Optimization of conditions for protease production by Chryseobacterium taeanense TKU001

A protease-producing bacterium was isolated and identified as Chryseobacterium taeanense TKU001. An extracellular metalloprotease with novel properties of solvent- and surfactant-stable was purified from the culture supernatant of C. taeanense TKU001 with shrimp shell wastes as the sole carbon/nitrogen source. The optimized condition for protease production was found when the culture was shaken at 37 degrees C for 3 days in 50 mL of medium containing 0.5% shrimp shell powder (SSP) (w/v), 0.1% K2HPO4, and 0.05% MgSO4.7H2O. Two extracellular proteases (FI and FII) were purified and characterized, and their molecular weights, pH and thermal stabilities were determined. The molecular masses of TKU001 protease FI and FII determined by SDS-PAGE and gel filtration were approximately 41 kDa and 75 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU001 protease FI were 8, 60 degrees C, pH 6-9, and 60 degrees C, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU001 protease FII were 7, 60 degrees C, pH 7-9, and 50 degrees C, respectively. TKU001 protease FI and FII were both inhibited completely by EDTA, indicating that the TKU001 protease FI and FII were metalloproteases. TKU001 protease FI and FII retained more than 75% of its original protease activity after preincubation for 10 days at 4 degrees C in the presence of 25% most tested organic solvents. Additionally, the TKU001 protease FI retained 79%, 80%, and 110% of its original activity in the presence of 2% Tween 20, 2% Tween 40, and 2% Triton X-100, respectively. However, at the same condition, the activity of TKU001 protease FII retained 100%, 100%, and 121% of its original activity, respectively. This is the first report of C. taeanense being able to use shrimp shell wastes as the sole carbon/nitrogen source for proteases production. The novelties of the TKU001 protease include its high stability to the solvents and surfactants. These unique properties make it an ideal choice for application in detergent formulations and enzymatic peptide synthesis.     

Conversion of squid pen by Serratia ureilytica for the production of enzymes and antioxidants

Two proteases (P1 and P2) and a chitinase (C1) were purified from the culture supernatant of Serratia ureilytica TKU013 with squid pen as the sole carbon/nitrogen source. The molecular masses of P1, P2 and C1 determined by SDS-PAGE were approximately 50 kDa, 50 kDa and 60 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of P1, P2 and C1 were (pH 10, 40 degrees C, pH 7-11, and <50 degrees C), (pH 10, 40 degrees C, pH 8-11, and <40 degrees C) and (pH 6, 50 degrees C, pH 5-8, and <50 degrees C), respectively. P1 and P2 were inhibited by Mg(2+), EDTA and C1 was inhibited completely by Cu(2+). The antioxidant activity of TKU013 culture supernatant was 72% per mL (DPPH scavenging ability). With this method, we have shown that squid pen wastes can be utilized and have revealed its hidden potential in the production of functional foods.     

Purification and characterization of a serine protease extracellularly produced by Aspergillus fumigatus in a shrimp and crab shell powder medium

A fungus with protease and chitinase activities was isolated from the soil. It has been identified as Aspergillus fumigatus Fresenius TKU003. A. fumigatus TKU003 produced proteases and chitinases when it was grown in a medium containing shrimp and crab shell powder (SCSP) of marine waste. An extracellular protease was purified from the culture supernatant of A. fumigatus TKU003. The molecular weight of TKU003 protease was 124 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The pI for TKU003 protease was 8.3. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU003 protease was pH 8, 40 °C, 6–10, and 50 °C, respectively. The activity of the enzyme was strongly inhibited by PMSF. TKU003 serine protease, same as most other serine proteases of A. fumigatus, belongs to protease with alkaline pI. The unique characteristics of TKU003 protease is its high molecular weight.     

Conversion and degradation of shellfish wastes by <Emphasis Type="Italic">Serratia</Emphasis> sp. TKU016 fermentation for the production of enzymes and bioactive materials

A chitosanase and a protease were purified from the culture supernatant of Serratia sp. TKU016 with shrimp shell as the sole carbon/nitrogen source. The molecular masses of the chitosanase and protease determined by SDS–PAGE were approximately 65 and 53 kDa, respectively. The chitosanase was inhibited completely by Mn²⁺, but the protease was enhanced by all of tested divalent metals. The optimum pH, optimum temperature, pH stability, and thermal stability of the chitosanase and protease were (pH 7, 50°C, pH 6–7, <50°C) and (pH 8–10, 40°C, pH 5–10, <50°C), respectively. SDS (2 mM) had stimulatory effect on TKU016 protease activity. The result demonstrates that TKU016 protease is SDS-resistant protease and probably has a rigid structure. Besides, TKU016 culture supernatant (2% SPP) incubated for 2 days has the highest antioxidant activity, the DPPH scavenging ability was about 76%. With this method, we have shown that shrimp shell wastes can be utilized and it’s effective in the production of enzymes, antioxidants, peptide and reducing sugar, facilitating its potential use in biological applications and functional foods.     

Microbial reclamation of squid pen for the production of a novel extracellular serine protease by Lactobacillus paracasei subsp paracasei TKU012

A protease-producing bacterium, strain TKU012, was isolated from infant vomited milk and identified as Lactobacillus paracasei subsp paracasei. Strain TKU012 produced protease when it was grown in a medium containing squid pen powder of marine waste. An extracellular protease was purified from culture supernatant by DEAE-Sepharose and Sephacryl S-100 chromatography. A protease, purified 77-fold to homogeneity in an overall yield of 11%, has a molecular weight of about 49 kDa estimated by SDS-PAGE. The protease was maximally active at pH 10 and 60 degrees C and showed substrate specificity to casein and gelatin. The protease retains 21% and 91% activity in the presence of Tween 20 (2% w/v) and SDS (2mM), respectively. The enzyme activity was reduced in the presence of PMSF and showed 23% sequence coverage rate with metalloprotease of Serratia marcescens. This is the first report of extracellular proteases purified from lactobacilli.     

Purification and characterization of chitinases and chitosanases from a new species strain Pseudomonas sp. TKU015 using shrimp shells as a substrate

A chitinase (CHT1) and a chitosanase (CHS1) were purified from the culture supernatant of Pseudomonas sp. TKU015 with shrimp shell wastes as the sole carbon and nitrogen source. The optimized conditions of this new species strain (Gen Bank Accession Number EU103629) for the production of chitinases were found to be when the culture was shaken at 30 degrees C for 3 days in 100 mL of medium (pH 8) containing 0.5% shrimp shell powder (SSP) (w/v), 0.1% K2HPO4, and 0.05% MgSO(4).7H2O. The molecular weights of CHT1 and CHS1 determined by SDS-PAGE were approximately 68 kDa and 30 kDa, respectively. The optimum pH, optimum temperature, pH stability, and the thermal stability of CHT1 and CHS1 were pH 6, 50 degrees C, pH 5-7, <50 degrees C and pH 4, 50 degrees C, pH 3-9, <50 degrees C, respectively. CHT1 was inhibited completely by Mn2+ and Fe2+, and CHS1 was inhibited by Mn2+, Cu2+, and PMSF. CHT1 was only specific to chitin substrates, whereas the relative activity of CHS1 increased when the degree of deacetylation of soluble chitosan increased.     

Bioconversion of squid pen by Lactobacillus paracasei subsp paracasei TKU010 for the production of proteases and lettuce growth enhancing biofertilizers

A protease-producing bacterium, strain TKU010, was isolated from infant vomited milk and identified as Lactobacillus paracasei subsp. paracasei. A surfactant-stable protease, purified 64-fold from the third day culture supernatant to homogeneity in an overall yield of 11%, has a molecular weight of about 49,000. The enzyme degraded casein and gelatin, but did not degrade albumin, fibrin, and elastin. The enzyme activity was increased about 1.5-fold by the addition of 5 mM Ba²⁺. However, Fe²⁺ and Cu²⁺ ions strongly inhibited the enzyme. The enzyme was maximally active at pH 10 and 60 °C and retained 94% and 71% activity in the presence of Tween 20 (2% w/v) and SDS (2 mM), respectively. The result of identification of TKU010 protease showed that nine tryptic peptides were identical to Serratia protease (serralysin) (GenBank accession number gi999638) with 35% sequence coverage. In comparison with the tryptic peptides of L. paracasei subsp. paracasei TKU012 protease, TKU010 protease possessed two additional peptides with sequences of AATTGYDAVDDLLHYHER and QTFTHEIGHALGLSHPGDYNAGEGNPTYR. The fourth day culture supernatant of TKU010 showed maximal activity of about 5-fold growth enhancing effect on lettuce weight, which was not shown with L. paracasei subsp paracasei TKU012.     

Degradation of chitin and production of bioactive materials by bioconversion of squid pens

Serratia marcescens TKU011, a protease- and chitosanase-producing bacterium, the optimized condition for protease and chitosanase production was found after the media were heated at 121 °C for 120 min and the culture was shaken at 25 °C for 5 days in 100 mL of medium containing 1% squid pen powder (SPP) (w/v), 0.1% K₂HPO₄, and 0.05% MgSO₄. An extracellular metalloprotease with novel properties of solvent stable, and alkaline was purified from the culture supernatant of S. marcescens TKU011 with squid pen wastes as the sole carbon/nitrogen source. The enzyme was a monomeric protease with a molecular mass of 48–50 kDa by SDS–PAGE and gel filtration chromatography. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU011 protease were 8, 50 °C, pH 5–11, and <40 °C, respectively. Besides protease and chitosanase, with this method, deproteinization of squid pen for β-chitin, the production of peptide and reducing sugar may be useful for biological applications.     

Fermented and enzymatic production of chitin/chitosan oligosaccharides by extracellular chitinases from Bacillus cereus TKU027

Two chitinases, Chi I and Chi II, were purified from the culture supernatant of Bacillus cereus TKU027 with shrimp head powder (SHP) as the sole carbon/nitrogen source. The molecular masses of Chi I and Chi II determined using SDS-PAGE were approximately 65kDa and 63kDa, respectively. Chi I toward various surfactants showed high stability, such as SDS, Tween 20, Tween 40 and Triton X-100, and these surfactants were stimulator of Chi I chitinase activity. Concomitant with the production of Chi I and Chi II, chitin oligosaccharides were also observed in the culture supernatant, including chitobiose, chitotriose, chitotetrose and chitopentose at concentrations of 0.44mg/mL, 0.08mg/mL, 0.09mg/mL and 0.43mg/mL, respectively. Chitosan with 60% deacetylation was degraded by TKU027 crude enzyme to prepare chitooligosaccharides. MALDI-TOF MS analysis of the enzymatic hydrolyzates indicated that the products were mainly chitooligosaccharides with degree of polymerization (DP) in the 4-9 range.     

Purification of a thermostable chitinase from Bacillus cereus by chitin affinity and its application in microbial community changes in soil

A thermostable chitinase was purified by chitin affinity from the culture supernatant of Bacillus cereus TKU028 with shrimp head powder (SHP) as the sole carbon/nitrogen source. TKU028 chitinase was purified using a one-step affinity adsorbent system, and the molecular mass of TKU028 chitinase (approximately 40 kDa) was then determined using SDS-PAGE. The enzyme was stable for 60 min at temperatures below 60 °C and stable over a broad pH range of 4–9 for 60 min. In addition, the temporal changes of a bacterial community in mangrove river sediment of the Tamsui River with added SHP were also analysed by PCR–denaturing gradient gel electrophoresis to investigate the effects of B. cereus TKU028 on the degradation of SHP. The 6-week incubation sample of SHP and B. cereus TKU028-amended mangrove river sediment displayed the highest amount of biomass, reducing sugar and total sugar, and some variance of bacterial community composition existed in the soils.     

Purification, characterization, and cDNA cloning of rice class III chitinase

Several chitinases were expressed in a rice cell suspension culture and detected in the medium. One of them, designated as RCB4, was isolated 248 fold from the culture filtrate to homogeneity by 70% ammonium sulfate precipitation, DEAE-cellulose, CM-cellulose, Sephadex G-75 column chromatography, and native gel slicing. RCB4 had a molecular mass of 32 kDa by SDS-PAGE. The optimum temperature was 40 degrees C, and 96% of its activity still remained at 60 degrees C. The optimum pH was 4, and 95% of its activity was maintained at pH 2. Using a substrate (GlcNAc)6, the Km and Vmax values of RCB4 were 0.53 mM and 11.1 mM/min, respectively. The N-terminal and internal amino acid sequences of RCB4 were determined to be VNSNLFRDYIGA and MALWA, respectively. A cDNA (C12523) clone that contained the N-terminal and internal amino acid sequences of RCB4 was obtained, sequenced, and renamed RCB41. RCB41 encoded 307 amino acid protein with a signal peptide of 25 amino acids and showed a 45% similarity to gladiolus chitinase GBC-a, one of the class III chitinase family. The expression of RCB4l in E. coli showed that RCB41 encodes a chitinase.     

Biodegradation of shellfish wastes and production of chitosanases by a squid pen-assimilating bacterium, <Emphasis Type="Italic">Acinetobacter</Emphasis><Emphasis Type="Italic">calcoaceticus</Emphasis> TKU024

Two chitosanases (CHSA1 and CHSA2) were purified from the culture supernatant of Acinetobacter calcoaceticus TKU024 with squid pen as the sole carbon/nitrogen source. The molecular masses of CHSA1 and CHSA2 determined by SDS-PAGE were approximately 27 and 66 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of CHSA1 and CHSA2 were (pH 6, 50°C, pH 4–10, <90°C) and (pH 7, 60°C, pH 6–11, <70°C), respectively. CHSA1 and CHSA2 had broad pH and thermal stability. CHSA1 and CHSA2 were both inhibited by EDTA and were inhibited completely by 5 mM Mn²⁺. CHSA1 and CHSA2 degraded chitosan with DD ranging from 60 to 98%, and also degraded some chitin. The most susceptible substrate was 60% deacetylated chitosan. Furthermore, TKU024 culture supernatant (1.5% SPP) incubated for 5 days has the most reducing sugars (0.63 mg/ml). With this method, we have shown that shellfish wastes may have a great potential for the production of bioactive materials.     

A novel nattokinase produced by Pseudomonas sp. TKU015 using shrimp shells as substrate

A nattokinase was purified from the culture supernatant of Pseudomonas sp. TKU015 with shrimp shell wastes as the sole carbon/nitrogen source. The molecular masses of TKU015 nattokinase determined by SDS-PAGE and gel filtration were approximately 21 and 24 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU015 nattokinase were 7, 50 °C, pH 4–11, and less than 50 °C, respectively. TKU015 nattokinase was inhibited completely by PMSF, indicating that the TKU015 nattokinase was serine protease. The results of peptide mass mapping showed that two tryptic peptides of the nattokinase were identical to a chitin binding protein from Bacillus cereus ATCC 14579 (GenBank accession number gi30020946) with 23% sequence coverage. With this method, Pseudomonas sp. TKU015 produces a nattokinase/fibrinolytic enzyme and may be considered as a new source for thrombolytic agents.     

Purification and characterization of two bifunctional chitinases/lysozymes extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium.

Two extracellular chitinases (FI and FII) were purified from the culture supernatant of Pseudomonas aeruginosa K-187. The molecular weights of FI and FII were 30,000 and 32,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 60,000 and 30,000, respectively, by gel filtration. The pIs for FI and FII were 5.2 and 4.8, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of FI were pH 8, 50 degrees C, pH 6 to 9, and 50 degrees C; those of FII were pH 7, 40 degrees C, pH 5 to 10, and 60 degrees C. The activities of both enzymes were activated by Cu2+; strongly inhibited by Mn2+, Mg2+, and Zn2+; and completely inhibited by glutathione, dithiothreitol, and 2-mercaptoethanol. Both chitinases showed lysozyme activity. The purified enzymes had antibacterial and cell lysis activities with many kinds of bacteria. This is the first report of a bifunctional chitinase/lysozyme from a prokaryote.     

Purification and characterization of a thermophilic and acidophilic chitinase from Microbispora sp. V2

AIM: Purification and characterization of a chitinase from Microbispora sp. V2. METHODS AND RESULTS: The chitinase from Microbispora sp. V2 was purified to homogeneity by gel filtration chromatography with 4.6% recovery. It had a molecular weight of 35 kDa and showed maximum activity towards p-nitrophenyl-beta-d-N,N'-diacetylchitobiose, indicating a chitobiosidase activity. The enzyme had a pH optimum of 3.0 and temperature optimum of 60 degrees C. It was stable in a wide pH range from 3.0 to 11.0, retaining 61% activity at pH 3.0 and 52% activity at pH 11.0. It retained 71% activity at 30 degrees C and 45% activity at 50 degrees C, up to 24 h. The enzyme activity was not inhibited by any of the metal ions tested except Hg2+, in the presence of which only 10% activity was retained. CONCLUSIONS: The 35 kDa chitinase from Microbispora sp. V2 has an acidic pH optimum and a high temperature optimum. It is fairly stable and active, and degrades chitin efficiently, although the growth of the culture and enzyme production is slow. SIGNIFICANCE AND IMPACT OF THE STUDY: This report is the first detailed study of a chitinase from Microbispora sp. V2, isolated from hot springs. The chitinase from Microbispora sp. V2 may have potential applications in the recycling of chitinous wastes, particularly due to its thermophilic and acidophilic character. Studies at molecular level may provide further insight on the chitinolytic system of Microbispora spp. with respect to the number and types of chitinases and their regulation.     

Purification and characterization of a novel isozyme of chitinase from Bombyx mori

75-kDa chitinase, which showed potential as a biocontrol agent against Japanese pine sawyer, was characterized after purification from the integument of the fifth instar larvae of Bombyx mori by chromatography on diethylaminoethyl (DEAE)-Toyoperal 650 (M), hydroxylapatite, and Fractogel EMD DEAE 650 (M) columns. The optimum pH was 6.0 toward N-acetylchitopentaose (GlcNAc5) and 10 toward glycolchitin. The optimum temperature was 60 degrees C toward GlcNAc5 and 25 degrees C toward glycolchitn. The enzyme was stable at pH 7-10 and below 40 degrees C. Kinetic analysis and reaction-pattern analysis using glycolchitin and N-acetylchitooligosacchraides as substrates indicated that 75-kDa chitinase is an endo- or random-type hydrolytic enzyme to produce the beta anomeric product and that it prefers the longer N-acetylchitooligosaccharides, suggesting, together with the N-terminal amino acid sequence, that the 75-kDa chitinase belongs to family 18 of glycosyl hydrolases.     

Optimization of culture conditions for the production of haloalkaliphilic thermostable protease from an extremely halophilic archaeon Halogeometricum sp. TSS101

AIMS: Isolation and screening of extreme halophilic archaeon producing extracellular haloalkaliphilic protease and optimization of culture conditions for its maximum production. METHODS AND RESULTS: Halogeometricum sp. TSS101 was isolated from salt samples and screened for the secretion of protease on gelatin and casein plates containing 20% NaCl. The archaeon was grown aerobically in a 250 ml flask containing 50 ml of (w/v) NaCl 20%; MgCl(2) 1%; KCl 0.5%; trisodium citrate 0.3%; and peptone 1%; pH 7.2 at 40 degrees C on rotary shaker. The production of enzyme was investigated at various pH, temperatures, NaCl concentrations, metal ions and different carbon and nitrogen sources. The partially purified protease had activity in a broad pH range (7.0-10.0) with optimum activity at pH 10.0 and a temperature (60 degrees C). The enzyme was thermostable and retained 70% initial activity at 80 degrees C. Maximum protease production occurred at 40 degrees C in a medium containing 20% NaCl (w/v) and 1% skim milk powder after 84 h in shaking culture. Enzyme secretion was observed at a broad pH range of 7.0-10.0. Addition of CaCl(2) (200 mmol) to the culture medium enhanced the production of protease. Protein rich flours proved to be cheap and good alternative source for enzyme production. Different osmolytes were tested for the growth and production of haloalkaliphilc protease and found that betaine and glycerol enhanced growth without secretion of the protease. Immobilization studies showed that whole cells immobilized in 2% alginate beads were stable up to 10 batches and able to secrete the protease, which attained maximum production within 60 h under shaking conditions. CONCLUSIONS: Halogeometricum sp. TSS101 secreted an extracellular haloalkaliphilic and thermostable protease. The optimum conditions required for maximum production are 20% NaCl, 1% skim milk powder and temperature at 40 degrees C. Addition of CaCl(2) (200 mmol) enhanced the enzyme production. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of haloalkaliphilic protease. SIGNIFICANCE AND IMPACT OF THE STudy: The low cost protein rich flours were used as an alternative carbon and nitrogen sources for enzyme production. Immobilization of halophilic cells in alginate beads can be used in continuous production of halophilic enzyme. The halophilic and thermostable protease from Halogeometricum sp. TSS101 is good source for industrial applications and can be a suitable source for preparation of fish sauce.     

Purification and characterization of extracellular lipases from <Emphasis Type="Italic">Pseudomonas monteilii</Emphasis> TKU009 by the use of soybeans as the substrate

A lipase-producing bacterium was isolated and identified as Pseudomonas monteilii TKU009. A lipase (F2) and lipase-like materials (F1) were purified from the culture supernatant of P. monteilii TKU009 with soybean powder as the sole carbon/nitrogen source. The molecular mass of F1 and F2 was estimated to be 44 kDa by SDS-PAGE and gel filtration. The optimum pH, optimum temperature, and pH and thermal stabilities of F2 were 7, 40°C, 8–11, and 50°C; and of F1 were 6, 40°C, 6–7, and 50°C, respectively. F2 was completely inhibited by EDTA and slightly by Mg²⁺, Fe²⁺, Mn²⁺, and SDS. F1 was completely inhibited by EDTA and Fe²⁺ and strongly by Zn²⁺, Mn²⁺, Ca²⁺, Mg²⁺, and SDS. The activities of both the enzymes were enhanced by the addition of non-ionic surfactants Triton X–100 and Tween 40, especially for F1. F2 preferably acted on substrates with a long chain (C10–C18) of fatty acids, while F1 showed a broad spectrum on those with chain length of C4–C18. The marked activity of F2 in organic solvents makes it an ideal choice for application in a water-restricted medium including organic synthesis. Li-June Ming is a visiting Professor at the National Cheng Kung University.     

A novel nonionic surfactant- and solvent-stable alkaline serine protease from <Emphasis Type="Italic">Serratia</Emphasis> sp. SYBC H with duckweed as nitrogen source: production,purification, characteristics and application

A novel nonionic surfactant- and hydrophilic solvent-stable alkaline serine protease was purified from the culture supernatant of Serratia sp. SYBC H with duckweed as nitrogen source. The molecular mass of the purified protease is about 59 kDa as assayed via SDS-PAGE. The protease is highly active over the pH range between 5.0 and 11.0, with the maximum activity at pH 8.0. It is also fairly active over the temperature range between 30 and 80°C, with the maximum activity at 40°C. The protease activity was substantially stimulated by Mn²⁺ and Na⁺ (5 mM), up to 837.9 and 134.5% at 40°C, respectively. In addition, Mn²⁺ enhanced the thermostability of the protease significantly at 60°C. Over 90% of its initial activity remained even after incubating for 60 min at 40°C in 50% (v/v) hydrophilic organic solvents such as DMF, DMSO, acetone and MeOH. The protease retained 81.7, 83.6 and 76.2% of its initial activity in the presence of nonionic surfactants 20% (v/v) Tween 80, 25% (v/v) glycerol and Triton X-100, respectively. The protease is strongly inhibited by PMSF, suggesting that it is a serine protease. Washing experiments revealed that the protease has an excellent ability to remove blood stains.     

Stability of thermostable alkaline protease from Bacillus licheniformis RP1 in commercial solid laundry detergent formulations

The stability of crude extracellular protease produced by Bacillus licheniformis RP1, isolated from polluted water, in various solid laundry detergents was investigated. The enzyme had an optimum pH and temperature at pH 10.0–11.0 and 65–70 °C. Enzyme activity was inhibited by PMSF, suggesting that the preparation contains a serine-protease. The alkaline protease showed extreme stability towards non-ionic (5% Tween 20% and 5% Triton X-100) and anionic (0.5% SDS) surfactants, which retained 100% and above 73%, respectively, of its initial activity after preincubation 60 min at 40 °C.

The RP1 protease showed excellent stability and compatibility with a wide range of commercial solid detergents at temperatures from 40 to 50 °C, suggesting its further application in detergent industry. The enzyme retained 95% of its initial activity with Ariel followed by Axion (94%) then Dixan (93.5%) after preincubation 60 min at 40 °C in the presence of 7 mg/ml of detergents. In the presence of Nadhif and New Det, the enzyme retained about 83.5% of the original activity. The effects of additives such as maltodextrin, sucrose and PEG 4000 on the stability of the enzyme during spray-drying and during subsequent storage in New Det detergent were also examined. All additives tested enhanced stability of the enzyme.