Wash Performance Analysis Research Articles

Alkaline Thermo- and Oxidant-Stable Protease from Bacillus pumilus Strain TNP93: Laundry Detergent Formulations.

The study aims to produce a detergent-compatible and alkaline thermophilic protease from a Bacillus strain and to investigate its usability as a detergent bio-additive. The protease-producing bacterium was identified as Bacillus pumilus strain TNP93 according to the 16S rRNA sequence. The bacterium optimally synthesized the protease at 40°C and pH 10 in 40h. The raw protease displayed its optimum activity at pH 10 and 60°C and its stability between pH 6-13 and 30-100°C for 24h. The molecular mass of the proteolytic band was estimated to be about 85kDa. The protease was not inhibited by any of the metal ions used (Ba2+, Ca2+, Co2+, Cu2+, Mg2+, Mn2+, Zn2+). 97 and 90% of its original activity with 5mM PMSF and EDTA remained. The activity was measured as 84, 124, and 95%, respectively, in the presence of 1% concentrations of Tween 20, Tween 80, and Triton X-100. In addition, all of its activity was preserved when the enzyme was exposed to 5% H2O2. The end products of casein were detected as tyrosine, aspartic acid, glycine, and cysteine by thin-layer chromatography. Considering the wash performance analysis, the mix of 1% commercial detergent and enzyme almost removed all of the protein-based stains (blood and egg yolk albumin). These remarkable findings indicate that the alkaline, thermo-, and oxidant-stable TNP93 protease is a valuable candidate for usage as a biological additive in various laundry detergents.

Detergent-stable amylase production by Paenibacillus lactis strain OPSA3 isolated from soil; optimization by response surface methodology

This study aimed to isolate thermostable, alkaliphilic, and detergent-tolerant amylase-producing bacteria. Pure isolates from environmental samples were screened on a starch-based medium (pH 11), and selected isolates were identified using cultural and molecular techniques. Product optimization studies were conducted, and secreted amylase was partially purified using 40% (w/v) saturation ammonium sulfate at 4 °C. The wash performance of concentrated amylase was analyzed. A novel isolate, Paenibacillus lactis OPSA3, was selected for further studies. The isolate produced amylase optimally when grown on banana peels and soybean extracts, which are agro-wastes. Optimization by Response surface Methodology resulted in a 2.1-fold increase in alkaliphilic amylase production. A 2.46-fold purification was achieved, with an enzyme activity yield of 79.53% and specific activity of 26.19 Umg−1. Wash performance analysis using the amylase supplemented with boiled commercial detergent (kiln®) showed good cleaning efficiency. The amylase has the potential for application as a component of green detergent.

Stain-removal potential of keratinase produced by Bacillus species isolated from feather waste

The aim of this study was to determine the potential of keratinases produced by Bacillus species isolated from poultry feather waste in stain removal. The effect of individual and combined detergent components on the keratinase activity of EZYKer-51, EZYKer-50 and EZYKer-53 enzymes produced from keratin degrading Bacillus licheniformis-K51, Bacillus subtilis-K50 and Bacillus sp.-K53 respectively was determined. EZYKer-51, which had the least enzyme inhibition in the presence of the detergent component was used as the enzyme component in detergent formulation and a wash performance analysis test was carried out to compare the stain removal capacity of DETKER 51 (detergent formulated using EZYKer-51 as enzyme component) with a commercial enzyme. Data were analyzed using descriptive statistics. EZYKer-51 showed a significantly higher (P≤0.05) enzyme activity in the presence of the detergent ingredients for both the crude and purified forms of the enzyme compared with the activities of EZYKer-50 and EZYKer-53, except for the activities (8.02±1.32 U/mL and 7.94±0.63 U/mL) in the presence of 15% LAS and 5% liquid paraffin. Visually, better stain removal was achieved when endogenous protease of commercial enzyme was replaced with EZYKer-51. These results suggest that keratinase EZYKer-51 may be a useful alternative for applications in detergent formulation.

Production, partial purification and efficacy of keratinase from Bacillus halotolerans L2EN1 isolated from the poultry farm of Himachal Pradesh as a potential laundry additive

To augment the keratinolytic ability of Bacillus halotolerans L2EN1 isolated from the poultry farm (Nahan, District Sirmour) of Himachal Pradesh, different cultural conditions were optimised using One Variable at a Time (OVAT) approach accompanied by Response Surface Methodology (RSM). Optimisation (OVAT) results revealed that after 3rd day of incubation, maximum enzyme activity was attained at 45 °C, pH 11.0 with 12.5% of inoculum size in the presence of Mn2 + and EDTA in the production medium. Sucrose (1.5%) and yeast extract (2.0%) were observed to be best carbon and nitrogen sources, respectively. A significant increase of 73.19 per cent in the keratinase activity was observed using Central Composite Design (CCD) of RSM. The SDS-PAGE results revealed that crude keratinase is a heterotetramer made up of four polypeptide chains with molecular weights of 17, 37, 40 and 60 kDa. Partial purification by 90 − 100 percent ammonium sulphate gave maximum keratinase production of 22.66 U/mL with purification of 1.68 and yield of 11.47 per cent. The enzyme showed compatibility with different commercial detergents and retained its activity in the order: Reshma (97.77%) > Speed (93.44%) > Tide (79.93%) >Ariel (70.18%) > Surf excel (67.98%) at 50 °C after 1 h of incubation. Wash performance analysis demonstrated that washing with tap water at 18, 28, 35 and 45 °C for 30, 45 and 60 min removed some amount of blood stains from the cotton cloth pieces. However, replacement of detergent’s enzyme (Reshma) with crude keratinase achieved complete blood stain removal under same conditions, suggesting its suitability as a potential cleaning additive in detergents for the removal of blood (proteinaceous) stains for long washing cycles (1 h).

Highly Efficient Computationally Derived Novel Metagenome α-Amylase With Robust Stability Under Extreme Denaturing Conditions.

α-Amylases are among the very critical enzymes used for different industrial purposes. Most α-amylases cannot accomplish the requirement of industrial conditions and easily lose their activity in harsh environments. In this study, a novel α-amylase named PersiAmy1 has been identified through the multistage in silico screening pipeline from the rumen metagenomic data. The long-term storage of PersiAmy1 in low and high temperatures demonstrated 82.13 and 71.01% activities after 36 days of incubation at 4 and 50°C, respectively. The stable α-amylase retained 61.09% of its activity after 180 min of incubation at 90°C and was highly stable in a broad pH range, showing 60.48 and 86.05% activities at pH 4.0 and pH 9.0 after 180 min of incubation, respectively. Also, the enzyme could resist the high-salinity condition and demonstrated 88.81% activity in the presence of 5 M NaCl. PersiAmy1 showed more than 74% activity in the presence of various metal ions. The addition of the detergents, surfactants, and organic solvents did not affect the α-amylase activity considerably. Substrate spectrum analysis showed that PersiAmy1 could act on a wide array of substrates. PersiAmy1 showed high stability in inhibitors and superb activity in downstream conditions, thus useful in detergent and baking industries. Investigating the applicability in detergent formulation, PersiAmy1 showed more than 69% activity after incubation with commercial detergents at different temperatures (30–50°C) and retained more than 56% activity after incubation with commercial detergents for 3 h at 10°C. Furthermore, the results of the wash performance analysis exhibited a good stain removal at 10°C. The power of PersiAmy1 in the bread industry revealed soft, chewable crumbs with improved volume and porosity compared with control. This study highlights the intense power of robust novel PersiAmy1 as a functional bio-additive in many industrial applications.

Caracterización de la proteasa Geobacillus stearothermophilus para su uso en la industria de detergentes

Cloning of protease gene from a thermophilic strain of Geobacillus stearothermophilus (B-1172) was carried out in E. coli BL 21, and its expression was studied. The expressed protease was purified followed by its identification. A 16.9 folds purification with 55.68% recovery of the protease was achieved by ammonium sulfate precipitation and gel filtration chromatography. The protease specific activity was 120 U mg−1. The purified enzyme remained stable at 90°C at a pH range 6-9. Its interaction with EDTA, different metal ions, inhibitors, surfactants and detergents was also mapped. Its interaction with EDTA showed no significant effect on the activity of the enzyme confirming its metaloprotease nature. Metal ions i.e. Ca2+, Mg2+, Ni2+, Cd2+, Cu2+, Zn2+ showed no significant effect on the stability of protease. Its compatibility was checked with different commercial detergent (6 mg/mL) such as Surf Excel Arial, Bonus, wheel and Shine. It retained more than 80% proteolytic activity in all detergents after incubation at 50°C for 1 h. Wash performance analysis of the protease of G. stearothermophilus showed good results of de-staining of blood sample at various temperatures. Therefore, recombinant protease could prove as good candidate for commercial use in detergents.

Purification and Characterization of Metalloprotease from Serratia marcescens PPB-26 and Its Application for Detergent Additive

In this study, the extracellular metalloprotease from Serratia marcescens PPB-26 was purified to homogeneity via ethanol fractionation and DEAE-cellulose column chromatography. Thus, a 3.8-fold purification was achieved with a 20% yield and specific activity of 76.2 U/mg. The purified protease was a 50-kDa monomer whose optimum pH and temperature for activity were 7.5 and 30℃ respectively; however, it was found to remain active in the 5-9 pH range and up to 40℃ for 6 h. The protease had a half-life of 15 days at 4℃, an optimum reaction time of 10 min, and an optimum substrate (casein) concentration of 0.25%. Furthermore, the Michaelis constant (Km) and reaction velocity (Vmax) of the protease were calculated to be 0.28% and 111.11 μmoles/(min·mg)-1, respectively. The protease was stable when subjected to metal ions (2 mM), showing increased activity with most (especially CoCl2 and MgSO4 (30.54% increase)). It was also stable when exposed to oxidizing agents, bleaching agents, and detergents (5% v/v for 60 min). It retained 93% of its activity in non-ionic detergents (Tween-20, Tween-80, and Triton X-100). Moreover, wash performance analysis in commercial detergents (Ariel and Tide) showed that not only was the protease capable of protein stain removal, but also reduced cleaning time by 80% when added to detergents. Thus, the Serratia marcescens PPB-26 metalloprotease appears to be a promising new candidate as a laundry additive in the detergent industry.

Thermo and alkali stable β-mannanase: Characterization and application for removal of food (mannans based) stain

The present work aims to characterize thermo and alkali stable β-mannanase (ManSS11) from newly isolated Klebsiella pneumoniae SS11 and its food stain (mannan based) removal efficiency. The enzyme, ManSS11 was stable over a wide range of pH and temperature. The highest activity of ManSS11 was observed at pH 9.0 and temperature, 70 °C, with t1/2 of 135.91 min at the same temperature while, >70% of its initial activity was retained at pH 7.0–10.6. It was purified to 5.50-fold homogeneity with a final recovery of 9.6% and a specificity of 7573.57 U/mg protein. Purified ManSS11 was visible as a single protein band with a molecular weight of ~45 kDa. The kinetic parameters of Km, Vmax and kcat were 1.66 mg/mL, 833.33 μmolmL−1 min−1 and 1190.47 s−1 respectively. The compatibility of β-mannanase with different detergents together with wash performance test confirmed its potential usability in laundry sector. Wash performance analysis confirmed that the enzyme displayed great efficiency in the removal of stains caused by mannan containing foods.

Production, purification and biochemical characterization of a novel detergent-stable serine alkaline protease from Bacillus safensis strain RH12

A novel extracellular protease (SAPRH) was hyper-produced (9000 U/mL) from Bacillus safensis RH12, a newly isolated enzyme from a Tunisian offshore oil field. The enzyme was purified to homogeneity, using salt-precipitation, heat-treatment and FPLC anion-exchange chromatography. The purified enzyme was a monomer of molecular mass of ~28 kDa. The NH2-terminal 23 amino-acid sequence of SAPRH showed high homology with those of Bacillus-proteases. SAPRH displayed optimal activity at pH 9 and 60 °C. It was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), indicating that it belongs to the serine-proteases family. Moreover, SAPRH was extremely stable at a broad range of temperature and pH retaining 85% of its activity at 50 °C and 75% at pH 11. The enzyme exhibited excellent stability and compatibility with surfactants and commercial detergents, revealing 90% stability with SDS and 100% stability with Class commercial laundry detergent. One of the most distinctive properties is its catalytic efficiency, which is higher than that of Alcalase 2.5 L, typeDX (commercial enzyme) and SAPB from B. pumilus CBS. Interestingly, the results of the wash performance analysis demonstrated considerably good de-staining at 40 °C for 30 min with low supplementation (500 U/mL). Accordingly, such a protease could be considered as a good detergent-additive in detergent industry.

Molecular Cloning and Characterization of a Novel α-Amylase from Antarctic Sea Ice Bacterium Pseudoalteromonas sp. M175 and Its Primary Application in Detergent.

A novel cold-adapted and salt-tolerant α-amylase gene (amy175) from Antarctic sea ice bacterium Pseudoalteromonas sp. M175 was successfully cloned and expressed. The open reading frame (ORF) of amy175 had 1722 bp encoding a protein of 573 amino acids residues. Multiple alignments indicated Amy175 had seven highly conserved sequences and the putative catalytic triad (Asp244, Glu286, and Asp372). It was the first identified member of GH13_36 subfamily which contained QPDLN in the CSR V. The recombinant enzyme (Amy175) was purified to homogeneity with a molecular mass of about 62 kDa on SDS-PAGE. It had a mixed enzyme specificity of α-amylase and α-glucosidase. Amy175 displayed highest activity at pH 8.0 and 25°C and exhibited extreme salt-resistance with the maximum activity at 1 M NaCl. Amy175 was strongly stimulated by Mg2+, Ni2+, K+, 1 mM Ca2+, 1 mM Ba2+, 1 mM Pb2+, 1 mM sodium dodecyl sulphate (SDS), and 10% dimethyl sulfoxide (DMSO) but was significantly inhibited by Cu2+, Mn2+, Hg2+, 10 mM β-mercaptoethanol (β-ME), and 10% Tween 80. Amy175 demonstrated excellent resistance towards all the tested commercial detergents, and wash performance analysis displayed that the addition of Amy175 improved the stain removal efficiency. This study demonstrated that Amy175 would be proposed as a novel α-amylase source for industrial application in the future.

Purification and characterization of an organic solvent-tolerant and detergent-stable lipase from Haloferax mediterranei CNCMM 50101

The present study investigates the purification and biochemical characterization of an extracellular lipase (HML) from Haloarchaea Haloferax mediterranei strain ATS1, isolated from the Sebkha (Medea, Algeria). The pure protein was obtained with ammonium sulfate precipitation (40–70%)-dialysis and UNO Q-6 FPLC, and characterized biochemically. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer, with a molecular mass of 45,011.09 Da. It showed optimum lipase activity at pH 7 and 60 °C. HML showed a higher specific activity on triacylglycerols with long-chains fatty acids, indicating that HML is a true lipase. This enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggested its belonging to the serine lipase family. The Km and Vmax for HML toward olive oil were 1.01 mM and 1195 U/mg, respectively. Compared to Lipolase, HML displayed an elevated organic solvent tolerance, an outstanding stability to surfactants, oxidizing, and auxiliary agents, a considerable compatibility with various commercialized laundry detergents, and wash performance analysis revealed that it could remove oil-stains effectively. Overall, HML has a number of attractive properties that make it a potential promising candidate for the synthesis of non-aqueous peptides and detergent formulations.

Purification, biochemical, and molecular characterization of novel protease from Bacillus licheniformis strain K7A

A novel extracellular alkaline protease, called SAPHM, from Bacillus licheniformis strain K7A was purified by four steps procedure involving heat treatment (30 min at 70 °C) followed by ammonium sulfate precipitation (40–70%)-dialysis, UNO Q-12 FPLC, and ZORBAX PSM 300 HPLC, and submitted to biochemical characterization assays. The purified enzyme is a monomer of molecular mass of 30,325.12 Da. It was completely inhibited by phenylmethanesulfonyl fluoride (PMSF)and diiodopropyl fluorophosphates (DFP), which strongly suggested its belonging to the serine protease family. Its sequence of the 26 NH2-terminal residues showed high homology with those of Bacillus proteases. The purified enzyme was optimally active at pH 10 and temperature 70 °C. Its catalytic efficiency was higher than those of Alcalase and Thermolysin. SAPHM exhibited excellent stability to detergents and wash performance analysis revealed that it could remove blood-stains effectively. Data suggest also that SAPHM may be considered as potential candidate for future applications in non-aqueous peptide biocatalysis because it possesses an elevated organic solvent resistance. The sapHM gene encoding SAPHM was cloned, sequenced, and expressed in Escherichia coli strain BL21(DE3)pLysS. The biochemical properties of the extracellular purified recombinant enzyme (rSAPHM) were similar to those of native one. The deduced amino acid sequence showed strong homology with other Bacillus proteases. The highest sequence identity value (97%) was obtained with APRMP1 protease from Bacillus licheniformis strain MP1, with only 9 aa of difference.

Proteolytic and amylolytic enzymes from a newly isolated Bacillus mojavensis SA: Characterization and applications as laundry detergent additive and in leather processing

The present work aims to study the simultaneous production of highly alkaline proteases and thermostable α-amylases by a newly isolated bacterium Bacillus mojavensis SA. The optimum pH and temperature of amylase activity were 9.0 and 55°C, respectively, while those of the proteolytic activity were 12.0 and 60°C, respectively. Both α-amylase and protease enzymes showed a high stability towards a wide range of pH and temperature. Furthermore, SA crude enzymes were relatively stable towards non-ionic (Tween 20, Tween 80 and Triton X-100) and anionic (SDS) surfactants, as well as oxidizing agents. Both activities were improved by the presence of polyethylene glycol 4000 and glycerol. Additionally, the crude enzymes showed excellent stability against various solid and liquid detergents. Wash performance analysis revealed that the SA crude enzymes exhibited a remarkable efficiency in the removal of a variety type of stains, such as blood, chocolate, coffee and oil. On the other side, SA proteases revealed a potential dehairing activity of animal hide without chemical assistance or fibrous proteins hydrolysis. Thus, considering their promising properties, B. mojavensis SA crude enzymes could be used in several biotechnological bioprocesses.

Strain improvement studies on Microbacterium foliorum GA2 for production of α-amylase in solid state fermentation: Biochemical characteristics and wash performance analysis at low temperatures.

Microbacterium foliorum GA2, an alkali-tolerant bacterium, was randomly mutated using UV radiation and sodium azide to obtain a mutant with a higher cold-active extracellular amylolytic activity. A mutant, designated as MFSD20, was selected owing to its higher amylase activity at 20°C. Under optimized conditions, amylase production was achieved best with raw banana peels (5000 units) in solid-state fermentation (SSF). The enzyme was purified by salt precipitation and chromatographic methods and afterwards characterized biochemically. The purified enzyme showed maximal activity at temperatures between 15-25°C and at pH 8.0. Interestingly, this mutant biocatalyst (MFSD20) displays higher catalytic activity under conditions of low temperature (4°C) and high pH (10.0), in the presence of SDS (0.1 and 1%), and exhibited 85% and 50% requirement of divalent metallic ions Ca2+ and Mg2+, respectively. This mutant enzyme extract in combination with "Wheel detergent" was highly effective in the removal of tomato sauce and chocolate stains from white cotton fabric was demonstrated by ~50% additional reflectance compared with detergent alone, in a wash performance analysis at 20 ± 2°C. The features shown by mutant M. foliorum GA2 make it a promising candidate for industrial applications involving starch degradation at low temperatures.

Surfactant- and oxidant-stable alkaline proteases from Bacillus invictae: Characterization and potential applications in chitin extraction and as a detergent additive

A newly alkaline proteases producing strain was isolated from sea water. The strain was identified as Bacillus invictae on the basis of biochemical characteristics and 16S rRNA sequence analysis. The crude protease activity showed an optimal activity at approximately 60°C and in wide pH interval ranging from 9.0 to 11.0. At least six clear caseinolytic protease bands were observed in a zymogram. Phenylmethylsulfonyl fluoride (PMSF), a serine-protease inhibitor, was found to inhibit completely the protease activity. The crude alkaline proteases showed high stability toward solid and liquid detergents. Furthermore, wash performance analysis revealed that the crude enzyme could effectively remove blood stain when added to commercial detergent. In addition, the crude proteases were found to be effective in the deproteinization of shrimp shell waste. The percent of protein removal after 3h of hydrolysis at 50°C with an E/S ratio of 10U/mg of protein or after fermentation by the strain were about 76% and 82%, respectively. Thus, the results of the present study showed that the crude proteases of B. invectae could be effectively used in several industrial applications, as an eco-friendly agent.

Characterization of a novel protease from Aeribacillus pallidus strain VP3 with potential biotechnological interest

The present study investigates the purification and physico-chemical characterization of an extracellular protease from the Aeribacillus pallidus strain VP3 previously isolated from a geothermal oil-field (Sfax, Tunisia). The maximum protease activity recorded after 22h of incubation at 45°C was 3000U/ml. Pure enzyme, designated as SPVP, was obtained after ammonium sulfate fractionation (40–60%)-dialysis followed by heat-treatment (70°C for 30min) and UNO Q-6 FPLC anion-exchange chromatography. The purified enzyme is a monomer of molecular mass about 29kDa. The sequence of the 25 NH2-terminal residues of SPVP showed a high homology with those of Bacillus proteases. The almost complete inhibition by PMSF and DIFP confirmed that SPVP is a member of serine protease family. Its optima of pH and temperature were pH 10 and 60°C, respectively. Its half-life times at 70 and 80°C were 8 and 4h, respectively. Its catalytic efficiency was higher than those of SAPCG, Alcalase Ultra 2.5L, and Thermolysin type X. SPVP exhibited excellent stability to detergents and wash performance analysis revealed that it could remove blood-stains effectively and high resistance against organic solvents. These properties make SPVP a potential candidate for applications in detergent formulations and non-aqueous peptide biocatalysis.

An Alkaline Protease from Bacillus pumilus MP 27: Functional Analysis of Its Binding Model toward Its Applications As Detergent Additive.

A proteolytic strain of Bacillus pumilus MP 27 was isolated from water samples of Southern ocean produced alkaline protease. Since protease production need expensive ingredients, an economically viable process was developed by using low cost carbon source, wheat straw, supplemented with peptone. This protease was active within temperature ranges 10–70°C at pH 9. This process was optimized by response surface methodology using a Box Bekhman design by Design Expert 7.0 software that increased the protease activity to 776.5 U/ml. Moreover, the enzyme was extremely stable at a broad range of temperature and pH retaining 69% of its activity at 50°C and 70% at pH 11. The enzyme exhibited excellent compatibility with surfactants and commercial detergents, showing 87% stability with triton X-100 and 100% stability with Tide commercial detergent. The results of the wash performance analysis demonstrated considerably good de-staining at 50 and 4°C with low supplementation (109 U/ml). Molecular modeling of the protease revealed the presence of serine proteases, subtilase family and serine active site and further docking supported the association of catalytic site with the various substrates. Certainly, such protease can be considered as a good detergent additive in detergent industry with a possibility to remove the stains effectively even in a cold wash.

Biochemical characterization of a detergent-stable serine alkaline protease from Caldicoprobacter guelmensis

Caldicoprobacter guelmensis isolated from the hydrothermal hot spring of Guelma (Algeria) produced high amounts of extracellular thermostable serine alkaline protease (called SAPCG) (23,000U/mL). The latter was purified by ammonium sulphate precipitation, UNO Q-6 FPLC and Zorbex PSM 300 HPLC, and submitted to biochemical characterization assays. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer, with a molecular mass of 55,824.19Da. The 19 N-terminal residue sequence of SAPCG showed high homology with those of microbial proteases. The enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggested its belonging to the serine protease family. It showed optimum protease activity at pH 10 and 70°C with casein as a substrate. The thermoactivity and thermostability of SAPCG were enhanced in the presence of 2mM Ca2+. Its half-life times at 80 and 90°C were 180 and 60min, respectively. Interestingly, the SAPCG protease exhibited significant compatibility with iSiS and Persil, and wash performance analysis revealed that it could remove blood-stains effectively. Overall, SAPCG displayed a number of attractive properties that make it a promising candidate for future applications as an additive in detergent formulations.

Purification and Characterization of Highly Alkaline Lipase from Bacillus licheniformis MTCC 2465: and Study of its Detergent Compatibility and Applicability

AbstractAn extracellular alkaline lipase from Bacillus licheniformis MTCC 2465 has been studied and analyzed for its applicability as a detergent additive. The lipase obtained from Bacillus licheniformis MTCC 2465 was purified by ammonium sulfate fractionation and gel filtration chromatography. The enzyme was precipitated with a 60 % saturated ammonium sulfate fraction showed 6.73 fold purification with the fold purification of 10.4 and specific activity of 398 U/mg of protein with gel filtration chromatography. The optimal pH and temperature for activity were 10 and 60 °C respectively. The enzyme was found to be stable in the pH range of 8–11 with 90 % retention of activity at pH 11. The enzyme retained 90 % activity at 60 °C and 70 % of activity at 70 °C for 1 h. The enzyme activity was maximally enhanced by Hg2+ followed by Co2+ and Fe2+. The lipase was found to be stable in organic solvents with maximum stability in acetone followed by ethanol. The lipase exhibited remarkable stability in the presence of commercial detergents and found to be stable in bleaching agents. Wash performance analysis resulted in improvement of 10 % more grease removal ability of the present lipase in comparison to commercially available lipase.

Chromosomal Integration에 의해 제조한 Bacillus clausii C5 유래의 alkaline protease의 세제 첨가제 응용성

Bacillus clausii I-52 which produced SDS- and H₂O₂-tolerant extracellular alkaline protease (BCAP) was isolated from heavily polluted tidal mud flat of West Sea in Incheon, Korea and stable strain (transformant C5) of B. clausii I-52 harboring another copy of BCAP gene in the chromosome was developed using the chromosome integration vector, pHPS9-fuBCAP. When investigated the production of BCAP using B. clausii transformant C5 through pilot-scale submerged fermentation (500 L) at 37℃ for 30 h with an aeration rate of 1 vvm and agitation rate of 250 rpm, protease yield of approximately 105,700 U/mL was achieved using an optimized medium (soybean meal 2%, wheat flour 1%, sodium citrate 0.5%, K₂HPO₄ 0.4%, Na₂HPO₄ 0.1%, NaCl 0.4%, MgSO₄ · 7H₂O 0.01%, FeSO₄· 7H₂O 0.05%, liquid maltose 2.5%, Na₂CO₃ 0.6%). The enzyme stability of BCAP was increased by addition of polyols (10%, v/v) and also, the stabilities of BCAP towards not only the thermal-induced inactivation at 50℃ but also the SDS and H₂O₂-induced inactivation at 50℃ were enhanced. Among the polyols examined, the best result was obtained with propylene glycol (10%, v/v). The BCAP supplemented with propylene glycol exhibited extreme stability against not only the detergent components such as α-orephin sulfonate (AOS) and zeolite but also the commercial detergent preparations. The granulized enzyme of BCAP was prepared with approximately 1,310,000 U/g of granule. Wash performance analysis using EMPA test fabrics revealed that BCAP granule exhibited high efficiency for removal of protein stains in the presence of anionic surfactants as well as bleaching agents. When compared to Savinase 6T ⓡ and Everlase 6T ⓡ manufactured by Novozymes, BCAP under this study probably showed similar or higher efficiency for the removal of protein stains. These results suggest that the alkaline protease produced from B. clausii transformant C5 showing high stability against detergents and high wash performance has significant potential and a promising candidate for use as a detergent additive.