Characterization of a novel amylolytic enzyme encoded by a gene from a soil-derived metagenomic library.
It has been estimated that less than 1% of the microorganisms in nature can be cultivated by conventional techniques. Thus, the classical approach of isolating enzymes from pure cultures allows the analysis of only a subset of the total naturally occurring microbiota in environmental samples enriched in microorganisms. To isolate useful microbial enzymes from uncultured soil microorganisms, a metagenome was isolated from soil samples, and a metagenomic library was constructed by using the pUC19 vector. The library was screened for amylase activity, and one clone from among approximately 30,000 recombinant Escherichia coli clones showed amylase activity. Sequencing of the clone revealed a novel amylolytic enzyme expressed from a novel gene. The putative amylase gene (amyM) was overexpressed and purified for characterization. Optimal conditions for the enzyme activity of the AmyM protein were 42 degrees C and pH 9.0; Ca2+ stabilized the activity. The amylase hydrolyzed soluble starch and cyclodextrins to produce high levels of maltose and hydrolyzed pullulan to panose. The enzyme showed a high transglycosylation activity, making alpha-(1, 4) linkages exclusively. The hydrolysis and transglycosylation properties of AmyM suggest that it has novel characteristics and can be regarded as an intermediate type of maltogenic amylase, alpha-amylase, and 4-alpha-glucanotransferase.
Yun
Jiae
J
Department of Food Science and Technology, School of Agricultural Biotechnology, Center for Agricultural Biomaterials, Seoul National University, Seoul, South Korea.
Kang
Seowon
S
Park
Sulhee
S
Yoon
Hyunjin
H
Kim
Myo-Jeong
MJ
Heu
Sunggi
S
Ryu
Sangyeol
S
D000595:Amino Acid Sequence; D000681:Amylases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015698:Genomic Library; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology
D004269:DNA, Bacterial; D000681:Amylases