PURIFICATION AND PROPERTIES OF DETERGENT-COMPATIBLE EXTRACELLULAR ALKALINE PROTEASE FROMScopulariopsisspp.

Abstract

A fungal alkaline protease of Scopulariopsis spp. was purified to homogeneity with a recovery of 32.2% and 138.1 U/mg specific activity on lectin-agarose column. The apparent molecular mass was 15 ± 1 kD by sodium dodecyl sulfate polyacryalamide gel electrophoresis (SDS-PAGE). It was a homogenous monomeric glycoprotein as shown by a single band and confirmed by native PAGE and gelatin zymography. The enzyme was active and stable over pH range 8.0-12.0 with optimum activity at pH 9.0. The maximum activity was recorded at 50°C and remained unaltered at 50°C for 24 hr. The enzyme was stimulated by Co(2+) and Mn(2+) at 10 mM but was unaffected by Ba(2+), Mg(2+), Cu(2+), Na(+), K(+), and Fe(2+). Ca(2+) and Fe(3+) moderately reduced the activity (∼18%); however, a reduction of about 40% was seen for Zn(2+) and Hg(2+). The enzyme activity was completely inhibited by 5 mM phenylmethylsulfonyl fluoride (PMSF) and partially by N-bromosuccinimide (NBS) and tocylchloride methylketone (TLCK). The serine, tryptophan, and histidine may therefore be at or near the active site of the enzyme. The protease was more active against gelatin compared to casein, fibrinogen, egg albumin, and bovine serum albumin (BSA). With casein as substrate, Km and Vmax were 4.3 mg/mL and 15.9 U/mL, respectively. An activation was observed with sodium dodecyl sulfate (SDS), Tween-80, and Triton X-100 at 2% (v/v); however, H2O2 and NaClO did not affect the protease activity. Storage stability was better for all the temperatures tested (-20, 4, and 28 ± 2°C) with a retention of more than 85% of initial activity after 40 days. The protease retained more than 50% activity after 24 hr of incubation at 28, 60, and 90°C in the presence (0.7%, w/v) of commercial enzymatic and nonenzymatic detergents. The Super Wheel-enzyme solution was able to completely remove blood staining, differing from the detergent solution alone. The stability at alkaline pH and high temperatures, broad substrate specificity, stability in the presence of surfactants and oxidizing and bleaching agents, and excellent compatibility with detergents clearly suggested the use of the enzyme in detergent formulations.

A novel alkaline protease from alkaliphilic Idiomarina sp. C9-1 with potential application for eco-friendly enzymatic dehairing in the leather industry

“…AprA exhibits higher optimal pH (10.5) and better stability under alkaline conditions than many previously reported alkaline proteases from Bacillus 4 , 6 , 8 , 9 , 15 , 16 , 27 , 33 , 51 , 56 – 61 , Vibrio 19 , Aspergillus 21 , 62 , 63 , Thermoactinomyces 24 , 26 , Streptomyces 25 , Thermus 28 , Pseudoalteromonas 41 , Alteromonas 42 , Stenotrophomonas 44 , Trametes 52 , Termitomyces 54 , Virgibacillus 64 , Caldicoprobacter 65 , Hirsutella 66 , Scopulariopsis 67 , and Penicillium 68 (Table S1 ). Furthermore, AprA also retains high activity and stability over a wide range of pH (7.0–11.5) and temperature (40–70 °C), several metal ions, surfactants and some oxidizing and reducing agents.…”

SHG10 keratinolytic alkaline protease from Bacillus licheniformis SHG10 DSM 28096: Robust stability and unusual non‐cumbersome purification

“…EMB9, and B. licheniformis 3C5, displaying perceivable activity at extremists pH(s) (10-12) and temperatures (60-75°C) [16,24,[39][40][41][42]. Like other proteases reported in the literature [25,[43][44][45]49], SHG10 keratinolytic protease exhibited similar profile for pH stability under wide range of pH (6-10) for extended hours. This pH stability profile would in turn impose an additional industrial value of this enzyme.…”

“…Like other alkaline proteases reported in the literature [9,11,25,38,44,49,51], SHG10 keratinolytic protease is a serine protease type as majority of its activity (86%) was lost in presence of 3 mM PMSF. In contrast, EDTA, a neutral protease inhibitor, did not impose an inhibitory effect on enzyme activity but stimulated the activity at a concentration of 10 mM.…”

“…profundum BK-P23, Pseudalteromonas sp.129-1, S. koyangensis TN 650, B. licheniformis 3C5, Beauveria sp. MTCC 5184, Scopulariopsis spp., B. circulans BM15, and B. licheniformis NCIM 2042; where their activity had not affected adversely but slightly stimulated by non-ionic detergents (Tween 20, Tween 80, and Triton X-100) [9,25,26,39,[42][43][44][45][46]. Unlike some alkaline proteases previously reported in the literature that being moderately SDS inhibited (BM15 protease retained 70 and 56% of its activity in presence of 0.05 and 0.4% SDS, respectively) [45] or completely SDS inhibited (proteases from Oerskovia xanthineolytica TK-1 and Streptomyces sp.…”

A New Subtilase-Like Protease Deriving from Fusarium equiseti with High Potential for Industrial Applications

“…The enzyme should also tolerate variability in water hardness. The use of fungal proteases in washing applications has been reported in only a few cases [47][48][49]. However, the recombinant Fe protease showed excellent performance in stain removal when tested in a laundry detergent application.…”