Characterization Of A Novel Glucose/Salt-Enhanced Thermophilic β-glucosidase

β-glucosidase is a rate-limiting enzyme in the hydrolysis of cellulose. Thefeedback inhibition of cellobiose to cellulose can be cut down by β-glucosidase.Furthermore, there are some significant applications of β-glucosidase in the foodaroma, physiological oligosaccharides and its derivatives. Source of β-sugar enzymeis very broad, such as bacteria, archaea, fungi, plants and animals. The β-glucoses ofdiversity sources are different in enzyme activity, optimum temperature, optimum pH,substrate specificity, inhibition of activation and so on. It is an important researchfield of enzyme engineering to discover new enzyme sources and find out a variety ofnew enzymes having superior performance in the above-mentioned factors.Acidothermus cellulolyticus11B is from Yellowstone Park, which grows at55℃, takes advantage of fiber and expresses abundant glycoside hydrolases.ABK51908.1is predicted to encode β-glucosidase (AcBg). The full-length of Bglgene is1437bp, encoding484amino acids, GC being62%of the total base withoutsignal peptide and belonging to glycoside hydrolase1family, which estimatedmolecular mass is53.0kDa. The maximum rate of amino acid identity of AcBg is59%. Homology modeling and molecular docking display that Glu178，Pro236，Asn307，Tyr309，Asn310，Glu382，Glu436are very conservative and are involvedin catalyzing or binding between β-glucosidase and substrate. The Bgl gene wassuccessfully cloned into the pET-20plasmid by the method of molecular biology, andthen expressed abundant protein in engineering bacteria. We obtain a relatively purerecombinant β-glucosidase through many times purifications. The result of enzymaticcharacterization show that substrates of AcBg is a variety of cellooligosaccharides andAcBg can hydrolyze other aromatic groups glucose, that is why AcBg is a β-glucosidase. The optimum pH and temperature of Recombinant enzyme is7.0and 70℃. The hydrolysis activity to pNPGlc is12.1U/mg under optimal conditions.Recombinant enzyme is an protein of high thermal stability, which half-life is8hoursat70℃. HPLC analysis showed that the enzyme is a progressive exoglycosidases.AcBg also has a role in the synthesis of oligosaccharides under certain conditions.The study found that the enzyme can be activated by monovalent cations. Theenzyme activity is by about2.6times and2.2times in the condition of5M NaCl andKCl. The enzyme activity remained more two times than unsalted at nearly saturatedsalt concentrations. Divalent metal ions inhibit enzyme activity varying degrees, suchas Ni2+, Zn2+, Fe2+, Mn2+, etc. Other divalent metal ions have no significant effect onenzyme activity. The study also found that enzyme is activated under glucosecondition. The enzyme activity reaches maximum under0.2M glucose condition,which is1.9times than not added glucose. AcBg shows typical characteristics of thesalt-activated and glucose-activated. Kinetic analysis showed that Kmdoes not alter insalt-activated process, but kcat/Kmchanges from the original71.0to158.4. Butglucose alters Kmof the enzyme. The Kmof the enzyme is0.21mM without glucose.The Kmof the enzyme is0.9mM under0.2M glucose condition. The Kmof theenzyme is5.7mM under1.0M glucose condition. The kcat/Kmalso change a lot. Thekcat/Kmis71.0without glucose. The kcat/Kmis37.0under0.2M glucose condition.The kcat/Kmis6.7under1.0M glucose condition. Circular dichroism and endogenousfluorescence analysis show that NaCl of high concentration does not affect thesecondary structure of the protein, but fluorescence intensity undergoes a drasticchange. NaCl of high concentration may change microenvironment the amino acid(Phe、Trp、Tyr), thereby increasing the enzyme activity.