Bifunctional recombinant fusion enzyme between maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase of thermophilic microorganism Metallosphaera hakonensis.
MhMTS and MhMTH are trehalose (alpha-D-glucopyranosyl- [1,1]-alpha-D-glucopyranose) biosynthesis genes of the thermophilic microorganism Metallosphaera hakonensis, and encode a maltooligosyltrehalose synthase (MhMTS) and a maltooligosyltrehalose trehalohydrolase (MhMTH), respectively. In this study, the two genes were fused inframe in a recombinant DNA, and expressed in Escherichia coli to produce a bifunctional fusion enzyme, MhMTSH. Similar to the two-step reactions with MhMTS and MhMTH, the fusion enzyme catalyzed the sequential reactions on maltopentaose, maltotriosyltrehalose formation, and following hydrolysis, producing trehalose and maltotriose. Optimum conditions for the fusion enzyme-catalyzed trehalose synthesis were around 70 degrees and pH 5.0-6.0. The MhMTSH fusion enzyme exhibited a high degree of thermostability, retaining 80% of the activity when pre-incubated at 70 degrees for 48 h. The stability was gradually abolished by incubating the fusion enzyme at above 80 degrees . The MhMTSH fusion enzyme was active on various sizes of maltooligosaccharides, extending its substrate specificity to soluble starch, the most abundant natural source of trehalose production.
Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Korea.
Ju-Seok
JS
Seo
Ju Hee
JH
An
Jong-Joo
JJ
Cheong
Yang Do
YD
Choi
Chung Ho
CH
Kim
D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005959:Glucosidases / Q000235:genetics / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013213:Starch / Q000378:metabolism; D016943:Sulfolobaceae / Q000201:enzymology* / Q000235:genetics; D014199:Trehalose / Q000096:biosynthesis*
D011993:Recombinant Fusion Proteins; D013213:Starch; D014199:Trehalose; D005964:Glucosyltransferases; C099084:maltooligosyl trehalose synthase; D005959:Glucosidases; C099156:maltooligosyl trehalose trehalohydrolase