| Wood-feeding termites are efficient lignocellulose decomposers in nature.They play a very important role in carbon recycling and energy flowing.The process to degrade lignocellulose is associated with symbiotic bacteria inhabited the intestinal tract of termites.Verrucomicrobia have been consistently detected in molecular surveys of the 16S rRNA genes targeting the microbial community of various termite species.Despite their ubiquity in higher and lower termite species,their functional roles remain elusive.In this thesis,we studied the diversity of Verrucomicrobia in the intestinal tract of Nasutitermes sp.and Reticulitermes chinensis Snyder.In order to infer the possible and probable ecological roles of verrucomicrobia in termites,the basic characteristics of TSB-47,a pure culture isolated from R.chinensis,were investigated.Then three?-glucosidase genes(GM1124,GM1951,GM3190)derived from Verrucomicrobia sp.TSB-47 were expressed three and the characterized were characterized..All of these may increase our understanding of the Verrucomicrobia and lay the foundation for further elucidating the symbiotic mechanisms between Verrucomicrobia and termites.The main results of this study are as follows:1.The diversity of Verrucomicrobia in the gut of Nasutitermes sp.and R.chinensisTaking Nasutitermes sp.and R.chinensis as our research target,Verrucomicrobia 16S rRNA gene libraries were constructed.Six clones of Verrucomicrobia were obtained from the two libraries respectively.For Nasutitermes sp.,the most dominant phylotype was clone Nver-1 that accounted for 53 percent of the total clones.These clones were clustered within the family Opitutaceae and were most closely related to Opitutus sp.PH5-10(96%16S rRNA gene identity),an isolate obtained from American cockroach.The second dominant phylotype was clone Nver-3 that accounted for 17 percent of the total clones,which was most closely related to strain TAV1(97%16S rRNA gene identity),an isolate obtained from R.flavipes.For R.chinensis,the most dominant phylotype was clone Rver-2 that occupied the percent 55 of the total clones.These clones were most closely related to Verrucomicrobia sp.TSB-47,a strain that was isolated from the same termite by our laboratory.Further phylogenetic analysis clearly indicated that most of the clones were the members of Verrucomicrobia Subphylum 4,only two clones fall into the cluster of Verrucomicrobia Subphylum 1.Most of the Verrucomicrobia clones were closely related to those of the other termites and formed a distinct cluster,indicating their unique origin and functions in natural system.2.Characteristzation of Verrucomicrobia strain TSB-47The preliminary studies were perfomed to characterize the Verrucomicrobia strain TSB-47 which was previously isolated from hindgut homogenates of R.chinensis.Strain TSB-47 is facultatively anaerobic.Growth occurs at 20-35 C(optimum,25 C),at pH 6.5-8.5(optimum,pH 7.5),and at NaCl concentrations of 0%-2.25%(w/v).The major fatty acids were Anteiso C15:0(37.56%),C16:0(33.21%)and Anteiso C17:0(12.36%).The respiratory quinone type was MK-7.Strain TSB-47 can grow with glucose or cellobiose as the sole carbon source.In nitrogen-free AM-5 medium,Strain TSB-47 could grow continuously for several generations,indicating that the strain could fix nitrogen efficiently.3.Expression of three ?-glucosidase genes of Strian TSB-47 and characterization of the recombinant enzymesThere are 14 p-glucosidase genes in the TSB-47 genome.We cloned and expressed three p-glucosidase genes(GM1124,GM195,GM3190)obtained from it.These genes were 1365 bp,2166 bp and 1356 bp in length,respectively.They were expressed inEscherichia coli BL21(DE3)and the expression product showed a molecular mass of 51KD,78.4KD,50.5KD by SDS-PAGE,respectively.The optimal pH for the activity of the purified recombinant enzyme GM1124-30a,GM1951-30a and GM3190-30a with pNPG as substrate were 5.0,6.0,5.5,respectively.And The optimal temperature for the activity of the purified recombinant enzyme GM1124-30a,GM1951-30a,GM3190-30a were 50?,50?,45?,respectively.The specific activity of the purified recombinant enzyme GM1124-30a,GM1951-30a,GM3190-30a were 2.55 U/mg,2.03 U/mg,8.8 U/mg,respectively.The activities of GM3190-30a were greater than the other two recombinant enzymes,therefore,we chose GM3190-30a for further studies to explore the other enzymatic properties.The Km and Vmax values were 23.4 mM and 13.7umol min-1·mg-1,respectively,with pNPG as substrate.1 mM and 5 mM various metal ions had different influence on the activity of GM3190-30a.At 1 mM,most tested metal ions showed a significant positive effect on the activity of ?-glucosidase 3190-30a,such as Fe3+,Al3+,Ni2+,Zn2+,Co+.Ba2+,Ca2+,Mn2+,K+had no effect on the activity of it.At 5 mM,Mg2+,Ba2+,Ca2+,Mn2+,K+,Co+are effective activators for enzyme activity,While Fe3+,Ni2+,2n2+showed a significant negative effect on the activity of the recombinant ?-glucosidase.At both concentrations,the activity of recombinant ?-glucosidase was signiifcantly inhibited by Cu2+.At the same time,the effects of different compounds on enzyme activity were determined.EDTA,acetone and high concentration of urea and low concentration of guanidine isothiocyanate and ethanol showed a significant positive effect on the activity of it;and the activity of recombinant ?-glucosidase was significantly inhibited by imidazole,methanol,high concentrations of guanidine isothiocyanate and ethanol.The activity of recombinant P-glucosidase was significantly inhibited by SDS.The hydrolysis pattern analysis by TLC indicated that the main products of cellobiose(G2)and cellotriose(G3)by GM3190-30a were glucose(G1)and cellobiose(G2).With the increasing of glucose concentration,the activity of the recombinant ?-glucosidase decreased.GM3190-30a showed a certain salt tolerance.When the concentration of NaCl and KCl reached 4M,more than 40%of enzyme activity was left.Meanwhile,by adding appropriate salt ions in the enzymatic reaction,its enzyme activity can be promoted. |
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