[Thermotoga neopolitina gene cluster, participating in degradation of starch and maltodextrins: expression of aglB and aglA gene in Escherichia coli, properties of recombinant enzymes].
The aglB and aglA genes from the starch/maltodextrin utilization gene cluster of Thermotoga neapolitana were subcloned into pQE vectors for expression in Escherichia coli. The recombinant proteins AglB and AglA were purified to homogeneity and characterized. Both enzymes are hyperthermostable, the highest activity was observed at 85 degrees C. AglB is an oligomer of identical 55-kDa subunits capable of aggregation. This protein hydrolyses cyclodextrins and linear maltodextrins to glucose and maltose by liberating glucose from the reducing end of the molecules, and it is a cyclodextrinase with alpha-glucosidase activity. The pseudo-tetrasaccharide acarbose, a potent alpha-amylase and alpha-glucosidase inhibitor, does not inhibit AglB but, on the contrary, acarbose is degraded quantitatively by AglB. Recombinant AglB is activated in the presence of CaCl2, KCl, and EDTA, as well as after heating of the enzyme. AglA is a dimer of two identical 54-kDa subunits, and it hydrolyses the alpha-glycoside bonds of disaccharides and short maltooligosaccharides, acting on the substrate from the non-reducing end of the chain. It is a cofactor-dependent alpha-glucosidase with a wide action range, hydrolysing both oligoglucosides and galactosides with alpha-link. Thereby, the enzyme is not specific with respect to the configuration at the C4 position of its substrate. For the enzyme to be active, the presence of NAD+, DTT, and Mn2+ is required. Enzymes AglB and AglA supplement one another in substrate specificity and ensure complete hydrolysis to glucose for the intermediate products of starch degradation.
Institute of Molecular Genetics, Russian Academy of Medical Sciences, Moscow, 123182 Russia.
N A
NA
Lunina
O V
OV
Berezina
B
B
Veith
V V
VV
Zverlov
I P
IP
Vorob'eva
L A
LA
Chekanovskaia
I S
IS
Khromov
G
G
Raash
V
V
Libel'
G A
GA
Velikodvorskaia
D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D016965:Gram-Negative Anaerobic Straight, Curved, and Helical Rods / Q000235:genetics*; D006868:Hydrolysis; D005810:Multigene Family; D011134:Polysaccharides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013213:Starch / Q000378:metabolism*
D029968:Escherichia coli Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; C008315:maltodextrin; D013213:Starch