Molecular cloning and characterization of trehalose biosynthesis genes from hyperthermophilic archaebacterium Metallosphaera hakonesis.
The trehalose (alpha-D-glucopyranosyl-[1,1]-alpha-D-glucopyranose) biosynthesis genes MhMTS and MhMTH, encoding a maltooligosyltrehalose synthase (MhMTS) and a maltooligosyltrehalose trehalohydrolase (MhMTH), respectively, have been cloned from the hyperthermophilic archaebacterium Metallosphaera hakonesis. The ORF of MhMTS is 2,142 bp long, and encodes 713 amino acid residues constituting a 83.8 kDa protein. MhMTH is 1,677 bp long, and encodes 558 amino acid residues constituting a 63.7 kDa protein. The deduced amino acid sequences of MhMTS and MhMTH contain four regions highly conserved for MTSs and three for MTHs that are known to constitute substrate-binding sites of starch-hydrolyzing enzymes. Recombinant proteins obtained by expressing the MhMTS and MhMTH genes in E. coli catalyzed a sequential reaction converting maltooligosaccharides to produce trehalose. Optimum pH of the MhMTS/MhMTH enzyme reaction was around 5.0 and optimum temperature was around 70 degrees C. Trehalose-producing activity of the MhMTS/ MhMTH was notably stable, retaining 80% of the activity after preincubation of the enzyme mixture at 70 degrees C for 48 h, but was gradually abolished by incubating at above 85 degrees C. Addition of thermostable 4-alpha-glucanotransferase increased the yield of trehalose production from maltopentaose by 10%. The substrate specificity of the MhMTS/MhMTH-catalyzed reaction was extended to soluble starch, the most abundant maltodextrin in nature.
Seo
Ju-Seok
JS
Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Korea.
An
Ju Hee
JH
Baik
Moo-Yeol
MY
Park
Cheon Seok
CS
Cheong
Jong-Joo
JJ
Moon
Tae Wha
TW
Park
Kwan Hwa
KH
Choi
Yang Do
YD
Kim
Chung Ho
CH
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D019641:DNA, Archaeal / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D019766:Genes, Archaeal; D005959:Glucosidases / Q000235:genetics / Q000378:metabolism; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism; D006004:Glycogen Debranching Enzyme System / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013213:Starch / Q000378:metabolism; D013379:Substrate Specificity; D016943:Sulfolobaceae / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D020124:Thermotoga maritima / Q000201:enzymology / Q000235:genetics; D014199:Trehalose / Q000096:biosynthesis* / Q000235:genetics
D017931:DNA Primers; D019641:DNA, Archaeal; D006004:Glycogen Debranching Enzyme System; D011994:Recombinant Proteins; D013213:Starch; D014199:Trehalose; D005964:Glucosyltransferases; C099084:maltooligosyl trehalose synthase; C020569:4 alpha-glucanotransferase; D005959:Glucosidases; C099156:maltooligosyl trehalose trehalohydrolase