A recombinant tannase from Lactobacillus plantarum, overexpressed in Escherichia coli, was
purified in a single step by metal chelate affinity chromatography on poorly activated nickel supports.
It was possible to obtain 0.9 g of a pure enzyme by using only 20 mL of chromatographic support.
The pure enzyme was immobilized and stabilized by multipoint covalent immobilization on highly
activated glyoxyl agarose. Derivatives obtained by multipoint and multisubunit immobilization were
500- and 1000-fold more stable than both the soluble enzyme and the one-point-immobilized
enzyme in experiments of thermal and cosolvent inactivation, respectively. In addition, up to 70 mg
of pure enzyme was immobilized on 1 g of wet support. The hydrolysis of tannic acid was optimized
by using the new immobilized tannase derivative. The optimal reaction conditions were 30% diglyme
at pH 5.0 and 4 C. Under these conditions, it was possible to obtain 47.5 mM gallic acid from 5 mM
tannic acid as substrate. The product was pure as proved by HPLC. On the other hand, the
immobilized biocatalyst preserved >95% of its initial activity after 1 month of incubation under the
optimal reaction conditionsThis work was supported by
Grants AGL2008-01052, AGL-2009-07625, Consolider INGENIO 2010
CSD2007-00063 FUN-C-FOOD(CICYT),RM2008-00002 (INIA), and
S-0505/AGR/000153 (CAM). J.A.C. is the recipient of a predoctoral
fellowship from the I3P-CSIC Program and FPI-MEC, and G.F.-L. and
L.B. are recipients of Ramon y Cajal postdoctoral contracts.Peer reviewe
