Naoki Tsuruoka

Collagenolytic Serine-Carboxyl Proteinase from Alicyclobacillus sendaiensis Strain NTAP-1: Purification, Characterization, Gene Cloning, and Heterologous Expression

ABSTRACT Enzymatic degradation of collagen produces peptides, the collagen peptides, which show a variety of bioactivities of industrial interest. Alicyclobacillus sendaiensis strain NTAP-1, a slightly thermophilic, acidophilic bacterium, extracellularly produces a novel thermostable collagenolytic activity, which exhibits its optimum at the acidic region (pH 3.9) and is potentially applicable to the efficient production of such peptides. Here, we describe the purification to homogeneity, characterization, gene cloning, and heterologous expression of this enzyme, which we call ScpA. Purified ScpA is a monomeric, pepstatin-insensitive carboxyl proteinase with a molecular mass of 37 kDa which exhibited the highest reactivity toward collagen (type I, from a bovine Achilles tendon) among the macromolecular substrates examined. On the basis of the sequences of the peptides obtained by digestion of collagen with ScpA, the following synthetic peptides were designed as substrates for ScpA and kinetically analyzed: Phe-Gly-Pro-Ala*Gly-Pro-Ile-Gly (kcat, 5.41 s−1; Km, 32 μM) and Met-Gly-Pro-Arg*Gly-Phe-Pro-Gly-Ser (kcat, 351 s−1; Km, 214 μM), where the asterisks denote the scissile bonds. The cloned scpA gene encoded a protein of 553 amino acids with a calculated molecular mass of 57,167 Da. Heterologous expression of the scpA gene in the Escherichia coli cells yielded a mature 37-kDa species after a two-step proteolytic cleavage of the precursor protein. Sequencing of the scpA gene revealed that ScpA was a collagenolytic member of the serine-carboxyl proteinase family (the S53 family according to the MEROPS database), which is a recently identified proteinase family on the basis of crystallography results. Unexpectedly, ScpA was highly similar to a member of this family, kumamolysin, whose specificity toward macromolecular substrates has not been defined.

Promotion by Collagen Tripeptide of Type I Collagen Gene Expression in Human Osteoblastic Cells and Fracture Healing of Rat Femur

Peptides produced by the enzymatic degradation of collagens are reported to have various activities of biological and medical interest. The mechanisms underlying their actions are, however, poorly understood. We have produced, by collagenase digestion of type I collagen, a highly purified, non-antigenic, and low allergenic tripeptide fraction (collagen tripeptide, Ctp). We report here the effects of Ctp on the in vivo bone fracture healing and in vitro calcification of osteoblastic cells. An oral administration of Ctp to rats with a femur fracture accelerated the fracture healing. Ctp apparently stimulated the calcification of human osteoblastic cells in culture. This osteotrophic effect was accompanied by a significant increase in type I collagen protein production and its mRNA levels. DNA microarray and quantitative RT-PCR analyses demonstrated that Ctp upregulated the bone-specific transcription factor, Osterix, suggesting that the induction of type I collagen gene expression by Ctp was mediated by upregulation of this factor.

Thermostable collagenolytic activity of a novel thermophilic isolate, Bacillus sp. strain NTAP-1.

We isolated an acidophilic thermophile belonging to the genus Bacillus, strain NTAP-1, which secreted a thermostable collagenolytic activity into the culture medium. The collagenolytic activity exhibited an optimum pH for Azocoll hydrolysis of pH 3.9 and was not completely inhibited by 10 mM ethylenediaminetetraacetic acid (residual activity, 63%), suggesting that Bacillus NTAP-1 produces a novel acid proteinase with highest activity for collagen. The collagenolytic activity was thermostable; more than 80% of the original activity was retained after incubation of the culture supernatant at pH 4.0 and 60 degrees C for 4 h.