Ogataea polymorpha maltaas: valgu ja selle mutantide iseloomustamine ja mikroplaadipõhise aktiivsuse analüüsi meetodi väljatöötamine

Pärmi Ogataea polymorpha maltaas MAL1 on α-glükosidaas, milles on optimeeritud kaks ensümaatilist aktiivsust, mille jaoks pagaripärmil on kahte tüüpi α-glükosidaasid – maltaasid ja isomaltaasid. MAL1 on laia substraadivaliku tõttu huvitav objekt α-glükosidaaside substraadispetsiifikat mõjutavate aminohapete uurimiseks. Lisaks suhkrute lõhustamisele võivad α-glükosidaasid neid ka transglükosüülida. Suhkrute transglükosüülimireaktsiooni produktid – oligosahhariidid – on sageli prebiootiliste või antikariogeensete omadustega. MAL1 võib oma laia substraadivaliku tõttu sünteesida huvitavaid oligosahhariide. Käesolevas töös uuriti MAL1 substraadispetsiifikat määravaid aminohappeid mutatsioonanalüüsi abil ning seati sisse metoodika, mille abil saab edaspidi sellealast uurimistööd teha odavamalt ja kiiremini. Samuti uuriti MAL1 transglükosüülimisvõimet ning -reaktsiooni produkte

Pärmide MAL klastrid ja valgud põhirõhuga Scheffersomyces stipitis’e α-glükosidaasidel

Pagaripärmi α-glükosiidsete suhkrute metabolismi ja selle eest vastutavaid α-glükosidaase on põhjalikult kirjeldatud, kuid mitte-konventsionaalsetele pärmidele on selles valdkonnas pööratud vähem tähelepanu. Käesoleva töö raames otsiti mitte-konventsionaalsete pärmide genoomist α-glükosiidsete suhkrute metabolismi eest vastutavaid geeniklastreid (MAL klastreid) ja uuriti nendes geeniklastrites kodeeritud valkude fülogeneesi. Samuti kirjeldati ühe mitte-konventsionaalse pärmi, Scheffersomyces stipitis’e, nelja α-glükosidaasi. Substraadivaliku poolest ei sarnanenud S. stipitis’e α-glükosidaasid pagaripärmi tänapäevaste α-glükosidaasidedega, vaid hoopis nende hüpoteetilise eellasvalguga, millel on laiem substraadivalik

Genome Mining of Non-Conventional Yeasts: Search and Analysis of MAL Clusters and Proteins

Genomic clustering of functionally related genes is rare in yeasts and other eukaryotes with only few examples available. Here, we summarize our data on a nontelomeric MAL cluster of a non-conventional methylotrophic yeast Ogataea (Hansenula) polymorpha containing genes for α-glucosidase MAL1, α-glucoside permease MAL2 and two hypothetical transcriptional activators. Using genome mining, we detected MAL clusters of varied number, position and composition in many other maltose-assimilating non-conventional yeasts from different phylogenetic groups. The highest number of MAL clusters was detected in Lipomyces starkeyi while no MAL clusters were found in Schizosaccharomyces pombe and Blastobotrys adeninivorans. Phylograms of α-glucosidases and α-glucoside transporters of yeasts agreed with phylogenesis of the respective yeast species. Substrate specificity of unstudied α-glucosidases was predicted from protein sequence analysis. Specific activities of Scheffersomycesstipitis α-glucosidases MAL7, MAL8, and MAL9 heterologously expressed in Escherichia coli confirmed the correctness of the prediction—these proteins were verified promiscuous maltase-isomaltases. α-Glucosidases of earlier diverged yeasts L. starkeyi, B. adeninivorans and S. pombe showed sequence relatedness with α-glucosidases of filamentous fungi and bacilli

Characterization of a Maltase from an Early-Diverged Non-Conventional Yeast Blastobotrys adeninivorans

Genome of an early-diverged yeast Blastobotrys (Arxula) adeninivorans (Ba) encodes 88 glycoside hydrolases (GHs) including two α-glucosidases of GH13 family. One of those, the rna_ARAD1D20130g-encoded protein (BaAG2; 581 aa) was overexpressed in Escherichia coli, purified and characterized. We showed that maltose, other maltose-like substrates (maltulose, turanose, maltotriose, melezitose, malto-oligosaccharides of DP 4‒7) and sucrose were hydrolyzed by BaAG2, whereas isomaltose and isomaltose-like substrates (palatinose, α-methylglucoside) were not, confirming that BaAG2 is a maltase. BaAG2 was competitively inhibited by a diabetes drug acarbose (Ki = 0.8 µM) and Tris (Ki = 70.5 µM). BaAG2 was competitively inhibited also by isomaltose-like sugars and a hydrolysis product—glucose. At high maltose concentrations, BaAG2 exhibited transglycosylating ability producing potentially prebiotic di- and trisaccharides. Atypically for yeast maltases, a low but clearly recordable exo-hydrolytic activity on amylose, amylopectin and glycogen was detected. Saccharomyces cerevisiae maltase MAL62, studied for comparison, had only minimal ability to hydrolyze these polymers, and its transglycosylating activity was about three times lower compared to BaAG2. Sequence identity of BaAG2 with other maltases was only moderate being the highest (51%) with the maltase MalT of Aspergillus oryzae