Expression and characterization of sweet potato invertase in Pichia pastoris.
An invertase cDNA (Ibbetafruct1) was cloned from sweet potato leaves and characterized. The deduced amino acid sequence of the Ibbetafruct1-encoded protein was closely related to vacuolar invertases and included the WECVD catalytic domain characteristic of them. An expression plasmid containing the coding region of Ibbetafruct1 under the control of the alcohol oxidase promoter was used to transform the methylotrophic yeast Pichia pastoris. The biochemical properties for the expressed recombinant enzyme, which was determined to be the acid beta-fructofuranosidase with an acidic pI value (5.1), were similar to those of vacuolar invertases purified from sweet potato. Periodic acid/Schiff staining and Con A-Sepharose gel-binding experiments revealed the recombinant invertase to be a glycoprotein containing glucose and/or mannose residues. Furthermore, the carbohydrate moiety appears to be a key determinant of the enzyme's sucrose hydrolysis activity, substrate affinity, and thermal stability.
Huang
Wen-Chin
WC
Department of Agricultural Chemistry, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei, Taiwan.
Wang
Ai-Yu
AY
Wang
Li-Ting
LT
Sung
Hsien-Yi
HY
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D027723:Ipomoea batatas / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D018515:Plant Leaves / Q000201:enzymology; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D014162:Transfection; D043324:beta-Fructofuranosidase
D018076:DNA, Complementary; D018744:DNA, Plant; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase