Characterization of induced Staphylococcus aureus bacteriophage SAP-26 and its anti-biofilm activity with rifampicin

Lytic bacteriophages (phages) have been investigated as treatments for bacterial infectious diseases. An induced phage, SAP-26, was isolated from a clinical isolate of Staphylococcus aureus. It belongs to the family Siphoviridae and its genome consists of double-stranded 41,207 bp DNA coding for 63 open reading frames. The phage SAP-26 showed a wide spectrum of lytic activity against both methicillin-resistant S. aureus and methicillin-susceptible S.aureus. Furthermore, combined treatment with a phage and antimicrobial agents showed a strong biofilm removal effect which induced structural changes in the biofilm matrix and a substantial decrease in the number of bacteria. Such a broad host range in S. aureus and biofilm removal activity of the phage SAP-26 suggests the possibility of its use as a therapeutic phage in combination with appropriate antimicrobial agent(s). Among the three antimicrobial agents combined with phage, the combination of rifampicin showed the best biofilm removal effect. To the authors' knowledge, this study showed for the first time that S. aureus biofilm could be efficiently eradicated with the mixture of phage and an antimicrobial agent, especially rifampicin.     

In Vitro Antibacterial Activity of 4-Phenyl-1-(2-phenyl-allyl)pyridinium bromide: A Novel Class of Pyridinium Based Antibacterial Compounds

The continuing increase in the incidence of multi drug resistant pathogenic bacteria and shortage of new antimicrobial agents are the prime driver in efforts to identify the novel antimicrobial classes. In vitro antibacterial activity of 4-phenyl-1-(2-phenylallyl) pyridinium bromide was tested against Gram positive Staphylococcus aureus, Streptococcus species, Bacillus subtilis, and Gram negative Klebsiella aerogenes and Escherichia coli using disk diffusion method. Among them S. aureus showed strong antibacterial activity (21.99 ± 0.03 mm) while E. coli showed very little activity (8.97 ± 0.06 mm) towards the compound. The MIC of 4-phenyl-1-(2-phenyl-allyl)-pyridinium bromide for 90% S. aureus was ≤20 μg/ml and was compared with phenoxymethylpenicillin, cloxacillin, erythromycin and vancomycin. When 4-phenyl-1-(2-phenyl-allyl)pyridinium bromide showed MIC at ≤20 μg/ml, all others showed MIC at ≤100 μg/ml. Strong antibacterial activity of 4-phenyl-1-(2-phenyl-allyl)pyridinium bromide against S. aureus indicates that there is a possibility to use it as an effective antibacterial agent.     

Purified Recombinant Phage Lysin LySMP: An Extensive Spectrum of Lytic Activity for Swine Streptococci

Bacteriophage lysin has attracted considerable attentions as possible antimicrobial agents for solution of antibiotic resistance. SMP was a Streptococcus suis serotype 2 bacteriophage isolated from nasal swabs of healthy Bama minipigs. The putative SMP bacteriophage lysin, designated LySMP, was recombinantly expressed in Escherichia coli BL21, and chromatographically purified. Treated with 0.8% of β-mercaptoethanol, LySMP exhibited an extensive lysin spectrum than those of whole phage against bacteria investigated. S. suis serotype 2, S. suis serotype 7 and S. suis serotype 9 strains were recovered from diseased pigs between 1998 and 2005 in China. Fifteen of seventeen strains of S. suis serotype 2 could be lysed, as well as S. suis serotype 7 and 9, Streptococcus equi ssp. zooepidemicus and Staphylococcus aureus. But E. coli and Salmonella enterica were not affected. Purified LySMP showed high degrading efficiency against PMSF or lysozyme treated cells comparing to PBS washed cells. Optimum pH and temperature conditions for the lysin were investigated by turbidity reduction assay. The lysin exerted efficient lysis activity at 37°C, pH 5.2. The turbidity of bacterium investigated was observed to decrease by 1.2–68% in 30 min. Result indicated that putative LySMP could be a candidate antimicrobial agent in controlling S. suis infection.     

Tertiary structure-related activity of tick defensin (persulcatusin) in the taiga tick, <Emphasis Type="Italic">Ixodes persulcatus</Emphasis>

Defensins are small cysteine-rich cationic proteins found in both vertebrates and invertebrates constituting the front line of host innate immunity. To examine the importance of the tertiary structure of tick defensin in its antimicrobial activity, we synthesized two types of the peptides with tertiary structure or primary one on basis of the information of the sequence in the defensin originated from the taiga tick, Ixodes persulcatus. Chemically synthesized peptides were used to investigate the activity spectrum against Staphylococcus aureus, Borrelia garinii and flora-associated bacteria. Both synthetic peptides showed antimicrobial activity against S. aureus in short-time killing within 1 h, but they do not show the activity against B. garinii, Stenotrophomonas maltophila and Bacillus spp., which were frequently isolated from the midgut of I. persulcatus. The teriary structure brought more potent activity to S. aureus than primary one in short-time killing. We also examined its antimicrobial activity by evaluation of growth inhibition in the presence of the synthetic peptides. Minimum inhibitory concentration (MIC) was ranged from 1.2 to 5.0 μg/ml in tertiary peptide and from 10 to 40 μg/ml in primary peptide, when 10 strains of S. aureus were used. From the curve of cumulative inhibition rates, MIC50 (MIC which half of the strains showed) to S. aureus is about 1.2 μg/ml in the peptide with tertiary structure and about 10 μg/ml in the linear one. Corynebacterium renale is 10 times or more sensitive to tertiary peptide than primary one. In conclusion, the presence of 3 disulfide bridges, which stabilize the molecule and maintain the tertiary structure, is considered to have an effect on their antimicrobial activities against Gram-positive bacteria such as S. aureus.     

Isolation and Characterization of Autolysin-Defective Mutants of Staphylococcus aureus That Form Cell Packets

Staphylococcus aureus produces multiple bacteriolytic enzymes (autolysins) and grows usually as a mixture of single cells, pairs, short chains, and irregular clusters. Autolysin-defective mutants that form cubic cell packets (Pa4A and PaH13) or grape-like clusters (Cu9S and CuD10) were isolated from S. aureus FDA 209P after mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine. The Pa4A mutant grown in nutrient broth formed cell packets consisting of 8–64 cells that appeared regularly arranged in three dimensions. Thin-section electron micrographs revealed that the packet cells were encased in an orderly manner within a thick peripheral wall and that their septa failed to split. Zymographic analysis of enzyme extracts from mutant Pa4A showed that it lacked the 33-kDa autolytic enzyme band present in the parent strain. Another mutant, Cu9S, formed grape-like clusters and showed a single autolytic enzyme band (33-kDa). The possibility that the 33-kDa autolytic enzyme is involved in splitting of the septum prior to cell separation in S. aureus is discussed. Received: 26 September 1996 / Accepted: 3 December 1996     

Biochemical characterization of a raw starch degrading <Emphasis Type="Italic">α</Emphasis>-amylase from the Indonesian marine bacterium <Emphasis Type="Italic">Bacillus</Emphasis> sp. ALSHL3

An Indonesian marine bacterial isolate, which belongs to genus of Bacillus sp. based on 16S rDNA analysis and was identified as Bacillus filicolonicus according to its morphology and physiology, produced a raw starch degrading α-amylase. The partially purified α-amylase using a maize starch affinity method exhibited an optimum pH and temperature of 6.0 and 60°C, respectively. The enzyme retained 72% of its activity in the presence of 1.5 M NaCl. Scanning electron micrographs showed that the α-amylase was capable of degrading starch granules of rice and maize. This α-amylase from Bacillus sp. ALSHL3 was classified as a saccharifying enzyme since its major final degradation product was glucose, maltose, and maltotriose.     

New insight in staphylococcin research: bacteriocin and/or bacteriocin-like inhibitory substance(s) produced by S. aureus AB188

Summary Staphylococcus aureus AB188 (a clinical isolate from wound pus) has been found to produce bacteriocins and/or bacteriocin-like inhibitory substance(s) tentatively termed staphylococcin Bac188. It has a broad activity spectrum against many Gram-positive (e.g. B. subtilis, S. aureus, E. faecalis), Gram-negative bacteria (e.g. E. coli, S. typhi and S. dysenteriae), and dermatophytes including Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton longi and Trichophyton rubrum. Interestingly, staphylococcin Bac188 also showed very potent activity against many clinical isolates of Mycobacterium tuberculosis. Staphylococcin Bac188 showed wide thermostability and remained stable over the wide pH range (pH 2–14). It was also resistant to treatment with chloroform, catalase, lipase and lysozyme, but showed sensitivity to proteinase K, trypsin and α-chymotrypsin suggesting its proteinaceous nature. Staphylococcin Bac188 revealed bactericidal effects on the S. aureus SS-1 sensitive strain as well as on E. coli and S. typhi, suggesting a similar mode of action on both Gram-negative and Gram-positive organisms. The antimicrobial, antidermatophytic and antimycobacterial activities expressed by S. aureus AB188 correlate with the production of a bacteriocin and/or bacteriocin-like inhibitory substance with properties similar to other staphylococcins reported earlier. To our knowledge, this is the first report describing such wide possible clinical applications of a bacteriocin and/or bacteriocin-like inhibitory substance produced by S. aureus AB188, suggesting further investigation for potential therapeutic development.     

Identification and Characterization of Novel Lipophilic Antimicrobial Peptides Derived from Naturally Occurring Proteins

Microbes are increasingly developing defensive mechanisms against known drugs via mutations. There are signs of emergence of superbugs immune to most known antibiotics available. The need for a new class of drugs to counteract this problem is of paramount importance for continued general well being of mankind. A new class of drugs, antimicrobial peptides, has not been fully exploited primarily due to high cytotoxicity, poor lipophilicity preventing systemic distribution and stability. We have synthesised 9-amino acid residue cationic peptides RH01 and RH02 lipidated with myristoyl and octyl groups respectively. These peptides exhibited potent antimicrobial activity and low cytotoxicity. The lipopeptide RH01 has antimicrobial activity against a broad range of microorganisms including bacteria, yeast and filamentous fungi with greatest activity toward Gram-positive bacteria, including S. aureus MRSA stain, MIC’s ranging between 2–8 μM. The MIC for Gram-negative bacteria was higher ranging from between 30–250 μM. RH01 also had antimicrobial activity towards fungi showing good activity against the pathogenic yeast Candida albicans but was less active towards the filamentous fungi Aspergillus niger. The antimicrobial activity of RH01 as a measure of Ki₍₅₀₎ for E. coli and S. aureus was 35–60 μM and 3–7 μM, respectively. In-house data showed the compound is bactericidal even at higher bacteria concentration. The octylated lipopeptide RH02 has similar activities towards S. aureus (3.3 μM) and E coli (53.3 μM) as the myristolated RH01. There was no haemolytic activity of the lipopeptide RH01 towards human blood. Acute intravenous toxicity study in mice showed that both RH01 and RH02 induced no macroscopic abnormalities at their highest non-lethal dose of 75 mg/kg and 150 mg/kg bodyweight, respectively.Australian Peptide Conference Issue.     

SD-8, a novel therapeutic agent active against multidrug-resistant Gram positive cocci

Anti-bacterial drug resistance is one of the most critical concerns among the scientist worldwide. The novel antimicrobial decapeptide SD-8 is designed and its minimal inhibitory concentration and therapeutic index (TI) was found in the range of 1–8 μg/ml and 45–360, respectively, against major group of Gram positive pathogens (GPP). The peptide was also found to be least hemolytic at a concentration of 180 μg/ml, i.e., nearly 77 times higher than its average effective concentration. The kinetics assay showed that the killing time is 120 min for methicillin-sensitive Staphylococcus aureus (MSSA) and 90 min for methicillin-resistant S. aureus (MRSA). Membrane permeabilization is the cause of peptide antimicrobial activity as shown by the transmission electron microscopy studies. The peptide showed the anti-inflammatory property by inhibiting COX-2 with a K _D and K _i values of 2.36 × 10⁻⁹ and 4.8 × 10⁻⁸ M, respectively. The peptide was also found to be effective in vivo as derived from histopathological observations in a Staphylococcal skin infection rat model with MRSA as causative organism.     

Contribution of bovine lactoferrin inter-lobe region to iron binding stability and antimicrobial activity against <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The investigation of the recombinant bovine lactoferrin-derived antimicrobial protein (rBLfA) demonstrates that the inter-lobe region of bovine lactoferrin contributes to iron binding stability and antimicrobial activity against Staphylococcus aureus. rBLfA containing N-lobe (amino acid residues 1–333) and inter-lobe region (residues 334–344) was expressed in Pichia pastoris at shaking flask and fermentor level. The recombinant intact bovine lactoferrin (rBLf) and N-lobe (rBLfN) were expressed in the same system as control. The physical–chemical parameters of rBLfA, rBLfN and rBLf including amino acid residues, molecular weight, isoelectric point, net positive charge and instability index were computed and compared. The simulated tertiary structure and the calculated surface net charge showed that rBLfA maintained original structure and exhibited a higher cationic feature than rBLf and rBLfN. The three proteins showed different iron binding stability and antimicrobial activity. rBLfA released iron in the pH range of 7.0–3.5, whereas rBLfN lost its iron over the pH range of 7.0–4.0 and iron release from rBLf occurred in the pH range of 5.5–3.0. However, the minimum inhibition concentration of rBLfA against S. aureus ATCC25923 was 6.5 μmol/L, compared with 12.5 and 25 μmol/L that of rBLfN and rBLf, respectively. These results revealed that S. aureus was more sensitive to rBLfA than rBLfN and rBLf. It appeared that the strong cationic character of inter-lobe region related positively to the higher anti-S. aureus activity.     

Isolation and characterization of a new bacteriophage MMP17 from <Emphasis Type="Italic">Meiothermus</Emphasis>

Thermophiles and their viruses are extraordinarily important because of their roles in processes of evolution, biogeochemistry and ecology. Species of the genus Meiothermus share close relationship with genus Thermus, but no Meiothermus bacteriophage has been reported yet. In this work, a new thermophilic bacteriophage named MMP17 (Meiothermus Myoviridae phage 17) was isolated from a Meiothermus strain and was further characterized. MMP17 was a typical myovirus with an icosahedral head (42 nm in diameter) and a tail (120 nm in length and 17 nm in width). Its DNA was about 33.5–39.5 kb in size. MMP17 was very stable at 55–60°C and pH 6–7. According to the one-step growth curve, the latent period and the burst period were 60 and 30 min, respectively. An average of 15 phage particles was released from each infected cell. Four major bands were detected in purified virion preparation by SDS-PAGE. As MMP17 was a thermophilic bacteriophage with lower production temperature, its characterization and the relationship between MMP17 and Thermus bacteriophages deserved for further study.     

Partial purification and properties of a laundry detergent compatible alkaline protease from a newly isolated <Emphasis Type="Italic">Bacillus</Emphasis> species Y

Alkaline protease production by a newly isolated Bacillus species from laundry soil was studied for detergent biocompatibility. From its morphological and nucleotide sequence (about 1.5 kb) of its 16S rDNA it was identified as Bacillus species with similarity to Bacillus species Y (Gen Bank entry: ABO 55095), and close homology with Bacillus cohnii YN-2000 (Gen Bank entry: ABO23412). Partial purification of the enzyme by ammonium sulfate (50–70% saturation) yielded 8-fold purity. Casein zymography and Sodium dodecylsulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) of the partially purified enzyme revealed two isozymes of molecular sizes approximately 66 kDa and 18 kDa, respectively. The enzyme was most active at pH 12 and 50°C. At pH 12 the enzyme was stable for 5 h and retained 60% activity. The enzyme retained 44% activity at 50°C up to 2 h. The protease showed good hydrolysis specificity with different substrates tested. The presence of Mn²⁺, Co²⁺ and ethylenediaminetetracetic acid (EDTA) showed profound increase in protease activity. The protease of Bacillus species Y showed excellent stability and compatibility with three locally available detergents (Kite, Tide and Aerial) up to 3 h retaining almost 70–80% activity and 10–20% activity at room temperature (30°C) and 50°C, respectively, indicating the potential role of this enzyme for detergent application.     

Staphylococcus aureus virulence attenuation and immune clearance mediated by a phage lysin‐derived protein

New anti‐infective approaches are much needed to control multi‐drug‐resistant (MDR) pathogens, such as methicillin‐resistant Staphylococcus aureus (MRSA). Here, we found for the first time that a recombinant protein derived from the cell wall binding domain (CBD) of the bacteriophage lysin PlyV12, designated as V12CBD, could attenuate S. aureus virulence and enhance host immune defenses via multiple manners. After binding with V12CBD, S. aureus became less invasive to epithelial cells and more susceptible to macrophage killing. The expressions of multiple important virulence genes of S. aureus were reduced 2.4‐ to 23.4‐fold as response to V12CBD. More significantly, V12CBD could activate macrophages through NF‐κB pathway and enhance phagocytosis against S. aureus. As a result, good protections of the mice from MRSA infections were achieved in therapeutic and prophylactic models. These unique functions of V12CBD would render it a novel alternative molecule to control MDRS. aureus infections.     

Extracellular production of beta-amylase by a halophilic isolate, <Emphasis Type="Italic">Halobacillus</Emphasis> sp. LY9

A moderately halophilic strain LY9 with high amylolytic activity was isolated from soil sample obtained from Yuncheng, China. Biochemical and physiological characterization along with 16S rRNA sequence analysis placed the isolate in the genus Halobacillus. Amylase production started from the post-exponential phase of bacterial growth and reached a maximum level during the early-stationary phase. The isolate LY9 was found to secrete the amylase, the production of which depended on the salinity of the growth medium. Maximum amylase production was observed in the presence of 10% KCl or 10% NaCl. Maltose was the main product of soluble starch hydrolysis, indicating a β-amylase activity. The enzyme showed optimal activity at 60°C, pH 8.0, and 10–12.5% of NaCl. It was highly active over broad temperature (50–70°C), NaCl concentration (5.0–20.0%), and pH (4.0–12.0) ranges, indicating its thermoactive and alkali-stable nature. However, activity dropped off dramatically at low NaCl concentrations, showing the amylase was halophilic. Ca²⁺ was found to stimulate the β-amylase activity, whereas ethylenediaminetetraacetic acid (EDTA), phenylarsine oxide (PAO), and diethyl pyrocarbonate (DEPC) strongly inhibited the enzyme, indicating it probably was a metalloenzyme with cysteine and histidine residues located in its active site. Moreover, the enzyme exhibited remarkable stability towards sodium dodecyl sulfate (SDS) and Triton X-100. This is the first report of β-amylase production from moderate halophiles. The present study indicates that the extracellular β-amylase of Halobacillus sp. LY9 may have considerable potential for industrial application owing to its properties.     

Molecular and enzymatic characterization of a subfamily I.4 lipase from an edible oil-degrader <Emphasis Type="Italic">Bacillus</Emphasis> sp. HH-01

An edible-oil degrading bacterial strain HH-01 was isolated from oil plant gummy matter and was classified as a member of the genus Bacillus on the basis of the nucleotide sequence of the 16S rRNA gene. A putative lipase gene and its flanking regions were cloned from the strain based on its similarity to lipase genes from other Bacillus spp. The deduced product was composed of 214 amino acids and the putative mature protein, consisting of 182 amino acids, exhibited 82% amino acid sequence identity with the subfamily I.4 lipase LipA of Bacillus subtilis 168. The recombinant product was successfully overproduced as a soluble form in Escherichia coli and showed lipase activity. The gene was, therefore, designated as lipA of HH-01. HH-01 LipA was stable at pH 4–11 and up to 30°C, and its optimum pH and temperature were 8–9 and 30°C, respectively. The enzyme showed preferential hydrolysis of the 1(3)-position ester bond in trilinolein. The activity was, interestingly, enhanced by supplementing with 1 mM CoCl₂, in contrast to other Bacillus lipases. The lipA gene seemed to be constitutively transcribed during the exponential growth phase, regardless of the presence of edible oil.     

The antibacterial and immunostimulative effect of chitosan-oligosaccharides against infection by <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolated from bovine mastitis

Based on our previous study evaluating the in vivo cure efficacy of chitosan on bovine mastitis, a more water-soluble chitosan-oligosaccharide (OCHT) with a high degree of deacetylation and low molecular weight was prepared to obtain high antibiotic efficacy. The growth of Staphylococcus aureus isolated from bovine mastitis was inhibited within 10 min of treatment with OCHT in concentrations ranging from 0.0001 to 0.5%. Additionally, electron microscopic observation indicated that the surface of the OCHT-treated bacteria was expanded, distorted, and lysed compared to that of the control bacteria. In mice, the proportion of monocytes was elevated, and the levels of interleukin-6 and interferon-γ sharply increased l h after the peritoneal inoculation of the OCHT (0.5 to 1 mg per mouse). Mice challenged intraperitoneally with S. aureus (2.5 × 10⁸ colony forming units) after oral treatment with OCHT (0.5 to 2 mg per day) for 7 days showed a higher survival rate (70–100%) than that of the control (10%). We suggest that the OCHT prepared in this study is a potential agent for the prevention and treatment of bovine mastitis based on its strong antibacterial activity against S. aureus as well as the immunostimulative effect it exhibits on murine infection by S. aureus. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of a new bacteriocin produced from a novel isolated strain of Bacillus lentus NG121

The new bacteriocin is produced from Bacillus lentus NG121 isolated from Khameera – a traditional fermented food from Himachal Pradesh, India which has been reported for the first time in the literature to produce bacteriocin and exhibited very high activity units of 20 × 10⁵ AU (Arbitrary Units)/ml. This bacteriocin was partially purified and was further characterized to assess its preservation characteristics. It showed strong antimicrobial activity against the most challenging and serious test indicators like Listeria monocytogens and Staphylococcus aureus. There was a drastic decrease up to 70% in viable cells of the indicators within the first 10 h of adding partially purified bacteriocin thus proving its bactericidal action. It could withstand the high heat of 100 °C for 10 min of heating time without losing any activity. A wide range of pH tolerance i.e. from 5.0–10.0 was expressed by this bacteriocin. It was found completely sensitive to proteolytic enzyme trypsin. The unique combination of all the above mentioned characteristics makes the bacteriocin of newly isolated Bacillus lentus NG121, a food grade bacteria, highly desirable for preservation of different food items in the food industry.     

Partial purification and characterization of thermostable esterase from the hyperthermophilic archaeonSulfolobus solfataricus

A thermostable esterase from the hyperthemophilic archaeonSulfolobus solfataricus was partially purified 590-fold with 16.2% recovery. The partially purified esterase had a specific activity of 29.5μmol min⁻¹ mg⁻¹ when the enzyme activity was determined usingp-nitrophenyl butyrate as a substrate. The apparent molecular weight was about 100 kDa, while the optimum temperature and pH for esterase were 75°C and 8.0, respectively. The enzyme showed high thermal stability and solvent tolerance in comparison to its mesophilic counterpart. The enzyme also showed chiral resolution activity for (S)-ibuprofen, indicating thatS. solfataricus esterase can be used for the production of commercially important chiral drugs.     

一株金黄色葡萄球菌噬菌体的裂解谱特异性和分子分类研究

[目的]从医院污水中分离金黄色葡萄球菌噬菌体,观察其形态,确证裂解谱特征并研究生物学和基因学特性,为噬菌体的临床应用奠定实验基础.[方法]将金黄色葡萄球菌ATCC25923作为宿主菌,采用双层琼脂平板法从医院污水中分离纯化噬菌体,电镜下观察形态并测定其最佳感染复数、一步生长曲线及裂解谱;全基因组测序并进行基因结构分析和...     

Production and characterization of a novel extracellular metalloproteinase by a newly isolated moderate halophile, <Emphasis Type="Italic">Halobacillus</Emphasis> sp. LY6

A moderately halophilic bacterium LY6 with high proteolytic activity was isolated. Biochemical and physiological characterization, along with 16S rDNA sequence analysis placed the isolate in the genus Halobacillus. The salinity of the culture medium strongly influenced the proteinase production of LY6. Maximum enzyme production was observed in the medium containing 5% Na₂SO₄ or 10% NaCl. Proteinase production was synchronized with bacterial growth and reached a maximum level during the mid-stationary phase. Enzyme purification was carried out by a simple approach including a combination of ammonium sulfate precipitation and Sephacryl S-100 gel filtration chromatography. SDS-PAGE and gelatin zymography analysis revealed it was a monomer with high molecular weight of 69 kDa. Optimal proteinase activity was obtained at pH 10.0, 40°C, and 10% NaCl. It was high active over broad temperature (30–80°C), pH (6.0–12.0), and NaCl concentration (0–25%) ranges, indicating its thermostable, alkali-stable, and halotolerant nature. Moreover, the enzyme activity was markedly enhanced by Ca²⁺ and Cu²⁺, but strongly inhibited by EDTA, PAO, and DEPC, indicating that it probably was a metalloproteinase with cysteine and histidine residues located in its active site.