Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 81:1836–1839 (2006) Continuous biotransformation of pyrogallol to purpurogallin using cross-linked enzyme crystals of laccase as catalyst in a packed-bed reactor J Jegan Roy and T Emilia Abraham ∗ Chemical Sciences Division, Regional Research Laboratory (CSIR), Trivandrum 695 019, India Abstract: Cross-linked enzyme crystals (CLEC) of laccase were prepared by crystallizing laccase with 75% (NH 4 ) 2 SO 4 and cross-linking using 1.5% glutaraldehyde. The cross-linked enzyme crystals were further coated with 1 mmol L -1 β -cyclodextrin by lyophilization. The lyophilized enzyme crystals were used as such for the biotransformation of pyrogallol to purpurogallin in a packed-bed reactor. The maximum conversion (76.28%) was obtained with 3 mmol L -1 pyrogallol at a residence time of 7.1 s. The maximum productivity (269.03 g L -1 h -1 ) of purpurogallin was obtained with 5 mmol L -1 pyrogallol at a residence time of 3.5 s. The productivity was found to be 261.14 g L -1 h -1 and 251.1 g L -1 h -1 when concentrations of 3 mmol L -1 and 7 mmol L -1 respectively were used. The reaction rate of purpurogallin synthesis was maximum (2241.94 mg purpurogallin mg -1 CLEC h -1 ) at a residence time of 3.5 s, when 5 mmol L -1 pyrogallol was used as the substrate. The catalyst to product ratio calculated for the present biotransformation was 1:2241. The CLEC laccase had very high stability in reuse and even after 650 h of continuous use, the enzyme did not lose its activity.  2006 Society of Chemical Industry Keywords: biotransformation; cross-linked enzyme crystals (CLEC) laccase; purpurogallin; packed-bed reactor; enzyme biocatalysis INTRODUCTION Biotransformations using enzymes represent a useful tool for the production of various chemicals such as pharmaceuticals, agrochemicals, fragrances and flavors, and consumer care products, because of their advantages such as regio-stereo- and chemo- selectivity. Immobilization 1 of enzymes is one of the techniques used by industry to bring down the cost of the process by reusing the enzymes in biotransformations. However, immobilization has a few disadvantages such as dilution of catalyst, instability in higher temperatures, and low activity loading 2 on the support (0.1–10% w/w). Cross- linked enzyme crystals (CLEC) technology 3 is one of the most exciting developments in the area of biocatalysis. Cross-linking 4 of enzyme crystals brings about both stabilization and immobilization of enzymes without dilution of activity. Cross-linked enzyme crystals are prepared by controlled precipitation of enzymes into micro-crystals followed by cross-linking using bifunctional reagents to form strong covalent bonds between the ε-amino groups of lysine residues. 5 In a CLEC, the lattice interactions in the enzyme crystal provide additional physical and thermal stability 6 when fixed by inter- and intra-molecular chemical cross-links. In an immobilized enzyme, the enzyme is linked by point attachment to a two-dimensional solid surface, but protein in cross-linked crystals is stabilized by links in all three dimensions of its structure. Hence, CLECs are highly active, recyclable and have good mechanical stability. Cross-linked enzyme crystals have the additional advantages 7 of good stability in solvents, and being stable under conditions of shear stress and various temperature and storage conditions. Cross-linked enzyme crystals are mainly used in biotransformations 8 because of the use of high con- centrations of organic solvents and substrates where the soluble enzyme cannot be used. Biotransforma- tions using CLEC enzymes have been performed for peptide synthesis, chiral resolution, chemo- and regio-selective transformation, carbon–carbon bond formation, reduction reactions and so forth. 9 Laccase (benzenediol:oxygen oxidoreductase (EC 1.10.3.2), a polyphenol oxidase, belongs to the family of blue multi-copper oxidases. It is one of the blue copper oxidases which catalyze the four-electron reduction of oxygen to water. Laccase can catalyze ortho- and para- diphenols and substituted phenols to ortho- and para- quinones respectively. Generally, laccase-catalyzed oxidation of pyrogallol to purpurogallin is used for the assay of laccase enzyme. No reports of the chem- ical synthesis of purpurogallin could be found in the literature. However, it was originally isolated from the ∗ Correspondence to: T Emilia Abraham, Chemical Sciences Division, Regional Research Laboratory (CSIR), Trivandrum 695 019, India E-mail: emiliatea@yahoo.com Contract/grant sponsor: CSIR, India (Received 16 February 2006; revised version received 2 May 2006; accepted 9 May 2006) DOI: 10.1002/jctb.1612  2006 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2006/$30.00