Properties Of ?-glucosidase CHU₃577 From Cytophaga Hutchinsonii

With the development of industry,the world's resources and energy gradually depleted and shortages,energy and environmental problems become a serious challenge to the survival and development of human.Solar energy is a very important energy on earth,and biomass resources is the world's most abundant carbon source as immobilized solar energy.Countries are committed to the development of biomass energy,of which the wood fiber accounting for 50%,and can be used as an important clean renewable resources in theory.However,the dense cellulose structure,dissolve difficulty,crystallization and other issues lead to its difficult to be degraded and use,and it becomes the bottleneck of cellulose research.The existing physical and chemical methods of the crystallization area destruction are energy consumption,environment pollution,and do not meet the needs of sustainable development.Therefore,it is important to study the microbial strains for the efficient degradation of cellulose.Cytophaga hutchinsonii is a widely distribute Gram-negative cellulolytic bacterium which belongs to the phylum Bacteroidetes.C.hutchinsonii can degrade crystalline cellulose rapidly and thoroughly,but it does not secrete cell-free cellulases,or has cellulosomes.Presumably it has an unique mechanism of cellulose degradation.There is little understanding of the degradation mechanism of C.hutchinsonii.There are many genes may encode cellulases in C.Hutchinsonii,including three GH5 family endo-cellulases,six GH9 family endo-cellulases and four GH3 family ?-glucosidase.Among them,?-glucosidase is the necessary enzyme in the degradation of cellulose,it plays a key regulatory role in cellobiose and short chain fiber oligosaccharides degradation and cellulose degradation rate.Further research of(3-glucosidase has important significance in improving the degradation efficiency of cellulose and clarifying the unique cellulose degradation mechanism in C.hutchinsonii.In C.hutchinsonii,the four ?-glucosides of the GH3 family were CHU 2268?Bg1A?,CHU₃784,CHU₂273?BgIB?and CHU 3577?Bg1C?,respectively.Our previous study found that BglA was a ?-glucosidase,and also was a fiber oligosaccharides that could degrade fiber oligosaccharides,BgIB is an algalophilic?-glucosidase,whereas CHU-3784 is inactive and CHU-3784 may be a pseudogen.In this paper,BglC focused on the study.A series of studies have been done around the p-glucosidase gene identification,cloning,heterologous expression,enzymatic properties and site-directed mutations and other aspects,including the following elements:1.Bioinformatics analysis and heterologous expression of BglC.The BglC gene is 2253 bp in length,encoding 751 amino acids.There is a GH3 conservative domain and NCBI is labeled as the GH3 family of ?-glucosidase?E-value of 4.95e-158?.Signal P predicted CHU₃577 without signal peptide sequence,no transmembrane structure.PSORTb predicted the protein located in intracellular.The BglC gene was ligated with pMAL-C2X plasmid in E.coli DH5a.The plasmid was transfected into E.coli JM109,and the highly expressed recombinant p-glucosidase was obtained after induction.2.Biochemical properties of BglC.?-pNPG was used as the substrate to study the biochemical properties of BglC,and found that the optimum reaction temperature was 42?,the optimum pH was 6.5,the stability was good at low temperature and the heat resistance was poor.Various metal ions influence on BglC enzymes before the final concentration is 10 mM was studied,we found that enzyme activity increased with the increase of Mg 2+,Mn2+,Fe3+ ion concentration,and then became stable.Among them,Mg2+ is the strongest.Ca2+ concentration has little effection on the enzyme activity.Ni2+and Cu2+ have inhibitory effect on enzyme activity.The impact of organic solvent on the enzyme activity found that methanol,ethanol,acetone,n-butanol and ethyl acetate will inhibit enzyme activity.BglA was also found to be highly tolerant to glucose as it retained 60%activity when the concentration of glucose was 100 times higher than that of the?-pNPG.3.The diversity of hydrolyzed substrates of BglC.Besides the property of ?-glucosidase,BglC can hydrolyze ?-1,3-glycosidic bond of laminaribiose and ?-1,4-glycosidic bond of cellobiose,and and both can carry out transglycosylation,but could not hydrolyze the maltose,isomaltose,?-pNPG,gentian disaccharide,honey disaccharide,pine disaccharide,almond glycosides or can carry out transglycosylation.When the cellobiose or ?-pNPG as the substrate,the hydrolysis activity is almost equivalent.BglC can hydrolyze cellobiose,cellotriose,cellotetrase and cellopentose,and can carry out transglycosylation.While glucose-free glycosylation.4.When the fiber was used as the substrate,the efficiency of the transglycosyl group was almost the same at below 45?,and the efficiency was higher than 45 0 C.Under the condition of pH weak acidity,the transglycosylation efficiency is very high and the weakly alkaline condition is almost free of transglycosylation,which is consistent with the enzymatic activity of ?-pNPG as substrate.As for transglycosylation BglC can also can carry out transglycosylation when cellobiose as substrate,the organic solvent of methanol ethanol concentration was 20%.Indicating that the organic solvent is relatively resistant to the transglycosylation effect when cellobiose as substrate.When the glucose concentration is 10 times the concentration of the cellobiose substrate,hydrolytic activity is about 90%of the original,but almost no transglycosylation occurs.We guess hydrolysis and transglycosylation is not reversible reaction.5.Site mutation of BglC amino acid site.The three-dimensional structure of BglC is similar to that of exo-hydrolytic enzyme 1EX1 |A from barley,which is more than 35%.After sequence alignment point mutations,the D303A and E513A were found to be the active sites.When the?-pNPG was used as substrate,the hydrolytic activity disappeared after the mutation of W443A,I336M,D122A,M268A,E188A/R,K224A,R185A/V,S271A,D303 A,K309F and E513A;We speculate that the above sites are important for the catalytic activity of BglC enzymes.the hydrolysis activity is reduced to varying degrees after the mutation of I336L,I336S,K309T,F171L,F171M,S271Y;and S82A,183 A,I336A increased hydrolysis activity.S82A is 116%of the original vitality,183 A is 130%of original vitality,I336A is 120%of the original hydrolytic activity.The above sites may be involved in the catalytic reaction of the enzyme.For the 1336 site,I336M enzyme activity lost,I336L,I336S decreased activity,I336A activity increased.Indicating that 1336 different amino acids at the same site act different effects of enzyme activity.The results are consistent with the results of hydrolysis of BglC with cellobiose as substrate.Indicating that the above changes in site affect the enzyme activity.When ?-pNPG as the substrate,the tolerance to glucose is little change as I336A,F171M,S82A site changes.While mutations in the I83A,K309T,I336S,I336L sites,the glucose tolerance was reduced.The above sites may be associated with glucose tolerance.Which I83A mutation after the enzyme activity increased,and glucose tolerance decreased,indicating that enzyme activity and glucose tolerance is not positively correlated.