Cloning, Expression And Characterization Of Cellobiase From Thermoanerobacterium Aotearoense

Global energy consumption and fossil fuel depletion have promoted the production of biofuels and bioproducts from lignocellulosic biomass since lignocellulosic material is the most abundantly renewable source of energy on Earth. Enzymatic hydrolysis of plant biomass converting cellulose to glucose is recognized as a critical step in the production of biofuels and platform molecules in the synthesis of chemicals from lignocellulosic biomass. Design of better cellulase cocktails with increased performance is one of the most important tasks in obtaining high yields of fermentable sugars from lignocellulosic biomass.Cellobiase(EC 3.2.1.21) is a glucosidase enzyme that hydrolyzes β-1,4 bonds linking two glucose or glucose-substituted molecules. It is an important component of the cellulase enzyme system. Cellobiase decrease cellobiose inhibition by hydrolyzing this disaccharide to produce glucose, thus allowing the cellulolytic enzymes to function more efficiently. Besides, β-glucosidase is used as a flavor enzyme to improve the flavor of wine, tea, fruit juice.Thermoanaerobacterium aotearoense P8G3#4 produced cellobiase(BGL) intracellularly when grown in liquid culture on cellobiose. The gene bgl, encoding cellobiase, was cloned and sequenced. Analysis revealed that the bgl contained an open reading frame of 1314 bp encoding a protein of 446 amino acid residues, and the product belonged to the glycoside hydrolase family 1 with the canonical Glycoside Hydrolase Family 1(GH1)(β/α)8 TIM barrel fold. Expression of p ET-bgl together with a chaperone gene cloned in vector p Gro7 in Escherichia coli dramatically enhanced the crude enzyme activity to a specific activity of 256.3 U/mg wet cells, which resulted in a 9.2-fold increase of that obtained from the expression without any chaperones. The purified BGL exhibited relatively high thermostability and p H stability with its highest activity at 60 °C and p H 6.0. In addition, the activities of BGL was remarkably stimulated by the addition of 5 m M Na+ or K+. The enzyme showed strong ability to hydrolyze cellobiose with a Km and Vmax of 25.45 m M and 740.5 U/mg, respectively. The BGL was activated by glucose at concentration varying from 50 to 250 m M, and tolerant to glucose inhibition with a Ki of 800 m M glucose. The supplement of the purified BGL to the sugarcane bagasse hydrolysis mixture containing a commercial cellulase resulted in about 20% enhancement of the released reducing sugars.These properties of the purified BGL should have important practical implication in its potential applications for better industrial production of glucose or bioethanol started from lignocellulosic biomass.