• Journal of Microbiology and Biotechnology
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• v.21 no.3
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• pp.256-262
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• 2011

A 20.9-kDa metalloprotease was isolated from dried fruiting bodies of the wild basidiomycete mushroom Lepista nuda. The N-terminal amino acid sequence of the protease was seen to be ATFVLTAATNTLFTA, thus displaying no similarity with the sequences of previously reported metalloproteases. The protease was purified using a procedure that entailed ion-exchange chromatography on CM-Cellulose, Q-Sepharose, and Mono S, and FPLC-gel filtration on Superdex 75. The protease functioned at an optimum pH of 7.0 and an optimum temperature of $50^{\circ}C$. It was also noted that the protease demonstrated a proteolytic activity of 1,756 U/mg toward casein. The $K_m$ of the purified protease toward casein was 6.36 mg/ml at a pH of 7.0 and with a temperature of $37^{\circ}C$, whereas the $V_{max}$ was 9.11 ${\mu}g\;ml^{-1}\;min^{-1}$. The activity of the protease was adversely affected by EDTA-2Na, suggesting that it is a metalloprotease. PMSF, EGTA, aprotinin, and leupeptin exerted no striking inhibitory effect. The activity of the protease was enhanced by $Fe^{2+}$, but was curtailed by $Cd^{2+}$, $Cu^{2+}$, $Hg^{2+}$, $Pb^{2+}$, $Zn^{2+}$, and $Fe^{2+}$ ions. The protease also exhibited inhibitory activity against HIV-1 reverse transcriptase with an $IC_{50}$ value of 4.00 ${\mu}M$. The $IC_{50}$ values toward hepatoma Hep G2 and leukemia L1210 cells in vitro were 4.99 ${\mu}M$ and 3.67 ${\mu}M$, respectively.

• Journal of Microbiology and Biotechnology
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• v.24 no.7
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• pp.969-978
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• 2014

The aprE2 gene with its prosequence from Bacillus subtilis CH3-5 was overexpressed in Escherichia coli BL21(DE3) by using plasmid pET26b(+). After IPTG induction, active and mature AprE2 was produced when cells were grown at $20^{\circ}C$, whereas inactive and insoluble enzyme was produced in a large amount when cells were grown at $37^{\circ}C$. The insoluble fraction was resuspended with 6 M guanidine-HCl and dialyzed against 2 M Tris-HCl (pH 7.0) or 0.5 M sodium acetate (pH 7.0) buffer. Then active AprE2 was regenerated and purified by a Ni-NTA column. Purified AprE2 from the soluble fraction had a specific activity of $1,069.4{\pm}42.4U/mg$ protein, higher than that from the renatured insoluble fraction. However, more active AprE2 was obtained by renaturation of the insoluble fraction. AprE2 was most stable at pH 7 and $40^{\circ}C$, respectively. The fibrinolytic activity of AprE2 was inhibited by PMSF, but not by EDTA and metal ions. AprE2 degraded $A{\alpha}$ and $B{\beta}$ chains of fibrinogen quickly, but not the ${\gamma}$-chain. AprE2 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe-pNA. The $K_m$ and $k_{cat}/K_m$ of AprE2 was 0.56 mM and $3.10{\times}10^4S^{-1}M^{-1}$, respectively.

• BMB Reports
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• v.37 no.2
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• pp.199-205
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• 2004

The six lumbrokinase fractions (F1 to F6) with fibrinolytic activities were purified from earthworm Lumbricus rubellus lysates using the procedures of autolysis, ammonium sulfate fractionation, and column chromatography. The proteolytic activities on the casein substrate of the six iso-enzymes ranged from 11.3 to 167.5 unit/mg with the rank activity orders of F2 > F1 > F5 > F6 > F3 > F4. The fibrinolytic activities of the six fractions on the fibrin plates ranged from 20.8 to 207.2 unit/mg with rank orders of F6 > F2 > F5 > F3 > F1 > F4. The molecular weights of each iso-enzyme, as estimated by SDS-PAGE, were 24.6 (F1), 26.8 (F2), 28.2 (F3), 25.4 (F4), 33.1 (F5), and 33.0 kDa (F6), respectively. The plasminogen was activated into plasmin by the enzymes. The optimal temperature of the six iso-enzymes was $50^{\circ}C$, and the optimal pH ranged from pH 4-12. The four iso-enzymes (F1-F4) were completely inhibited by PMSF. The two enzymes (F5 and F6) were completely inhibited by aprotinin, TLCK, TPCK, SBTI, LBTI, and leupeptin. The N-terminal amino acid (aa) sequences of the first 20 to 22 residues of each fraction had high homology. All six isoenzymes had identical aa residues 2-3 and 13-15. The N-terminal 21-22 aa sequences of the F2, F3, and F4 isoenzymes were almost the same. The N-terminal aa sequences of F5 and F6 were identical.

• Korean Journal of Microbiology
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• v.32 no.4
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• pp.291-296
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• 1994

A bacterial strain NO. AM-28, showing proteolytic activity against defatted soybean was isolated from domestic soil. The isolated strain was identified as Aeromonas hydrophila by both the biochemical tests using API kit and the analysis of cellular fatty acid profile with MIDI system. The protease production from A. hydrophila AM-28 was highly enhanced when it was cultivated in the medium containing glycerol as a carbon source, tryptone or $(NH_4)_2HPO_4$ as a nitrogen source, and $CaCl_2$ as a mineral source. The optimal pH and temperature for the enzyme was 8.0 and $65^{\circ}C$, respectively. The enzyme was stable up to $55^{\circ}C$ and at pH values ranging from 7.0 to 13.0. The enzyme activity was inhibited by phenylmethylsulfonyl fluoride and EDTA, indicating that serine residue and metal ions be involved in enzyme activity.

• Preventive Nutrition and Food Science
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• v.18 no.4
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• pp.273-279
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• 2013

Protease widely exists in the digestive tract of animals and humans, playing a very important role in protein digestion and absorption. In this study, a high protease-producing strain Planomicrobium sp. L-2 was isolated and identified from the digestive tract of Octopus variabilis. The strain was identified by physiological and biochemical experiments and 16S rDNA sequences analysis. A protease was obtained from the strain Planomicrobium sp. L-2 through ammonium sulfate precipitation, dialysis and enrichment, DEAE-Sephadex A50 anion-exchange chromatography, and Sephadex G-100 gel chromatography. The molecular weight and properties of the protease were characterized, including optimum temperature and pH, thermal stability, protease inhibitions and metal ions. According to our results, the protease from Planomicrobium sp. L-2 strain designated as F1-1 was obtained by three-step separation and purification from crude enzyme. The molecular weight of the protease was 61.4 kDa and its optimum temperature was $40^{\circ}C$. The protease F1-1 showed a broad pH profile for casein hydrolysis between 5.0~11.0. No residual activity was observed after incubation for 40 min at $60^{\circ}C$ and 60 min at $50^{\circ}C$. F1-1 protease was inhibited by $Mn^{2+}$, $Hg^{2+}$, $Pb^{2+}$, $Zn^{2+}$, and $Cu^{2+}$ ions, as well as PMSF, indicating that the protease F1-1 was a serine protease. Additionally, research basis provided by this study could be considered for industrial application of octopus intestinal proteases.

• Journal of Microbiology and Biotechnology
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• v.21 no.7
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• pp.667-674
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• 2011

The gene for esterase (rEst1) was isolated from a new species of genus Rheinheimera by functional screening of E. coli cells transformed with the pSMART/HaeIII genomic library. E. coli cells harboring the esterase gene insert could grow and produce clear halo zones on tributyrin agar. The rEst1 ORF consisted of 1,029 bp, corresponding to 342 amino acid residues with a molecular mass of 37 kDa. The signal P program 3.0 revealed the presence of a signal peptide of 25 amino acids. Esterase activity, however, was associated with a homotrimeric form of molecular mass 95 kDa and not with the monomeric form. The deduced amino acid sequence showed only 54% sequence identity with the closest lipase from Cellvibrio japonicus strain Ueda 107. Conserved domain search and multiple sequence alignment revealed the presence of an esterase/ lipase conserved domain consisting of a GXSXG motif, HGGG motif (oxyanion hole) and HGF motif, typical of the class IV hormone sensitive lipase family. On the basis of the sequence comparison with known esterases/ lipases, REst1 represents a new esterase belonging to the class IV family. The purified enzyme worked optimally at $50^{\circ}C$ and pH 8, utilized pNP esters of short chain lengths, and showed best catalytic activity with p-nitrophenyl butyrate ($C_4$), indicating that it was an esterase. The enzyme was completely inhibited by PMSF and DEPC and showed moderate organotolerance.

• Journal of Microbiology and Biotechnology
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• v.22 no.10
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• pp.1388-1394
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• 2012

A xylanase-producing thermophilic strain, Geobacillus sp. TC-W7, was isolated from a hot spring in Yongtai (Fuzhou, China). Subsequently, the xylanase gene that encoded 407 amino acids was cloned and expressed. The recombinant xylanase was purified by GST affinity chromatography and exhibited maximum activity at $75^{\circ}C$ and a pH of 8.2. The enzyme was active up to $95^{\circ}C$ and showed activity over a wide pH range of 5.2 to 10.2. Additionally, the recombinant xylanase showed high thermostability and pH stability. More than 85% of the enzyme's activity was retained after incubation at $70^{\circ}C$ for 90 min at a pH of 8.2. The activity of the recombinant xylanase was enhanced by treatment with 10 mM enzyme inhibitors (DDT, Tween-20, 2-Me, or TritonX-100) and was inhibited by EDTA or PMSF. Its functionality was stable in the presence of $Li^+$, $Na^+$, and $K^+$, but inhibited by $Hg^{2+}$, $Ni^{2+}$, $Co^{2+}$, $Cu^{2+}$, $Zn^{2+}$, $Pb^{2+}$, $Fe^{3+}$, and $Al^{3+}$. The functionality of the crude xylanase had similar properties to the recombinant xylanase except for when it was treated with $Al^{2+}$ or $Fe^{2+}$. The enzyme might be a promising candidate for various industrial applications such as the biofuel, food, and paper and pulp industries.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.852-858
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• 2008

An extracellular protease (Mc1) was isolated from the nematode-trapping fungus Monacrosporium cystosporium by gel filtration, anion-exchange, and hydrophobic interaction chromatographies. This protease had a molecular mass of approximately 38 kDa and displayed an optimal activity at pH 7-9 and $56^{\circ}C$ (over 30 min). Its proteolytic activity was highly sensitive to the serine protease inhibitor PMSF (phenylmethylsulfonylfluoride, 0.1 mM), indicating that it belonged to the serine-type peptidase group. The Michaelis constant ($K_m$) and $V_max$ for substrate N-Suc-Ala-Ala-Pro-Phe-pNA were $1.67{\times}10^{-4}\;M$ and 0.6071 $OD_{410}$ per 30 s, respectively. This protease could degrade a broad range of substrates including casein, gelatin, BSA (bovine serum albumin), and nematode cuticle. Moreover, the enzyme could immobilize the free-living nematode Panagrellus redivivus and the pine wood nematode Bursaphelenchus xylophilus, suggesting that it might playa role in infection against nematodes. The encoding gene of Mc1 was composed of one intron and two exons, coding for a polypeptide of 405 amino acid residues. The deduced amino acid sequence of Mcl showed 61.4-91.9% identity to serine proteases from other nematode-trapping fungi. Our results identified that Mcl possessed biochemical properties including optimal reaction condition and substrate preference that are different from previously identified serine proteases.

• Food Science and Biotechnology
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• v.17 no.4
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• pp.766-771
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• 2008

The proteolytic enzyme from Saccharomyces sp. B101 was purified to homogeneity by ammonium sulfate fractionation, ultrafiltration, diethyl aminoethyl (DEAE)-Sephadex A-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography from the culture supernatant of Saccharomyces sp. B101. The specific activity and the purification fold of the purified enzyme were 4,688.9 unit/mg and 18, respectively. The molecular weight of the purified enzyme was estimated to be 33 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the enzyme activity were pH 8.5 and $30^{\circ}C$, respectively. The enzyme activity was relatively stable in the pH range of 6.5-8.5 at below $35^{\circ}C$. The salt-tolerance and stability for the enzyme activity were relatively stable even at NaCl concentrations of 10 and 15%. The activity of enzyme was inhibited by $Ag^{2+}$ and $Fe^{2+}$, and activated by $Mn^{2+}$. In addition, the enzyme activity was potently inhibited by ethylenediaminetetraacetic acid (EDTA) and phenylmethyl sulfonylfluoride (PMSF). Based on these findings we concluded that the purified enzyme was a serine protease. Km and Vmax values for hammastein milk casein were 1.02 mg/mL and 278.38 unit/mL, respectively.

• Microbiology and Biotechnology Letters
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• v.49 no.2
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• pp.181-191
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• 2021

The present study addresses isolation, optimization, partial purification, and characterization of a haloalkaline serine protease from a newly isolated haloarchaeal strain isolated from Wadi El Natrun in Egypt. We expected that a two-step sequential statistical approach (one variable at a time, followed by response surface methodology) might maximize the production of the haloalkaline serine protease. The enzyme was partially purified using Hiprep 16/60 sephacryl S-100 HR gel filtration column. Molecular identification revealed the newly isolated haloarchaeon to be Natrialba hulunbeirensis strain WNHS14. Among several tested physicochemical determinants, casamino acids, KCl, and NaCl showed the most significant effects on enzyme production as determined from results of the One-Variable-At-A-time (OVAT) study. The BoxBehnken design localized the optimal levels of the three key determinants; casamino acids, KCl, and NaCl to be 0.5% (w/v), 0.02% (w/v), and 15% (w/v), respectively, obtaining 62.9 U/ml as the maximal amount of protease produced after treatment at 40℃, and pH 9 for 9 days with 6-fold enhancement in yield. The enzyme was partially purified after size exclusion chromatography with specific activity, purification fold, and yield of 1282.63 U/mg, 8.9, and 23%, respectively. The enzyme showed its maximal activity at pH, temperature, and NaCl concentration optima of 10, 75℃, and 2 M, respectively. Phenylmethylsulfonyl fluoride (PMSF, 5 mM) completely inhibited enzyme activity.