, are readily separated from reaction mixtures, are conve- nient to handle, and allow reduced effluent disposal problems [13]. In laboratory applications, NHases have commonly been used as immobilized whole cell biocatalysts entrapped in calcium alginate [14,15] or polyacrylamide gels [16]. A thermostable NHase was isolated from the moderate ther- mophile Geobacillus pallidus RAPc8 [17], the gene has been cloned and over-expressed in E. coli BL21 (DE3) [18]. The NHase con- sists of two subunits ( and ) having M r of 28 and 29 kDa where the functional structure is heterotetrameric () 2 [19]. The active site of the G. pallidus RAPc8 NHase, which is located at interface of the - and -subunits, contains a non-corrinoid cobalt (Co III ), chelated cysteine sulfinic acid residues and main-chain nitrogen 1381-1177/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.molcatb.2009.12.011