Collagenolytic Serine-Carboxyl Proteinase fromAlicyclobacillus sendaiensisStrain 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 (k cat , 5.41 s ؊1 ; K m , 32 M) and Met-Gly-ProArg*Gly-Phe-Pro-Gly-Ser (k cat , 351 s ؊1 ; K m , 214 M), 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.Collagen is an insoluble structural protein that accounts for approximately 30% of the total weight of animal proteins and is produced in large quantities as a by-product in livestock industries. It has recently been shown that the enzymatic degradation of collagen as well as that of gelatin, a denatured form of collagen, allows efficient utilization of these structural proteins. This degradation produces peptides, the collagen peptides, which have been shown to have several biological activities of industrial and medical interest (27), leading to the establishment of a wide variety of applications, e.g., an immunotherapeutic agent (7, 9), a moisturizer for cosmetics, a preservative (2), and seasoning and dietary materials (4).Generally, the enzymatic degradation of collagen is not affected by ordinary digestive proteinases but requires the collagen-specific, Zn 2ϩ -dependent metalloproteinases (collagenases). Although microbial collagenases have been found in a wide variety of mesophilic bacterial strains (3), the industrialscale application of known bacterial collagenases for collagen peptide production has been hampered because of their insufficient stability. In 2000...

The QM/MM Molecular Dynamics and Free Energy Simulations of the Acylation Reaction Catalyzed by the Serine-Carboxyl Peptidase Kumamolisin-As

“…Kumamolisin-As can cleave the -ProXaa*Yaa-sequence (here Xaa and Yaa denote the residues at the P 1 and P 1 ′ sites, respectively). One experimental observation that is of considerable interest is the fact that this enzyme generally has higher specificity for the substrates with a positively charged residue at the P 1 site than for those they do not carry such charge (3,9). For instance, it was found that the k cat value for a substrate containing the -ProArg*Gly sequence is ∼65-fold higher than for a substrate containing the -Pro-Gly*Gly sequence and the K M is ∼6.5-fold higher, leading to 10-fold increase of k cat /K M (3).…”

“…Kumamolsin-As belongs to the recently characterized family of serine-carboxyl peptidases (sedolisins) that were originally described by Murao, Oda, and co-workers about 20 years ago (1)(2)(3). Sedolisins are present in a wide variety of organisms, including archaea, bacteria, molds, slime molds (mixomycetes), amoebas, fishes, and mammals, and they are active at low pH and often high temperature.…”

Interstitial Collagen Catabolism

“…Although initially reported as being collagenolytic, the Ser proteases fibroblast activation protein/separase and trypsin-2 do not cleave interstitial collagens within their triple helices (20). 3 Additional interstitial collagenases include several that act under acidic conditions, such as Cynara cardunculus Asp protease cardosin A (22) and Alicyclobacillus sendaiensis Ser-carboxyl protease kumamolisin-As/ScpA (23), as well as the Cys proteases ginger (Zingiber officinale) GP2 and GP3 (24) and Fasciola hepatica FhCL2 and FhCL3 (25). GP2 hydrolyzes type I collagen at three distinct sites (Table 1) (24).…”

Biosynthesis of industrial enzymes by free and immobilized Alicyclobacillus in different matrices and the use of ultrafiltration in the enzymes concentration

“…The result of the present study was close to that obtained by Lima et al, 35 in the production of collagenase enzyme by Candida albicans, which had a value of 7.06 U mL -1 . Nakayama et al, 36 studied the production of collagenase enzyme by Bacillus NATP-1, previously described as A. sendaiensis, 4 and obtained enzymatic activity of 24 U mL -1 through the use of oxygen and pH control during enzyme production in a potato dextrose broth medium. These same authors also evaluated enzyme production in the presence of collagen and gelatin to 1.5% (w/v), and found that this factor did not induce collagenase production.…”

“…1 Enzymes produced by such microorganisms have been studied for use in various industrial applications, including galactosidase, esterases, proteases and amylases, due to their thermostable characteristics. [2][3][4][5] The Alicyclobacillus acidocaldarius and Alicyclobacillus sendaiensis species have been evaluated for the production of both amylase and collagenase, respectively.…”