| Mannan is a type of plant polysaccharide with a complex structure,and its main chain is usually comprised of mannose and glucose units linked by ?-1,4 glycosidic bonds.According to the structural characteristics of the branched sugar units,it can be futher divided into pure mannan,glucomannan,galactomannan,and galactoglucomannan.Mannan showed unique physical and chemical properties,such as excellent gel-forming capablilites by abosorbing water,strong chemical inertness,and great biological safety,and therefore it is widely used in food,pharmaceutical,and textile industries.In contrast,mannooligosaccharides showed a variety of important bioactivities,such as anti-tumor,regulating immunity,and promoting cell division effects.Recent studies showed that the biological activity of mannooligosaccharides isclosely related to the composition of sugar units and their degrees of polymerization.Mannanase is a type of glycoside hydrolases(GHs),which can hydolyze different types of glycosidic bonds among mannan polysaccharide molecules,thereby generating oligosaccharide products with smaller molecular weights.Compared with chemical or physical methods,enzymatic degradation of mannan polyosaccharides to produce oligosaccharides showed the advantages of more mild reaction conditions,stronger controllability,clearer substrate preference,and redicatable oligosaccharide products.However,tool-like ?-mannanases capable of directly preparing oligosaccharide fragments with designed structure properties are still currently rare.In-depth research on the exploration and development of related enzyme resources,molecular characterization,and discovery of associated catalytic properties has become an important preliminary basis.Mannan polysaccharides are comprised of various sugar units,thereby their complete enzymatic degradation requires the synergy of multiple enzymes.For example,endotype ?-mannanase(EC 3.2.1.78)can randomly cleave P-1,4 glycosidic bonds in the main chain of mannan,therefore it has been is the current research focus on in current mannanase studies,mainly focusing on focuses focused on the exploration of ?-mannanase resources,crystal structure discovery,and various molecular modification of mannanase to improve the biochemical characteristics and enzymatic properties of the enzyme mutants.However,there are relatively few studies on the mechanisms for P-mannanases' substrate preference,substrate action modes,and the oligosaccharide-yielding propertis,in particular on their inner relationships associated with catalytic mechanisms,which have limitted the accurate application of?-mannanase as tool in the direct oligosaccharide preparation or the sequencing of associated sugar chains.Flammeovirga sp.strain MY04,a polysaccharide-degrading marine bacterium,can grow excellently on more than ten types of polysaccharides including mannan,agarose,alginate,so which means an abudunt mannanase bacterial resource strain of mannanases.Herin,the whole genome of MY04 strain was molecularly mined,and two potential ?-mannanases genes,man01929 and man02066 were obtained respectively.The sequence properties of Man01929 and Man02066 proteins were analyzed using associated bio-soft tools.Construction of the expression vectors,heterologous expression,and purification of recombinant enzymes were performed with the focus on the comparative studies of ?-mannanases rMan01929 and rMan02066 e.g.,their biochemical characteristics,substrate preference,substrate(polysaccharide and oligosaccharide)degradation modes,and oligosaccharide-yielding properties,etc.,to clarify their relevant inner relationships.In this thesis,we also performed site-directed mutagenesis based on the three-dimensional structure modeling of the ?-mannanase Man01929,to discover potential mechanisms associated to the enzyme's substrate preference,oligosaccharide-yielding properties,and protein structure properties.Possible transducer mechanism of Man01929 and its series of mutants which can catalyze mannan both as a glycoside hydrolase and as a glycoside transferase were discussed.The research contents,primary results,and key conclusions are listed as the following:1.Studies on the biochemical characteristics,substrate preference and enzymatic properties of ?-mannanases Man01929 and Man02066(1)Sequence properties of P-mannanases Man01929 and Man02066The ?-mannanase Man01929 has 932 amino acids,a theoretical molecular weight of 103.1 kDa,an isoelectric point of 4.25,and no signal peptide.It is predicted that Man01929 has five functional modules,e.g.,the N-terminus contains a GH5 catalytic module(from D51-Y291),the Coagulation factor 5/8 C-terminal module the amino acid at positions(I447-E585),2 PKD/Chitinase modules,binding module 64(CBM64)(P592-P675,P682-W766,and(P772-E845),and the Secretion system C-terminal sorting domain(V859-Q932),and the Secretion system C-terminal sorting domain.Among elucidated enzymes,Man01929 showed the greatest sequence identity to Man5A from Blue Mussel Mytilus edulis,36.79%.Phylogenetic tree analysis indicated that Man01929 belongs to the 10th subfamily of the GH5 superfamily.The ?-mannanase Man02066 has 378 amino acids,the theoretical molecular weight of 44.1 kDa,the isoelectric point of 5.49,and that the amino acid at the N-terminal 1-22 position is a type I signal peptide.Man02066 is comprised of only one module,i.e.,the GH26 catalytic module(Glycoside hydrolase family 26 domain).Among elucidated enzymes,the greatest sequence identity of 42.82%with CjMan26C,a ?-mannanase from Cellvibrio japonicus.Phylogenetic tree analysis showed that Mna02066 belongs to the GH26 family.(2)Studies on the biochemical characteristics of recombinant enzymes rMan01929 and rMan02066The optimal temperatures for the degradation of KGM and LBG by rMan01929 are 40? and 50?,and the optimal pH values are 8.0 and 5.0 respectively.The recombinant enzyme rMan01929 showed thermal stability within 0-40? and tolerance between the pH values 5.0-10.0;the addition of metal ions Co2+to the reaction system of different substrates showed variable different effects on the activity of rMan01929,e.g.,Which can promote the activity of rMan01929 when the substrate is KGM,while when the substrate is LBG,the enzyme activity is significantly inhibited.The optimal temperatures of rMan02066 for the degradation of KGM and LBG are the same,40?,and the optimal pH value is 6.0;the recombinant enzyme rMan02066 showed a certain thermal stability at 0-30 ?,which was greatly affected by pH,and therefore Man02066 is sensitive to acid and alkali environments;NaCl(0-1M)can promote the enzyme activity of rMan02066 variously when substrates changed,for it can significantly increase the enzyme activity up to 148%when degrading LBG,while up to 120%when reacted with KGM.In summary,the biochemical characteristics of p-mannanase Man01929 and Man02066 are quite different,which will vary according to different substrates.(3)Studies on the catalytic properties of the recombinant enzymes rMan01929 and rMan02066The recombinant enzyme rMan01929:1)The enzyme rMan01929 can degrade KGM and LBG which are linked by ?-1,4 glycosidic bonds in the main chain,whereas can hardly degrade guar gum and xylan,and therefore the optimal substrate of Man01929 among tested polysaccharides is KGM;2)The enzyme rMan01929 is an endo-type ?-mannanase when degrading the polysaccharide KGM and LBG;3)Each size-defined oligosaccharide fractions were purified from the final oligosaccharide products by gel filtration chromatography,and the resulting 1H-NMR spectrum showed type signal peaks at 5.05 ppm,a typical position for a HI signals of mannose units at the reducing end.Furthermore,the reducing ends of each final oligosaccharide product fractions were mainly identified as mannose units;4)The smallest substrate of the enzyme rMan01929 is M4 and the smallest product is M1;5)When rMan01929 degrades a series of mannan associated oligosaccharide substrates,e.g,various size-defined pure mannan oligosaccharide chains and their 2-AB-labelled products,the enzyme degrade the sugar chains from the reducing end to produce smaller size-defined oligosaccharide products.Furthermore,the size of small oligosaccharide product will increase apparel to the size of substrate chain.Therefore,Man01929 showed a typical variable endotype degradation mode toward various mannan substrates.Recombinase rMan02066 is different from rMan01929:1)The enzyme rMan02066 is a disaccharide-producing exolytic mode.However,rMan02066 degraded KGM to produce a series of size-defined oligosaccharide fractions as the final main products,due to the low activity to KGM and the presence of glucose units in the KGM backbone.2)The smallest substrate of rMan02066 is M3 and the smallest product is Ml,with M2 as the primary oligosaccharide products during the degradation;3)The enzyme rMan02066 continously yields disaccharide units,identified as M2,from the non-reducing end of the substrate chains until one mannose or one mannobiose unit remains in the final sugar chain product,with a moalr ratio equal to the substrate chain.In summary,both ManO 1929 and Man02066 are ?-mannanases.While differently,Man01929 is an endolytic ?-mannanase of the GH5-F10 family with certain thermostability and extensive pH tolerance,and thus it can be used to prepare oligosaccharide fragments with mannose residues as the reducing end,which represents a novel tool-like enaymatic property.Man02066 is a mannobiose-yielding exolytic ?-mannanase of the GH26 family and it is not resistant to acid and alkali environments.2.Site-directed gene mutations of the P-mannanase Man01929 and studies on associated catalytic mechanismsHomologous modeling of the P-mannanase Man01929 was performed using EfMan(PDB NO.5y6t)as template.After homology modeling,the crystal structure of Man01929 is molecularly docked with mannohexaose(M6).Using the PyMOL software,the crystal structure of the protein is analyzed and the potential sugar active site residues were predicted.The results showed that Man01929 is organized in a typical(?/?)gTIM barrel structure,which belongs to the Clan-A superfamily.The protein contains an open catalytic cavity,the length of which is about 25A-30A,with six subsites.Man01929 has two essential catalytic site residues,i.e.,Glu172(acid/base catalysis)and Glu289(nucleophilic catalysis),and twelve potential glycosyl binding site residues(Asp118,Met119,Asp124,Glu172,Trp182,Trp218,Ser219,Tyr221,Asp263,Glu268,Ile269,Glu323).In order to characterize the function of the above-mentioned glycosyl binding site residues,the recombinant plasmid pET30a-Man01929 was used as a template for the site-directed gene mutation performation of catalytic site residues(Glu172,Glu289)and-4(Asp124,Glu323),-3(Met119,Asp118),+2(Tyr221,Glu268,Ile269)selected subsite residues,which were thereafter subjected to further heterologous gene expression and recombinant protein purification.The changes between Man01929 and its protein mutants in their enzymatic properties were comparatively analyzed,and associated catalytic mechanisms were discussed.This study demonstrated that the key catalytic site residues of ?-mannanase Man01929 were Glu172(acid/base catalysis)and Glu289(nucleophilic catalysis);Furthermore,the-4(Asp124,Glu323),-3(Met119,Asp118)subsites amino acid residues not only played essential roles in the recognition and binding to,in particular to the non-reducing end of,the substrate sugar chain by Man01929,but also they showed key effects toward the substrate preferences,particularly the selectivity,recoginition,and action against galactomannan.Notably,the Asp118 and Asp124 site residues showed a significant effect on determining the degrees of polymerization of the final main mannan degradation products,and the protein mutants D118A,D118E,and D124Y of Man01929 produced larger size-defined oligosaccharide fractions as the final main products.This study also discovered that when the Asp124 residue of Man01929 was changed into Tyr124,the resulted enzyme mutants showed strong glycosyl hydrolase while additional weak glycosyltransferase activities,indicating that the increases of the electron cloud density at the 124th site residue whill benefit the enzyme mutants to initially yield oligosaccharide products as a glycoside hydrolase,and thereafter use them as actived glycoside donors and receptors to synthesis new oligosaccharide chanis,as a glycosyl transferase(GT).Thus,this study provided novel insight into the conversion regulation mechanisms of mannanses between GH and GT activities.In this thesis,we systematically analyzed the biochemical characteristics,substrate action modes,oligosaccharide-yielding properties,and associated catalytic mechanisms of ?-mannanase Man01929 and Man02066 from Flammeovirga sp.strain MY04.To our knowledge,we primarily discovered that the biochemical characteristics and substrate degradation modes of ?-mannanases Man01929 and Man02066 are significantly affected by the structures of substrates.Compared to the glycosyl binding site residues that bound to the reducing end of substrate sugar chains,site residues that bound to the non-reducing end of substrates play a more important role in the specific recognition and binding to the substrates,and thus affecting and even determining the substrate preferences and activities of an enzyme.Notably,Man01929 can be used to directly prepare oligosaccharide product fractions which are enriched with mannose at the reducing ends,meaning a novel important tool-like value.In this thesis,a novel protein mutation method for changing the glycoside hydrolase activity of the GH5 family endolytic ?-mannanase Man01929 into a glycosyltransferase activity has been established,and the underlying mechanism was prenimately discovered by gene mutation and protein characterization studies.In summary,this thesis aims to provide a certain new theoretical reference and technical pathes for the resource exploration and enzyme improvements of tool-type mannanases. |
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