Molecular cloning of the structural gene for alkaline elastase YaB, a new subtilisin produced by an alkalophilic Bacillus strain

Abstract

Alkaline elastase YaB is an extracellular serine protease of the alkalophilic Bacillus strain YaB. We cloned the structural gene, ale, and determined the nucleotide sequence. The mature enzyme (268 amino acids) was preceded by a putative signal sequence and a prosequence (27 and 83 amino acids, respectively). The mature enzyme was 55 % homologous to subtilisin BPN'. Almost all the positively charged residues are predicted to be on the surface of the molecule, which would facilitate binding to elastin. The P1 substrate site-related sequences differed between alkaline elastase YaB and subtilisin BPN'.To identify enzymes that digest elastin efficiently, we screened for microbial enzymes among alkalophilic bacteria, because local denaturation of elastin under alkaline conditions might facilitate its enzymatic digestion. Alkaline elastase YaB secreted by the alkalophilic Bacillus strain YaB was thus selected and purified (25). The enzyme was characterized as a serine protease of about 25,000 daltons. Chemical modification studies revealed that certain histidine and tyrosine residues are involved in the enzymatic activity (21). The enzyme has the peculiar characteristic of a very high optimum pH for digestion of casein and elastin and an elastolytic activity comparable to that of porcine pancreatic elastase (25). The substrate specificity was examined by cutting oxidized insulin A and B chains (23) and by using synthetic peptide substrates (24). The enzyme has a P1 substrate preference for Ala, in contrast to that of subtilisin BPN' for Tyr, and it efficiently hydrolyzes succinyl-AlaAla-Ala-p-nitroanilide, a specific substrate of porcine elastase (24). Furthermore, the enzyme binds to elastin below its isoelectric point (pl 10.6) (22). These features encouraged us to characterize the enzyme as a microbial elastase.To determine the molecular mechanism of the extremely high optimum pH and high elastolytic activity of the enzyme, we cloned the structural gene and analyzed its nucleotide and deduced amino acid sequences.Bacillus subtilis DB104 (nprEI8 nprR2 AaprE3 his-101) (9) has lesions in the structural genes for neutral and alkaline proteases and was a gift from Roy H. Doi and Fujio Kawamura. Escherichia coli MC1061 and JM109 were used as the host strains for cloning. All bacterial strains were propagated at 37°C in nutrient broth (Eiken Chemical Co., Tokyo, Japan) or Luria broth (11) with vigorous aeration or on media solidified with 1.5% agar. Media were s pplemerted with 100 p.g of ampicillin per ml, 10 ,ug of kana.nycin pei :nl, or 20 .'g of tetracycline per ml when necessary. pL"U18 and pHY300PLK were purchased from Takara Shuzo Co. (Kyoto, Japan).To detect the production of alkaline elastase, elastin plates and skim milk plates were used. Elastin plates were made by overlaying 5 ml of 1% elastin (Sigma Chemical Co., St. Louis, Mo.) and 0.7% agar on basal agar plates containing * Corresponding author. 0.8% nutrient broth and 1.5% agar. Skim milk plates contained 0.8% nutrient broth, 1.5% agar, and 1% skim milk....

Intracellular Localization and Trafficking of Serine Proteinase AhSub and Cysteine Proteinase AhCP of Acanthamoeba healyi

“…The roles of preand propeptides of subtilisin trafficking in bacteria have been studied by biochemical and molecular biological investigations. The prepeptide functions as the signal sequence required for protein secretion across the cytoplasmic membrane, and the propeptide has been proposed to function as an intramolecular chaperone for production of active subtilisin (1,7,22,26). The role of pre-or propeptide in the protozoan subtilases was analyzed in PfSUB-1 (21).…”

The sequence of a subtilisin‐type protease (aerolysin) from the hyperthermophilic archaeum Pyrobaculum aerophilum reveals sites important to thermostability

“…DY, subtilisin DY (Betzel et al, 1993); SUBT. BPN', subtilisin BPN' (Pantoliano et al, 1989); ISP-I, ISP-1 (Rufo et al, 1990); B. ALCALOPHILUS, subtilisin from Bacillus alcalophilus (van der Laan et al, 1992); ELASTASE YAB, elastase YaB (Kaneko et al, 1989); BSUB MINOR PROT., minor protease from B. subtilis (Sloma et al, 1991); Aqualysin I, aqualysin I from Thermus aquaticus (Terada et al, 1990); PROTEINASE K, proteinase K from Tritirachium album (Gunkel & Gassen, 1989); Predictprotein, secondary structure prediction of Predictprotein algorithm for aerolysin; Thermitase 2", Proteinase K 2", Carlsberg 2", BPN' 2", secondary structures of thermitase, proteinase K, subtilisin Carlsberg, and subtilisin BPN'. a Numbering is based on the first amino acid in the P. aerophilum aerolysin sequence alignment, rather than the position of the initiator methionine.…”

Molecular Cloning, Nucleotide Sequence, and Expression of the Structural Gene for Alkaline Serine Protease from AlkaliphilicBacillussp. 221

“…It was pointed out that Gly166 formed the bottom of P 1 pocket of subtilisin BPN'. 2 7) On the basis of this finding, Kaneko et al 7 ) suggested that the deletion of four amino acids at the region between 161 and 164 might change the conformation of P 1 pocket and deduced that this distortion correlated with the P 1 preference of YaB elastase for Ala, in contrast to that of subtilisin BPN' for Tyr. Since 221 protease, which shared the same deletion, also had high elastolytic activity28,29) and similar cleavage pattern on oxidized insulin B-chain,30) this conformational change of the P 1 pocket might also happen in this protease.…”

“…amylosacchariticus 6 ) have been cloned and sequenced. Recently, the genes encoding alkaline serine proteases from alkaliphilic Bacillus strains YaB 7 ) and PB92 8 ) have also been cloned and sequenced. Nucleotide and amino acid sequences of these subtilisin-type enzymes share significant homology although these enzymes are distinct from each other in their enzymatic and physicochemical properties.…”