Levansucrases encoded in the genome of Pseudomonas syringae pv. tomato DC3000: heterologous expression, biochemical characterization, mutational analysis and spectrum of polymerization products
Date
2012-08-27
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Abstract
Suhkrud on maakeral äärmiselt levinud molekulid ning neil on väga oluline roll elusorganismides. Fruktoosijääkidest koosnevaid suhkruid nimetatakse fruktaanideks ja nad on organismidele põhiliselt varuaineteks. Mõned taimed, näiteks sigur ja maapirn sisaldavad oma juurtes väga palju fruktaani. Fruktaanidele on leitud ka rakendusi näiteks toidutehnoloogias prebiootikumide, emulgaatorite või magusainetena ja meditsiinis vereplasma asendajana. Prebiootikumidena toimivad eelkõige lühiahelalised fruktaanid ehk fruktooligosahhariidid (FOS). FOS-id stimuleerivad kasulike piimhappebakterite hulka ja aktiivsust jämesooles, parandades sellega organismi tervist, pikaahelalised fruktaanid aga turgutavad immuunsüsteemi ja toimivad vähivastaselt.
Fruktaane sünteesivad fruktosüültransferaaside abil paljud taime-, seene- ja bakteriliigid. Oma doktoritöös uurisin taimi nakatava bakteri Pseudomonas syringae levaansukraase. Need on põnevad ensüümid, mis sünteesivad sahharoosist, aga ka veel odavamast toorainest, suhkrupeedi melassist, polümeerset fruktaani – levaani ja FOS-e. Huvitaval kombel on sellel mikroobil levaansukraase kodeerivaid geene kolm (lsc1, lsc2, lsc3), kuigi enamus levaani tootvaid baktereid saab hakkama ühega. Kui panna ükskõik milline neist kolmest mainitud DNA lõigust soolekepikesse (Escherichia coli), siis sünteesitakse täiesti töökorras ensüüm. Kui selline soolekepike kasvab sahharoosiga tardsöötmel, siis kattub tema koloonia ohtra limaga – levaaniga. Näitasime esmakordselt, et lisaks sahharoosile võivad pseudomoonastest pärit levaansukraasid kasutada ka rafinoosi ja stahhüoosi, mida leidub palju näiteks sojaoas. Minu doktoritöö peategelane, valk Lsc3 on väga aktiivne ning äärmiselt stabiilne katalüüsija – tema töövõime säilis kõrgel temperatuuril ja ka pikaaegsel säilitamisel. Sellised omadused teevad Lsc3-st valgu, mida võiks kasutada fruktaanide biotehnoloogilisel tootmisel, sest tööstuslikus protsessis on biokatalüsaatori efektiivsus ja stabiilsus ülioluline. Uudse kiibipõhise molekulide massi määramise meetodiga uurisime, milliseid fruktaane Lsc3 sünteesib. Avastasime, et ensüüm võib fruktoosijääke liita mitmetele erinevatele molekulidele nagu ksüloos, ksülobioos, ksülitool ja galakturoonhape, tekitades segusuhkruid. Segusuhkrud on huvitavad, sest neile ennustatakse uudseid bioloogilisi omadusi ja toimeid, näiteks võiksid nad olla eriti tõhusad prebiootikumid.
Levansucrases are bacterial enzymes belonging to family 68 of glycoside hydro¬lases (GHs). They catalyze hydrolysis of their substrate but also have prominent fructosyl transferase activity. The main substrate for levansucrases is sucrose which is major disaccharide in plants. Raffinose, likewisely abundant in some plants, is also used as a substrate. Spectrum of reaction products of levan¬sucrases comprises highly polymeric levan and fructooligosaccharides (FOS) of various degree of polymerization (DP). The entity of levansucrase reaction products depends on the enzyme and its origin, but also on reaction conditions enabling manipulation of the product spectrum. Levansucrases share highly similar five-blade β-propeller fold with other GH68 and 32 enzymes including bacterial inulosucrases, plant and microbial invertases, fructan exohydrolases and fructosyl transferases. In this thesis, levansucrases Lsc2 and Lsc3 from a plant pathogenic bac¬terium Pseudomonas syringae pv. tomato were expressed in a bacterial host Escherichia coli, purified and characterized. As a comparison, levansucrase LscA from P. chlororaphis subsp. aurantiaca was studied. The main results of this thesis are summarized as follows: 1. Lsc2 and Lsc3 of P. syringae pv. tomato were expressed with high yield in a bacterial host Escherichia coli exerting two expression systems. The first system relies on maltase gene promoter PMAL from a methylotrophic yeast Hansenula polymorpha. We verified that functionality of PMAL in E. coli is caused by the presence of σ70-like boxes in the eucaryotic promoter. The PMAL was shown to have suitable strength in E. coli providing a sufficient amount of catalytically active protein of interest. Due to its dual activity, it can be used as a promoter shared by yeasts and bacteria in heterologous protein expression trials. A pURI3 vector-based expression system was ad¬justed to obtain mutant and wild-type N-terminally His-tagged Lsc3 proteins. 2. P. syringae pathovars are exceptional among other bacterial species because they possess up to three levansucrase alleles in their genomes. We showed that all three lsc genes (lsc1, lsc2, lsc3) of P. syringae pv. tomato DC3000 encode functional levansucrase proteins, if expressed from a heterologous promoter in E. coli. 3. Enzymology and biochemistry of Lsc2 and Lsc3 was addressed and com¬pared with that of LscA from P. chlororaphis subsp. aurantiaca. All three proteins were shown to use sucrose, raffinose and stachyose as substrates. Low hydrolytic activity towards levan was also recorded. Affinities for sucrose of Lsc3, Lsc2 and LscA were similar, the Km values being around 20 mM. The maximum reaction velocity and catalytic efficiency of LscA was much lower than that of Lsc2 and Lsc3 proteins. Polymerization properties of the enzymes differed. At low sucrose concentration, Lsc3 polymerized much more effectively than LscA. At high substrate concentration, the difference in transfructosylating activity was evened out, but the FOS spectrum was still different – the LscA produced more high-DP FOS than Lsc3 or Lsc2. 4. As a novel feature for levansucrases of pseudomonads, this study shows the ability of Lsc3 and LscA to produce heterooligofructans (HOF) by trans¬fructosylating nonconventional fructosyl acceptors. For the first time, levansucrases were shown to transfructosylate D-sorbitol, D-galacturonic acid, D-mannitol, xylitol, methyl-α-D-glycopyranoside and a disaccharide xylobiose. Novel high-throughput nanoESI HCT mass spectrometry method was implemented and optimized to specify the HOF and conventional FOS. 5. Lsc2 and Lsc3 were shown to be stabile and catalytically active proteins that preserved their activity at various pH and temperature values. They also tolerated presence of several metal ions and detergents. Those characteristics are essential for extracellular proteins and they are important for enzymes to be used in industry. As we showed that the levansucrases of P. syringae pv. tomato can produce biotechnologically promising products levan, FOS and HOF from a cheap substrate, sucrose, they should certainly be regarded as feasible biocatalysts for technological approaches. 6. Whereas no data on structure-function relationships among levansucrases of Pseudomonas bacteria were available, mutational analysis of Lsc3 was initiated. Asp62, Asp219 and Glu303 were predicted as catalytic triad residues of Lsc3. Mutation analysis of Lsc3 specified Thr302 and His321 as residues implicated in substrate binding and transfructosylation reaction possibly belonging to the +1 subsite of the Lsc3 active centre.
Levansucrases are bacterial enzymes belonging to family 68 of glycoside hydro¬lases (GHs). They catalyze hydrolysis of their substrate but also have prominent fructosyl transferase activity. The main substrate for levansucrases is sucrose which is major disaccharide in plants. Raffinose, likewisely abundant in some plants, is also used as a substrate. Spectrum of reaction products of levan¬sucrases comprises highly polymeric levan and fructooligosaccharides (FOS) of various degree of polymerization (DP). The entity of levansucrase reaction products depends on the enzyme and its origin, but also on reaction conditions enabling manipulation of the product spectrum. Levansucrases share highly similar five-blade β-propeller fold with other GH68 and 32 enzymes including bacterial inulosucrases, plant and microbial invertases, fructan exohydrolases and fructosyl transferases. In this thesis, levansucrases Lsc2 and Lsc3 from a plant pathogenic bac¬terium Pseudomonas syringae pv. tomato were expressed in a bacterial host Escherichia coli, purified and characterized. As a comparison, levansucrase LscA from P. chlororaphis subsp. aurantiaca was studied. The main results of this thesis are summarized as follows: 1. Lsc2 and Lsc3 of P. syringae pv. tomato were expressed with high yield in a bacterial host Escherichia coli exerting two expression systems. The first system relies on maltase gene promoter PMAL from a methylotrophic yeast Hansenula polymorpha. We verified that functionality of PMAL in E. coli is caused by the presence of σ70-like boxes in the eucaryotic promoter. The PMAL was shown to have suitable strength in E. coli providing a sufficient amount of catalytically active protein of interest. Due to its dual activity, it can be used as a promoter shared by yeasts and bacteria in heterologous protein expression trials. A pURI3 vector-based expression system was ad¬justed to obtain mutant and wild-type N-terminally His-tagged Lsc3 proteins. 2. P. syringae pathovars are exceptional among other bacterial species because they possess up to three levansucrase alleles in their genomes. We showed that all three lsc genes (lsc1, lsc2, lsc3) of P. syringae pv. tomato DC3000 encode functional levansucrase proteins, if expressed from a heterologous promoter in E. coli. 3. Enzymology and biochemistry of Lsc2 and Lsc3 was addressed and com¬pared with that of LscA from P. chlororaphis subsp. aurantiaca. All three proteins were shown to use sucrose, raffinose and stachyose as substrates. Low hydrolytic activity towards levan was also recorded. Affinities for sucrose of Lsc3, Lsc2 and LscA were similar, the Km values being around 20 mM. The maximum reaction velocity and catalytic efficiency of LscA was much lower than that of Lsc2 and Lsc3 proteins. Polymerization properties of the enzymes differed. At low sucrose concentration, Lsc3 polymerized much more effectively than LscA. At high substrate concentration, the difference in transfructosylating activity was evened out, but the FOS spectrum was still different – the LscA produced more high-DP FOS than Lsc3 or Lsc2. 4. As a novel feature for levansucrases of pseudomonads, this study shows the ability of Lsc3 and LscA to produce heterooligofructans (HOF) by trans¬fructosylating nonconventional fructosyl acceptors. For the first time, levansucrases were shown to transfructosylate D-sorbitol, D-galacturonic acid, D-mannitol, xylitol, methyl-α-D-glycopyranoside and a disaccharide xylobiose. Novel high-throughput nanoESI HCT mass spectrometry method was implemented and optimized to specify the HOF and conventional FOS. 5. Lsc2 and Lsc3 were shown to be stabile and catalytically active proteins that preserved their activity at various pH and temperature values. They also tolerated presence of several metal ions and detergents. Those characteristics are essential for extracellular proteins and they are important for enzymes to be used in industry. As we showed that the levansucrases of P. syringae pv. tomato can produce biotechnologically promising products levan, FOS and HOF from a cheap substrate, sucrose, they should certainly be regarded as feasible biocatalysts for technological approaches. 6. Whereas no data on structure-function relationships among levansucrases of Pseudomonas bacteria were available, mutational analysis of Lsc3 was initiated. Asp62, Asp219 and Glu303 were predicted as catalytic triad residues of Lsc3. Mutation analysis of Lsc3 specified Thr302 and His321 as residues implicated in substrate binding and transfructosylation reaction possibly belonging to the +1 subsite of the Lsc3 active centre.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Keywords
sahhariidid, ensüümid, ensüümitehnoloogia, biokeemilised aspektid, saccharides, enzymes, enzyme technology, biochemical aspects