Purification and characterization of aqualysin I (a thermophilic alkaline serine protease) produced by <i>Thermus aquaticus</i> YT‐1

Matsuzawa, Hiroshi; Tokugawa, Kazuhisa; Hamaoki, Masaru; Mizoguchi, Masashi; Taguchi, Hayao; Terada, Ichiro; Kwon, Suk‐Tae; Ohta, Takao

doi:10.1111/j.1432-1033.1988.tb13809.x

Cited by 106 publications

(62 citation statements)

References 29 publications

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“…These results also suggest that the two proteases are different from each other in terms of substrate specificity and kinetic values. AKP-Vm enzyme is active under alkali condition raging from pH 8.0-12.0, and the enzyme activity can be inhibited by inhibitors (Table II) such as PMSF and aprotinin that are known as inhibitors for serine proteases (21). These results support the fact that AKP-Vm belongs to the family of alkaline serine proteases.…”

Section: Discussionsupporting

confidence: 73%

Purification and characterization of a novel alkaline serine protease secreted by Vibrio metschnikovii

Park

Park²,

Park³

et al. 2011

Int J Mol Med

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Abstract.A novel extracellular alkaline serine protease secreted by Vibrio metschnikovii (V. metschnikovii) ATCC700040 cells was purified by three chromatographic steps and characterized in terms of enzymatic kinetics and substrate specificity. The purified enzyme (named AKP-Vm) was composed of a single polypeptide with an apparent molecular weight of 50 kDa on 12% SDS-polyacrylamide gel in the presence of CuCl 2 . The optimal temperature and the pH for the enzyme were found to be 37˚C and 9.5, respectively. However, the enzyme activity was inhibited by inhibitors such as PMSF and aprotinin. AKP-Vm could hydrolyze a peptide bond at the carboxyl side of the arginine residue, as revealed by its amidolytic activity toward a chromogenic substrate, Boc-Val-Pro-Arg-pNA. . AKP-Vm protease could cleave various blood coagulation-associated proteins, including fibrinogen, prothrombin and thrombin. In particular, the enzyme showed powerful fibrinogenolytic and fibrinolytic activities, as it could cleave all major chains of fibrinogen and also digest cross-linked fibrin. The results obtained suggest that AKP-Vm is a novel alkaline serine protease that can actively cleave fibrinogen and cross-linked fibrin.

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Section: Discussionsupporting

confidence: 73%

Purification and characterization of a novel alkaline serine protease secreted by Vibrio metschnikovii

Park

Park²,

Park³

et al. 2011

Int J Mol Med

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“…Because subtilisins are commercially valuable enzymes, extensive attempts to improve their activities and stabilities with proteinengineering technology have also been made (10, 55, 60). The subtilisin family includes subtilisins from (hyper)thermophiles (13,27,28,35) and psychrophiles (3,14,29,37). The crystal structures of some of them have been determined (2,4,50,57).…”

mentioning

confidence: 99%

Ca ²⁺ -Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis : the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

Pulido

Saito

Tanaka

et al. 2006

Appl Environ Microbiol

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Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family. T. kodakaraensis subtilisin in a proform (T. kodakaraensis pro-subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-subtilisin was inactive in the absence of Ca 2؉ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca 2؉ at 80°C. This maturation process was completed within 30 min at 80°C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-subtilisin‫)ء‬ but forms an inactive complex with T. kodakaraensis mat-subtilisin‫,ء‬ at lower temperatures. At 80°C, approximately 30% of T. kodakaraensis pro-subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin‫,ء‬ and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-subtilisin was inactive in the absence of Ca 2؉ but was activated upon incubation with Ca 2؉ at 80°C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-subtilisin‫,ء‬ indicating that T. kodakaraensis mat-subtilisin does not require T. kodakaraensis propeptide for folding. However, only ϳ5% of T. kodakaraensis mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-subtilisin‫ء‬ and noncompetitively inhibited its activity with a K i of 25 ؎ 3.0 nM at 20°C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-subtilisin molecules that are activated later by those that are activated earlier.Subtilisin-like serine proteases (subtilases) are widely distributed in various organisms, including bacteria, archaea, and eucaryotes (48). They are divided into six families (47). Of these, the structures and functions of the subtilisin family (EC 3.4.21.108), which is represented by subtilisin E from Bacillus subtilis (52), subtilisin BPNЈ from Bacillus amyloliquefaciens (61), and subtilisin Carlsberg from Bacillus licheniformis (25), have been most extensively studied. The crystal structures of these subtilisins have been determined (26,54,64). Because subtilisins are commercially valuable enzymes, extensive attempts to improve their activities and stabilities with proteinengineering technology have also been made (10, 55, 60). The subtilisin family includes subtilisins from (hyper)thermophiles (13,27,28,35) and psychrophiles (3,14,29,37). The crystal structures of some of them have been determined (2,4,50,57). These thermostable and thermolabile subtilisins have been regarded not only as good models for studying stability-activitystructure relationships of proteins, but also as potent...

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“…This deletion should also influence P1 specificity. Aqualysin I preferentially hydrolyzed Ala esters and showed some elastolytic activity (12).…”

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confidence: 99%

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

et al. 1989

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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....

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Purification and characterization of aqualysin I (a thermophilic alkaline serine protease) produced by Thermus aquaticus YT‐1

Cited by 106 publications

References 29 publications

Purification and characterization of a novel alkaline serine protease secreted by Vibrio metschnikovii

Purification and characterization of a novel alkaline serine protease secreted by Vibrio metschnikovii

Ca ²⁺ -Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis : the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

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

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Purification and characterization of aqualysin I (a thermophilic alkaline serine protease) produced by Thermus aquaticus YT‐1

Cited by 106 publications

References 29 publications

Purification and characterization of a novel alkaline serine protease secreted by Vibrio metschnikovii

Purification and characterization of a novel alkaline serine protease secreted by Vibrio metschnikovii

Ca 2+ -Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis : the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

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

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Ca ²⁺ -Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis : the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding