Alkaline a-L-rhamnosidase from Acrostalagmus luteo-albus: Production, purification and characterization

ROJAS, NATALIA LORENA; ELGUEA, LUCIA; FERNANDEZ, MARIA ELENA; ORTIZ, GASTON EZEQUIEL; CONTRERAS ESQUIVEL, JUAN CARLOS; CAVALITTO, SEBASTIÁN FERNANDO; VOGET, CLAUDIO ENRIQUE; HOURS, ROQUE ALBERTO

Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional . Mexico City, Mexico.

Congreso; First International Congress on Biotechnology and Bioengineerin; International initiatives for a Sustainable Development; 2008

&amp;amp;amp;amp;amp;amp;amp;lt;!-- /* Font Definitions */ @font-face {font-family:"Tms Rmn"; panose-1:2 2 6 3 4 5 5 2 3 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-format:other; mso-font-pitch:variable; mso-font-signature:3 0 0 0 1 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Tms Rmn"; mso-fareast-font-family:"Times New Roman"; mso-bidi-font-family:"Times New Roman"; mso-ansi-language:ES-TRAD; mso-fareast-language:ES;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --&amp;amp;amp;amp;amp;amp;amp;gt; <!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:ES-AR; mso-fareast-language:ES-AR;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> <!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:ES-AR; mso-fareast-language:ES-AR;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> a-L-rhamnosidase (Rha, EC 3.2.1.40) is an enzyme of considerable importance in food technology. Despite this industrial interest, only a few crude a-L-rhamnosidase preparations are commercially available so far, specifically the so-called hesperidinase and naringinase. Moreover, all these preparations, currently obtained from Aspergillus and Penicillium genera, contain contaminating b-D-glucosidase activities that can limit their exploitation. The aim of this research is to study the production, purification and characterization of an alkali-tolerant Rha expressed by the filamentous fungi Acrostalagmus luteo-albus (Link: Fr) Zare, Gams et Schroers LPS (cult # 748). In presence of rhamnose as sole carbon source, this fungus produces a Rha of 109 kDa molecular weight and a pI value of 4.6 determined by SDS PAGE and analytical isoelectric focusing, respectively. The biosynthesis of the enzyme was repressed by glucose. This enzyme was purified to homogeneity by chromatographic and electrophoretic techniques. Using p-nitrophenil-a-L-rhamnopiranoside as substrate, the enzyme activity shows pH and temperature optima of 8 and 55 ºC respectively. When tested towards p-nitrophenil-a-L-rhamnopiranoside the enzyme exhibited Michaelis-Menten kinectics with KM and Vmax values of 3.38 mM and 68.5 U ml-1. Divalents cations such as Ca+2, Mg+2, Mn+2, and Co+2 showed no effect over enzyme activity, whereas this was completely inhibited by Zn+2 at a concentration of 0.1 mM. The enzyme was able to release rhamnose from naringin. Sequencing, cloning and expression of this enzyme is now under investigation.