Identification of essential histidine residues in a recombinant alpha-amylase of thermophilic and alkaliphilic Bacillus sp. strain TS-23.
To understand the structure-function relationships of a truncated Bacillus sp. strain TS-23 alpha-amylase, each of His-137, His-191, His-239, His-269, His-305, His-323, His-361, His-436, and His-475 was replaced with leucine. The molecular masses of the purified wild-type and mutant enzymes were approximately 54 kDa. The specific activity of His323Leu and His436Leu was decreased by more than 52%, while His239Leu, His305Leu, and His475Leu showed activity similar to that of the wild-type enzyme. As compared with the wild-type enzyme, His323Leu and His436Leu exhibited a 62% decrease in the value of k(cat)/ K(m). Alterations in His-191, His-239, His-305, and His-475 did not cause a significant change in the K(m) or k(cat) values. At 70 degrees C, a decreased half-life was observed in His436Leu. These results indicate that His-137, His-269, and His-361 of Bacillus sp. strain TS-23 alpha-amylase are important for proper catalytic activity and that His-436 may contribute to the thermostability of the enzyme.
Chang
Chen-Tien
CT
Department of Food and Nutrition, Providence University, Shalu, Taichung 433-01, Taiwan.
Lo
Huei-Fen
HF
Chi
Meng-Chun
MC
Yao
Chia-Yu
CY
Hsu
Wen-Hwei
WH
Lin
Long-Liu
LL
D000595:Amino Acid Sequence; D001407:Bacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D002478:Cells, Cultured; D003470:Culture Media; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000032:analysis*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D003470:Culture Media; D011994:Recombinant Proteins; D006639:Histidine; D000516:alpha-Amylases