Enzyme and Microbial Technology 39 (2006) 215–221 Synthesis of p-hydroxyphenylglicine by cell extract from Agrobaterium radiobacter encapsulated in alginate capsules I. Aranaz, N. Acosta, A. Heras ∗ Instituto de Estudios Biofuncionales U.C.M., Departamento de F´ ısica Qu´ ımica II, Facultad de Farmacia, Universidad Complutense, Paseo Juan XXIII, Num. 1, 28040 Madrid, Spain Received 3 December 2004; received in revised form 30 September 2005; accepted 21 October 2005 Abstract A crude cell extract from Agrobacterium radiobacter was encapsulated in calcium-alginate capsules for the synthesis of p-hydroxyphenylglicine (p-HPG). The effect of the encapsulation process on the two enzymatic activities involved on the p-HPG synthesis was studied. The encapsulation process was optimised with regard to alginate concentration and ratio extract:alginate. The encapsulation efficiency was around a 60% and the derivatives showed a p-HPG synthesis yield of around a 50%. The optimum temperature was 55 ◦ C and the activation energy (E a ) of the whole process was 4.4 kcal/mol. The biocatalyst could be used six times without any activity loss. © 2005 Elsevier Inc. All rights reserved. Keywords: p-Hydroxyphenylglicine; Encapsulation; d-Hydantoinase; N-carbamyl-d-amino acid amydohydrolase; Alginate 1. Introduction The application of the d-amino acids in the synthesis of chemicals, such as semi-synthetic antibiotics, pesticides and hormones [1] has a great interest for the industry. The d-p- hydroxyphenylglycine (p-HPG) is a lateral chain of -lactam antibiotics, such as amoxicillin and cefadroxil. This molecule can be produced in a hydantoin-transforming reaction by starting from d-hydroxyphenylhydantoin (d-HPH). d-Specific hydan- toinase (E.C.3.5.2.2) converts d-HPH to N-carbamoyl-d-p- hydroxyphenylglycine (C-p-HPG) [2]. l-HPH racemices to d- HPH at basic pH. This intermediate is hydrolyzed to optically pure p-HPG, either chemically with NaNO 2 /HCl [3], or enzymatically with N- carbamyl-d-amino acid amydohydrolase (E.C.3.5.1), hereafter d-case. One of the problems associated with the use of the hydantoin- transforming reaction in the industry is the low thermo-stability and sensibility to the oxidative process of the second enzyme, d-case [4]. Encapsulation of enzymes in alginate gels is characterised by the very mild conditions in which the immobilisation procedure ∗ Corresponding author. Tel.: +34 91394 32 84; fax: +34 91394 32 84. E-mail address: aheras@pluri.ucm.es (A. Heras). is carried out and by its low cost and ease of use. Moreover, it is possible to immobilise several enzymes at the same time. Algi- nate has been used as a matrix for the immobilisation of lipase, glucose oxidase, tannase, tyrosinase and coimmobilisation of glucose oxidase and catalase [5–9]. Recently, Foster et al. have reported the first encapsulation of Agrobacterium tumefaciens extract in alginate capsules to synthesise glycine [10]. These authors studied the effect of encapsulation in the d-hydantoinase and the d-case activities but the whole process was not studied. In this paper, calcium-alginate capsules have been used as a matrix for the crude cell extract encapsulation for the synthesis of p-HPG. The effect of alginate concentration has been studied to find the most suitable conditions for the synthesis of p-HPG. 2. Materials and methods 2.1. Materials 2.1.1. Chemicals Sodium alginate (medium viscosity) and the bicinchoninic acid protein assay kit (Kit num. BCA-1) were supplied by Sigma. Methanol (HPLC-gradient) was purchased from Panreac. Other chemicals were of analytical grade. 2.1.2. Biological material Crude extracts from over-expressed Agrobacterium radiobacter were pre- pared as previously described [11]. Briefly, A. radiobacter was grown at 30 ◦ C 0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.10.022