Production and biochemical characterization of the recombinant enzyme arabinofuranosídeo arabinofuranohidrolase derived from Bacillus subtilis

• Grant number: 13/07518-5
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: May 01, 2013
• End date: October 31, 2013
• Field of knowledge: Physical Sciences and Mathematics - Chemistry
• Principal Investigator: Richard John Ward
• Grantee: Sthefani Balero dos Santos Peria
• Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

The plant cell wall consists of cellulose microfibrils embedded in an amorphous matrix composed of hydrated polymers and hemicellulose, pectin, lignin, and glycoprotein. Among these carbohydrates forming polymers that make up the structure of the plant cell wall are glucose, galactose, fucose, and mannose, with six carbons and xylose and arabinose with five carbons. In the group of pectins found ramnogalacturonanos, arabinan, galactan and arabinogalactan. These carbohydrates are arranged in the skeletons of polymers (substituted or unsubstituted) forming an entangled polymer complex comprising the plant cell wall. For this structure to be undone and carbohydrates are exposed, there are different methods - physical and chemical - among which stand out the enzymatic method for easy implementation by not generating toxic waste, and the high specificity. An enzyme capable of acting on the deconstruction of this substrate is heterogeneous arabinofuranosidase that targets arabinoses O-2 or O-3 monosubstituted. Thus, this work is presenting the proposal to study the enzyme arabinofuranosídeo arabinofuranohidrolase (or arabinofuranosidase) of Bacillus subtilis. This arabinofuranosidase comprises a structure with two domains: a binding domain for carbohydrate (carbohydrate-binding module - CBM) belonging to family 6, and a catalytic domain. Its structure is predominantly ²-sheets, with its crystal deposited in the Protein Data Bank under the code 3C7G. This project aims to clone, express, purify and biochemically characterize the arabinofuranosidase from Bacillus subtilis, as well as determine its kinetic parameters and their catalytic performance. With this data in hand, it is intended to investigate the ability to withstand high temperatures by thermal denaturation experiments and thermostability for possible application to various industrial processes that already use. (AU)