טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentEinat Naveh
SubjectStructure-function Studies on Arabinases from Geobacillus
stearothermophilus
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professor Shoham Yuval
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

The aim of this research was to characterize two arabinanases (AbnA and AbnB) and two arabinofuranosidases (AbfA and AbfB) from Geobacillus stearothermophilus T-6 and to reveal the arabinan degradation pathway in this strain. Arabinases are enzymes that hydrolyze the polysaccharide arabinan which is part of pectin found in the plant cell wall.  These enzymes are classified as a-L-arabinofuranosidases (EC 3.2.1.55) and endo-a-1,5- arabinanases (EC 3.2.1.99).  The enzymatic degradation of the plant cell wall is of great interest since it can serve as a renewable energy sources that contribute no net CO2 emission to the atmosphere.

In the framework of this research we have cloned, sequenced and over expressed two arabinanases, AbnA and AbnB, both belong to glycosyl hydrolases family 43. The abnA gene encodes for a 93 kDa extracellular arabinanase (AbnA) and the abnB gene encodes for a 35 kDa intracellular arabinanase (AbnB). The kinetic constants of AbnA on arabinan at 40oC were Km, kcat, and Kcat/Km of 0.94 mM, 71.6sec-1, 75mM-1sec-1, respectively. The hydrolysis of linear arabinan (unsubstituted arabinan) by AbnA provided Km, kcat, and Kcat/Km of 4.8 mM,  81.2 sec-1 and 17mM-1sec-1, respectively. AbnA exhibited an endo mode of action on native arabinan as well as on linear arabinan, whereas AbnB was inactive towards native substituted arabinooligosaccharides, arabinan and linear arabinan but was capable of hydrolyzing linear arabinooligosaccharides.  The Arabinofuranosidases AbfA and AbfB were not capable of hydrolyzing the arabinan backbone but removed the arabinose side chains.  These two enzymes appear to act on different bonds, presumably a-1,3 or a-1,2.  G. stearothermophilus T-6 arabinofuranosidases were also active towards arabinooligosaccharides (the products of arabinan hydrolyzed by AbnA) and this resulted in the release of arabinose not only from the substitutions but also from the main backbone, suggesting that these enzymes can also hydrolyze a-(1,5) bonds.  High resolution crystal structures of native AbnB and AbnB together with arabinotriose were obtained in the laboratory of G. Shoham at the Hebrew University, Jerusalem.  The enzyme exhibits a beta-propeller fold in which the substrate is positioned in the appropriate distance from the three catalytic residues. The results from this research provided a complete picture on the degradation path of arabinan by G. stearothermophilus T-6.  First, the extracellular AbnA cleaves high molecular weight native decorated arabinan into short substituted arabinooligosaccharides that can enter the cell via specific oligosaccharides ABC transporters. The intracellular arabinofuranosidases then remove the substitutions to provide short linear arabinooligosaccharides that are hydrolyzed by the intracellular AbnB into arabinose.