Purification and characterization of a thermostable phytate resistant alpha-amylase from Geobacillus sp. LH8.
A thermophilic and amylolytic bacterium (LH8) was isolated from the hot spring of Larijan in Iran at 65 degrees C. Identification of strain LH8 by 16S rDNA sequence analysis showed that LH8 strain belongs to the Geobacillus sp. with 99% sequence similarity with the 16S rDNA of Geobacillus thermodenitrificans. A new alpha-amylase (GA) was extracted from this strain and purified by ion-exchange chromatography. SDS-PAGE showed a single band with an apparent molecular mass of 52kDa. The optimum temperature and pH were 80 degrees C and 5-7, respectively. In the presence of Mn2+, Ca2+, K+, Cr3+ and Al3+, the enzyme activity was stimulated while Mg2+, Ba2+, Ni2+, Zn2+, Fe3+, Cu2+ and EDTA reduced the activity. The K(m) and V(max) values for starch were 3 mg ml(-1) and 6.5 micromol min(-1), respectively. The gene encoding alpha-amylase was isolated and the amino acid sequence was deduced. Comparison of GA and other alpha-amylase amino acid sequences suggested that GA has conserved regions that were previously identified in alpha-amylase family but GA exhibited some substitutions in the sequence. Its phytate resistant is an important property of this enzyme. 5 and 10 mM phytic acid did not inhibit this enzyme. Therefore, features of phytate resistant alpha-amylase from Geobacillus sp. LH8 are discussed.
Mollania
Nasrin
N
Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran.
Khajeh
Khosro
K
Hosseinkhani
Saman
S
Dabirmanesh
Bahareh
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability / Q000187:drug effects; D056506:Geobacillus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010833:Phytic Acid / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013050:Spectrometry, Fluorescence; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D008670:Metals; D012336:RNA, Ribosomal, 16S; D010833:Phytic Acid; D000516:alpha-Amylases