title: Properties of the inulinase gene levH1 of Lactobacillus casei IAM 1045; cloning, mutational and biochemical characterization.
abstract: Though some genetic features of lactobacillar fructan hydrolases were elucidated, information about their enzymology or mutational analyses were scarce. Lactobacillus casei IAM1045 exhibits extracellular activity degrading inulin. After partial purification of the inulin-degrading protein from the spent culture medium, several fragments were obtained by protease digestion. Based on their partial amino-acid sequences, oligonucleotide primers were designed, and its structural gene (levH1) was determined using the gene library constructed in the E. coli system. The levH1 gene encoded a protein (designated as LevH1), of which calculated molecular mass and pI were 138.8-kDa and 4.66, respectively. LevH1 (1296 amino-acids long) was predicted to have a four-domain structure, containing (i) an N-terminal secretion signal of 40 amino-acids, (ii) variable domain of about 140 residues whose function is unclear, (iii) a catalytic domain of about 630 residues with glycoside-hydrolase activity consisting of two modules, a five-blade β-propeller module linked to a β-sandwich module, (iv) a C-terminal domain of about 490 residues comprising five nearly perfect repeat sequences of 80 residues homologous to equivalents of other hypothetical cell surface proteins, followed by 37-residues rich in Ser/Thr/Pro/Gly, a pentad LPQAG (the LPXTG homologue). When overproduced in E. coli, the putative variable-catalytic domain region of about 770 residues exhibited exo-inulinase activity. Deletion analyses demonstrated that the variable-catalytic domain region containing two modules is important for enzymatic activity. Presence of eight conserved motifs (I-VIII) was suggested in the catalytic domain by comparative analysis, among which motif VIII was newly identified in the β-sandwich module in this study. Site-directed mutagenesis of conserved amino-acids in these motifs revealed that D198, R388, D389 and E440, were crucial for inulinase activity. Moreover, mutations of D502A and D683A in motif VI and VIII respectively caused significant decrease in the activity. These results suggested that the variable domain and β-sandwich module, besides the β-propeller module, are important for inulin-degrading activity of LevH1.
authors.affiliation: Molecular Biology Group, Faculty of Engineering, Toyama University, 3190 Gofuku, Toyama 930-8555, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Kuzuwa
authors.forename: Ken-ji
authors.initials: KJ
authors.lastname: Yokoi
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Kondo
authors.forename: Hisashi
authors.initials: H
authors.lastname: Kimoto
authors.forename: Ayanori
authors.initials: A
authors.lastname: Yamakawa
authors.forename: Akira
authors.initials: A
authors.lastname: Taketo
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Kodaira
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D007444:Inulin / Q000378:metabolism; D007780:Lacticaseibacillus casei / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D007444:Inulin; D006026:Glycoside Hydrolases; C021512:levanase; C023086:inulinase