Expression of cyclodextrinase gene from Paenibacillus sp. A11 in Escherichia coli and characterization of the purified cyclodextrinase.
The expression of the Paenibacillus sp. A11 cyclodextrinase (CDase) gene using the pUC 18 vector in Escherichia coli JM 109 resulted in the formation of an insoluble CDase protein in the cell debris in addition to a soluble CDase protein in the cytoplasm. Unlike the expression in Paenibacillus sp. A11, CDase was primarily observed in cytoplasm. However, by adding 0.5 M sorbitol as an osmolyte, the formation of insoluble CDase was prevented while a three-fold increase in cytoplasmic CDase activity was achieved after a 24 h-induction. The recombinant CDase protein was purified to approximately 14-fold with a 31% recovery to a specific activity of 141 units/mg protein by 40-60% ammonium sulfate precipitation, DEAE-Toyopearl 650 M, and Phenyl Sepharose CL-4B chromatography. It was homogeneous by non-denaturing and SDS-PAGE. The enzyme was a single polypeptide with a molecular weight of 80 kDa, as determined by gel filtration and SDS-PAGE. It showed the highest activity at pH 7.0 and 40 degrees C. The catalytic efficiency (k(cat)/K(m)) values for alpha-, beta-, and gamma- CD were 3.0 x 10(5), 8.8 x 10(5), and 5.5 x 10(5) M(-1) min(-1), respectively. The enzyme hydrolyzed CDs and linear maltooligosaccharides to yield maltose and glucose with less amounts of maltotriose and maltotetraose. The rates of hydrolysis for polysaccharides, soluble starch, and pullulan were very low. The cloned CDase was strongly inactivated by N-bromosuccinimide and diethylpyrocarbonate, but activated by dithiothreitol. A comparison of the biochemical properties of the CDases from Paenibacillus sp. A11 and E. coli transformant (pJK 555) indicates that they were almost identical.
Kaulpiboon
Jarunee
J
Biological Science PhD Program, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Pongsawasdi
Piamsook
P
D000818:Animals; D001407:Bacillus / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011108:Polymers / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D008670:Metals; D011108:Polymers; C024617:polyol; D006026:Glycoside Hydrolases; C048745:cyclomaltodextrinase