Functional Studies On PT-box Linker Of Cellulase And Enzymatic Properties Of Novel ?-Glycosidase

Cellulose,the most abundant carbohydrate in nature,can be hydrolyzed by cellulases into fermentable sugars and be used as a sustainable alternative energy source for fossil fuels.Cellulases play a key role in the degradation of lignocellulosic and many cellulases have been utilized for industrial production.Some unique properties of enzymes often are needed for industrial applications.So it is necessary to constantly develop new enzyme sources and research on the enzyme catalysis process for the finding recombinant enzymes with new functions.With developing of molecular biology and structural biology,the catalytic mechanism of cellulase and findings new enzyme source have been two important field of research.In this thesis,the functions of PT-box Linker and the enzymatic properties of novel glycosidases were studied,respectively.In the first part of the dissertation,the endo-cellulase AcCel12B from the thermophilic Bacterium Acidothermus cellulolyticus 11B was selected as the research object.The linker of AcCel12B was modified by molecular biology.AcCel12B consists of a catalytic domain?CD?and a carbohydrate-binding module?CBM?which are linked by a 48 amino acid linker region.The linker region of AcCel12B contains a PT-box region which links the CD and CBM by two non-conserved regions.The PT/S-box was separated into two parts by a glycine.The amino acid sequences of the two parts were axisymmetrically arranged with glycine as the axis of symmetry.To evaluate the function of the PT/S-box in cellulose hydrolysis,we defined a part of PT-box as one PT-box unit.The effect of linker on enzyme function is investigated by redesigning the numbers of PT-box units.In this thesis,cellulase AcCel12B and three mutants with different numbers of PT-box?no or 1-3 PT-box?were successfully constructed by molecular biology method.Those were successfully expressed purified.We found that the activity of cellulases on Avicel and recyclable amorphous cellulose?RAC?increased with the number of PT/S-box units by studying the properties of cellulase AcCel12B and three mutants.The activity of AcCel12B and all mutants on soluble cellulose?CMC?was no significant difference.The same phenomenon is found when the time course of enzymatic hydrolysis of RAC is studied.The activity of cellulase on solid RAC increased with the number of PT-box,but the activity of cellulase on soluble cellulose produced by hydrolysis is no significant difference.In order to explore the reason for the differences in the hydrolysis of cellulose by different cellulases with different PT-box numbers,we studied the adsorption and desorption of AcCel12B and three mutants from solid cellulose.We found that the desorptions of AcCel12B and its mutants from RAC and Avicel were significantly different.The energy of desorption of wild type and mutant AcCel12B from cellulose decreased with the number of PT-box units.The adsorption of AcCel12B and mutants to RAC and Avicel were not significantly different.According to the above studies,we conclude that AcCel12B containing more PT/S-box units was more easily desorbed and had more opportunity to hydrolyze cellulose than others,resulting in AcCel12B containing more PT/S-box units shows higher catalytic hydrolysis activity than others apparently.So the number of PT/S-box units in endocellulase affected the desorption of the enzyme,which possibly is responsible for the differences in the activity of wild type and mutant AcCel12B on Avicel and RAC.In the second part of the dissertation,a new?-glycoside hydrolase gene?AcBg?was successfully cloned from Acidothermus cellulolyticus 11B ATCC 43068.We expressed the gene in Escherichia coli BL21?DE3?.The recombinant protein AcBg was obtained.The?-glucosidase is an important enzyme in the process of cellulose degradation.So far,many?-glucosidases have been found.They are bacteria-derived,archaebacterial,and fungal-derived,but high Enzymes that act at concentrations of salt are rare and enzymes that can be activated by high concentrations of salt are more sparse.The?-glucosidase found in this pepar can be activated by diverse monosaccharides and have salt-tolerance,which is uncommon comparing with a variety of?-glucosidase that have been found.The recombinant protein?Ac Bg?belongs to glycoside hydrolase family 1?GH1?AcBg had an optimal pH and temperature of 7.0 and 70°C,respectively.The specific activity of AcBg under optimal conditions was 290 U/mg and 33 U/mg for p-nitrophenyl-?-D-glucopyranoside?pNPG?and cellobiose,respectively.AcBg hydrolyzed the oligosaccharide sequentially from the non-reducing end to produce glucose units according to the results of HPLC analysis.The enzyme activity was activated by monovalent cations.Its activity rose more than 2-fold when 5 M NaCl/KCl were added.Bivalent metal ions(such as Ni²⁺,Zn²⁺,Fe²⁺,Mn²⁺,etc.)inhibited the activity of enzyme in different degrees,other divalent metal ions had no significant effect on the enzyme activity.Ac Bg showed monosaccharide-stimulation properties.The activity of the?-glucosidase was remarkably enhanced in the presence of 0.2 M D-glucose?increased more than 1.9-fold?,0.1 M?-Methyl-D-glucose?increased more than 1.4-fold?and 1.0 M D-xylose?increased more than 1.9-fold?.The catalysis kinetics and structural changes in various concentrations of glucose were determined.The results indicated that glucose reduced substrate affinity and caused conformational changes.