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.
Seo
Ju-Seok
JS
Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Korea.
An
Ju Hee
JH
Cheong
Jong-Joo
JJ
Choi
Yang Do
YD
Kim
Chung Ho
CH
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