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

An 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.     

Production of thermostable alkaline lipase on vegetable oils from a thermophilic Bacillus sp. DH4, characterization and its potential applications as detergent additive

BACKGROUND: The alkaline lipase production on vegetable oils as sole carbon source, its characterization and effect of different commercial detergents and surfactants on enzymatic activity from thermophilic Bacillus sp. DH4 was investigated. RESULTS: The organism grew on mannose, but the amount of lipase secreted was significantly less than on vegetable oils. This study identified a simple substrate for lipase production and established the utility of groundnut oil for increasing the lipase yield. The enzyme was compatible with various ionic and non‐ionic surfactants as well as commercial detergents. Lipase activity was strongly inhibited by sodium dodecyl sulfate (SDS), but not by Triton X‐100 or Triton X‐114. The best assay conditions observed for this lipase was found to be pH 9.0 and 50 °C. The enzyme was stable at alkaline pH and considerable activity was observed at pH 10 and it retained 93% of the residual activity at 60 °C. The lipase showed a novel property of marked activation at alkaline pH. Wash performance analysis of commercial detergent for removal of fatty stains improved upon addition of lipase. CONCLUSION: The production on cheap vegetable oils, novel properties and resistance towards various surfactants and tolerance to commercial detergents make this lipase a potential additive for detergent formulations. Significance and impact of the study: Bacillus sp. produces alkaline and thermostable lipase on cheap vegetable oils and its compatibility can find use in the detergent industry. Copyright © 2008 Society of Chemical Industry     

Function of lipase in lipid soil removal as studied using fabrics with different chemical accessibility

Fatty stain removal is enhanced by the inclusion of lipase in washing compounds and leads to increased lipid removal from within the fibers. Cotton fabrics with varied morphology/chemistry were investigated to study the accessibility of soil in textiles to detergent and lipase. Three cotton fabrics (untreated, mercerized, and carboxymethylated cotton), differing in chemical accessibility, and Tencel^TM lyocell fabric, a microdenier manufactured cellulosic fiber, were subjected to three treatments—unwashed, washed with detergent, and washed with lipase—so as to understand further the effects of fiber morphology on lipase effectiveness. Both detergents and lipase removed more soil from the more chemically accessible and hydrophilic textiles. Lipase increased lipid removal for all fabrics and all morphological locations on the fiber, including fiber surfaces, interfiber capillaries, small capillaries, and the center of the yarn bundle. Lipase removed significant quantities of soil from the lumen in untreated and mercerized cotons; these fabrics showed the largest total increases in amount of lipid removed by lipase. When the fiber surfaces were smoother and the fiber structure was less open and not carboxymethylated, i.e., the mercerized cotton fabric, more lipase benefit was observed (72% of the residual soil left after washing with detergent was removed when lipase was added). The total soil removal from the mercerized cotton fabric by use of lipase was equal to that observed for the more open, hydrophilic carboxymethylated fabric and for the Tencel, which has no lumen or other morphological features of natural cotton such as crenulations. Lipase appeared to enhance lipid removal under conditions where removal by the detergent surfactant system was limited. Furthermore, we concluded that lipase acted to remove lipid soil from within the fibers by functioning at the interior surfaces of microfibrils and pores within the fiber structure at the lipid-water interface.     

Interactions Between Oil Substrates and Glucose on Pure Cultures of Ruminal Lipase-Producing Bacteria

The hydrolysis of free fatty acids from lipids is a prerequisite for biohydrogenation, a process that effectively saturates free fatty acids. Anaerovibrio lipolyticus 5s and Butyrivibrio fibrisolvens have long been thought to be the major contributors to ruminal lipolysis; however, Propionibacterium avidum and acnes recently have been identified as contributing lipase activity in the rumen. In order to further characterize the lipase activity of these bacterial populations, each was grown with three different lipid substrates, olive oil, corn oil, and flaxseed oil (3 %). Because different finishing rations contain varying levels of glycogen (a source of free glucose) this study also documented the effects of glucose on lipolysis. P. avidum and A. lipolyticus 5s demonstrated the most rapid rates (P < 0.05) of lipolysis for cultures grown with olive oil and flaxseed oil, respectively. A. lipolyticus, B. fibrisolvens, and P. avidum more effectively hydrolyzed flaxseed oil than olive oil or corn oil, especially in the presence of 0.02 % glucose. Conversely, P. acnes hydrolyzed corn oil more readily than olive oil or flaxseed oil and glucose had no effect on lipolytic rate. Thus, these bacterial species demonstrated different specificities for oil substrates and different sensitivities to glucose.     

Biochemical Properties of a New Cold-Active Mono- and Diacylglycerol Lipase from Marine Member Janibacter sp. Strain HTCC2649

Mono- and di-acylglycerol lipase has been applied to industrial usage in oil modification for its special substrate selectivity. Until now, the reported mono- and di-acylglycerol lipases from microorganism are limited, and there is no report on the mono- and di-acylglycerol lipase from bacteria. A predicted lipase (named MAJ1) from marine Janibacter sp. strain HTCC2649 was purified and biochemical characterized. MAJ1 was clustered in the family I.7 of esterase/lipase. The optimum activity of the purified MAJ1 occurred at pH 7.0 and 30 °C. The enzyme retained 50% of the optimum activity at 5 °C, indicating that MAJ1 is a cold-active lipase. The enzyme activity was stable in the presence of various metal ions, and inhibited in EDTA. MAJ1 was resistant to detergents. MAJ1 preferentially hydrolyzed mono- and di-acylglycerols, but did not show activity to triacylglycerols of camellia oil substrates. Further, MAJ1 is low homologous to that of the reported fungal diacylglycerol lipases, including Malassezia globosa lipase 1 (SMG1), Penicillium camembertii lipase U-150 (PCL), and Aspergillus oryzae lipase (AOL). Thus, we identified a novel cold-active bacterial lipase with a sn-1/3 preference towards mono- and di-acylglycerides for the first time. Moreover, it has the potential, in oil modification, for special substrate selectivity.     

Burkholderia multivorans SB6 Lipase as a Detergent Ingredient: Characterization and Stabilization

In this study, 265 bacterial isolates were collected from kitchen wastewater samples using Rhodamine B agar medium. Of these, 115 isolates were found to respond positively to the addition of commercial detergents. Using 16S rRNA sequence analysis, the isolate demonstrating the high stability towards commercial detergents was identified as Burkholderia multivorans. An SB6 lipase with a molecular mass of 70 kDa was purified from B. multivorans. The purified enzyme showed optimal activity at pH 9.0 and 40 °C and remained stable in the presence of various metal ions, surfactants, and oxidizing agents. The addition of boron compounds improved the pH stability and thermostability of the enzyme, which displayed stability against some commercial detergents; moreover, this stability increased when boron compounds were added to the incubation medium as stabilizers. These properties make SB6 lipase an ideal choice as an additive in detergent formulations.     

Detergent Compatible Extracellular Lipase from Streptomyces cellulosae AU‐10: A Green Alternative for the Detergent Industry

Enzymes can decrease the environmental and economic load of detergent products by reducing the amount of chemicals used in detergents and by allowing washing at ambient temperatures. In this study, Streptomyces cellulosae AU‐10 (GenBank accession number: MG780240) lipase was purified 7.08‐fold with 68% yield using an aqueous 2‐phase system. The Streptomyces sp. AU‐10 lipase showed maximal activity at pH 9.0 and 40 °C. Hundred percent activities were measured in the pH range from 9.0 to 11.0 for 1 h. The enzyme was also highly stable at 30–50 °C. The values of K_m and V_max were calculated as 0.34 mM and 0.83 mM min^?1, respectively. The lipase has high hydrolytic activity for olive oil and sunflower oil. The effect of ethylenediamine tetraacetic acid on the enzyme has shown that the lipase is a metalloenzyme. The activity increased in the presence of Fe²⁺, Cu²⁺, and various boron compounds. The enzyme has shown a good stability not only with surfactants but also with oxidizing agents. In addition, activities in the presence of Omo, Ariel, Tursil, Pril, and Fairy were measured as 108.8%, 115.6%, 98.35%, 140.4%, and 107.6%, respectively. Considering its remarkable ability, the S. cellulosae AU‐10 lipase can be considered as a potential additive in the detergent industry.     

Production of a halotolerant lipase from Halomonas sp. strain C2SS100: Optimization by response-surface methodology and application in detergent formulations

The aim of this work was to optimize the production of a new lipase by a halotolerant bacterial strain Halomonas sp. C2SS100, by means of the response-surface methodology (RSM). The process parameters having the most significant effect on lipase production were identified using the Plackett–Burman screening design-of-experiments. Then, Box–Behnken design was applied to optimize lipase activity and the quadratic regression model of the lipase production was built. Indeed, the lipase yield was increased, and the value obtained experimentally (39 ± 2 U/ml) was very close to the rate predicted by the model (40.3 U/ml). Likewise, optimization of parameters by RSM resulted in 2.78-fold increase in lipase activity. These findings provide the first report on lipase production and optimization by a halotolerant bacterial strain belonging to Halomonas genus. Afterward, the biochemical properties of the produced lipase were studied for apply in oil stains removal. The crude lipase showed a maximum activity at 60°C and at pH ranging from 7 to 10. It displayed an important stability at high temperature, pH, and NaCl. Interestingly, this bacterial lipase exhibited a prominent stability toward some commercial solid and liquid detergents after 30 min of incubation at 50°C. The capability of the crude lipase to eliminate stain was ascertained on polycotton fabric pieces stained with lubricating oil. Whether with the addition of hot water alone or of a commercially available detergent, lipase is able to considerably boost the elimination of oil stains. The actual findings highlight the capacity of Halomonas sp. lipase for energy-efficient biocatalytic application.     

Study of detergency. I. Effect of the concentration and the kind of detergent in hard water

The effect of concentration and kind of detergent, and nature of oil-on-oil removal efficiency in hard water was studied. Maximum oil removal efficiencies are shown at the same water hardness for a given oil. As the concentration of detergents increases, the water hardness at maximum oil removal efficiency increases and the sharpness of the oil removal peak is lessened. A linear relationship between concentration of detergent and water hardness at maximum oil removal efficiency was found. This relationship is dependent on the nature of the oily soil and kind of detergent.     

The Use of Boron Compounds for Stabilization of Lipase from Pseudomonas aeruginosa ES3 for the Detergent Industry

This study aimed to characterize a lipase that is highly active and stable under typical washing conditions for use as a detergent ingredient by investigating the effects of various boron compounds on lipase stabilization under different conditions. In addition, the antimicrobial activity of the boron compounds used in enzyme stabilization was examined in order to obtain an effective antimicrobial detergent. A lipase‐producing bacterium was isolated from kitchen wastewater samples using Rhodamine‐B Agar medium and identified as Pseudomonas aeruginosa based on 16S rDNA sequence analysis. The ES3 lipase obtained from P. aeruginosa was purified, and the purified enzyme was found to have a molecular mass of 40 kDa. The enzyme showed optimal activity at pH 9.0–10.0 and 40 °C and remained stable in the presence of various metal ions, surfactants and oxidizing agents. Moreover, the pH stability and thermostability of the enzyme was improved by the addition of boron compounds, which, when used as stabilizers in the incubation media, also increased the stability of the enzyme towards commercial detergents. Furthermore, the enzyme displayed properties comparable with the commercial product Lipolase^®, which has shown excellent stability towards various commercial detergents. Finally, boron compounds used to stabilize the lipase were found to possess antimicrobial properties, suggesting that detergents incorporating these compounds will also exhibit antimicrobial activity when washing clothes and dishes.     

Effects of Different Oil Sources and Residues on Biomass and Metabolite Production by Yarrowia lipolytica YB 423-12

Yarrowia lipolytica is known to have the ability to assimilate hydrophobic substrates like triglycerides, fats, and oils, and to produce single-cell oils, lipases, and organic acids. The aim of the present study was to investigate the effects of different oil sources (borage, canola, sesame, Echium, and trout oils) and oil industry residues (olive pomace oil, hazelnut oil press cake, and sunflower seed oil cake) on the growth, lipid accumulation, and lipase and citric acid production by Y. lipolytica YB 423-12. The maximum biomass and lipid accumulation were observed with linseed oil. Among the tested oil sources and oil industry residues, hazelnut oil press cake was the best medium for lipase production. The Y. lipolytica YB 423-12 strain produced 12.32 ± 1.54 U/mL (lipase activity) of lipase on hazelnut oil press cake medium supplemented with glucose. The best substrate for citric acid production was found to be borage oil, with an output of 5.34 ± 0.94 g/L. The biotechnological production of valuable metabolites such as single-cell oil, lipase, and citric acid could be achieved by using these wastes and low-cost substrates with this strain. Furthermore, the cost of the bio-process could also be significantly reduced by the utilization of various low-cost raw materials, residues, wastes, and renewable resources as substrates for this yeast.     

Detergency study of the synergism between oily and particulate soil on polyester/cotton fabric

The removal of multiphase, multicomponent soils from fibrous substrates depends upon the nature of the soil mixture, the order of application, wash temperature, and type of detergent formulation. By studying these factors, we investigated the synergism between residual oil (triolein) and particulate soil (clay) on a durable press polyester/cotton fabric after laundering with four different detergents at wash temperatures of 27 and 49 C. To probe the interaction between clay and oil, fabric specimens were soiled with clay only, triolein only, clay followed by an application of triolein, and triolein followed by an application of clay. Four detergent formulations were used to launder the soiled fabrics, including one unbuilt liquid and three powdered detergents with different builder systems. The amount of residual oil (triolein) was determined by radiotracer technique, and the quantities of clay were determined by measuring aluminum by neutron activation. Reflectance measurements were used to calculate fabric whiteness. The soil distributions on and within the textile structure were obtained by scanning electron microscopy using backscattered electron images, secondary electron images and X-ray mapping. Osmium tetroxide was used to tag the oil, while silicon was the elemental tag for clay in the microscopic analysis. Results of the four factors studied can be summarized as follows. (i) In agreement with observations by previous researchers, a mixture of clay and oil is more difficult to remove than either the oil or the clay applied singly. It appears that oil acts as a matrix to bind clay, forming a composite soil. (ii) The specimens that were soiled first with oil and then clay had more soil removed by laundering than the specimens soiled with clay and then oil. Detergency was limited by the encapsulation of clay by the oil and adsorbtion of oil by the clay. (iii) The built powdered detergents were temperature sensitive, while the unbuilt liquid detergent was not. (iv) The built powdered detergents removed more soil (oily and clay) than the unbuilt liquid detergent.     

Production, Purification and Partial Characterization of Four Lipases from a Thermophile Isolated from Deception Island

Four lipases were purified from ID17, a thermophilic bacterium belonging to Geobacillus genus isolated from Deception Island, Antarctica. Lipase activity was detected by opacity test and p-nitrophenyl laurate methods. Lipase production was better in a medium containing tryptone as the carbon and nitrogen source, without non-ionic detergents and pH 7.5. Proteins were ultrafiltered from supernatant and separated using anion exchange and size exclusion chromatography resulting in four distinct fractions with lipase activity (called Lip1–4). Purified lipases showed an optimal pH at 9.0, 9.5, 10.0 and 8.0 and temperature at 65, 70, 75 and 80 °C for Lip1–4, respectively. Lip1 and Lip2 showed higher activity using p-nitrophenol decanoate as substrate, whereas Lip3 and Lip4 prefer p-nitrophenol laurate. Based on their molecular weight Lip1 and Lip2 are trimeric and pentameric proteins, respectively, whereas Lip3 and Lip4 are monomeric proteins. Lip1 was exceptionally thermostable maintaining 70 % of its activity after incubating it at 70 °C for 8 h. Based on their characteristics, the four lipases obtained from ID17 are good candidates to understand the mechanisms of lipase stability and to be used in different types of industrial applications.     

Purification of Bacillus licheniformis Lipase and its Application as an Additive in Detergent for Destaining

In this study, we aimed to optimize the nutritional and environmental conditions for the production of a novel lipase (LBL) from Bacillus licheniformis (GenBank accession no. MT118724). This strain was characterized by morphological and biochemical assays and Sanger sequencing of 16S rDNA. The crude lipolytic activity reached a maximum level 7.5 U mL⁻¹ at 40 °C and pH 8.0 using olive oil as substrate. Additionally, the crude enzyme maintained 100% of its initial activity after incubation for 1 h at 50 °C and pH 9.0. It is mandatory to note that LBL lipase displayed appreciable stability over a wide pH range and extreme temperatures. After purification, the optimal lipolytic activity was observed at pH 8.0 and 40 °C. LBL was shown to be a monomeric protein with an estimated molecular weight of 40 kDa. This novel lipase exhibited high stability and excellent compatibility compared to lipase extracted from Thermomyces lanuginosa (Lipolase® from Novozymes, Denmark) toward various detergents. Washing performance analysis revealed that it efficiently removes tomato sauce stain from cotton cloth. All these interesting enzymatic properties favor this new lipase as a potent candidate for applications in detergent formulations.     

Studies on applications of lipolytic enzyme in detergency I. Effect of lipase fromCandida cylindracea on removal of olive oil from cotton fabric

To investigate the effect of lipolytic enzyme on removal of triglyceride soils in laundry, the removal of olive oil from cotton fabric was examined by washing with an aqueous solution of lipase fromCandida cylindracea with and without surfactants at various washing temperatures and times. It was proved that, at optimum conditions, the removal of olive oil with the addition of lipase was 15 to 20% higher than without lipase. Therefore, it might be expected that lipase will be applied in the laundry detergents in practice.     

A kinetic study of fabric detergency

The kinetics of soil removal were investigated under domestic laundry conditions by incorporating small swatches of four artificially soiled test fabrics into a standard load of clean cotton goods. This prevented soil redeposition from affecting the soil removal rate. Two runs were analyzed, one with an anionic and the other with a nonionic detergent. Assessment of the amount of soil remaining on the fabric was made by reflectivity measurements interpreted according to the Kubelka-Munk equation. First-order kinetics were found to prevail for periods ranging from the first 6 min of the wash cycle to the entire 20 min, depending upon test fabric and detergent. For these lengths of time, the rate of soil removal was directly proportional to the amount of soil remaining on the fabric. The 8 first-order rate constants had rather similar values, varying at most by a factor of 2.3. The average value, 0.109 min⁻¹, corresponds to a 6.4-min wash period for removing one-half of the soil and to a 21-min period for removing 90% of the soil from the soiled fabric. The magnitude of the response of the four artificially soiled test fabrics to the two detergents is compared and discussed in terms of the soiling materials. The nonionic detergent was more effective in cleaning a fabric soiled mainly with kaolin and wool fat, while the anionic detergent was more effective with a fabric containing large amounts of liquid oily soil plus carbon black and oleophilic bentonite.     

Hydrolytic Activity of Castor Bean Powder: Effect of Gum Arabic,Lipase and Oil Concentrations

Lipase activity from castor bean seed powders was evaluated in hydrolysis reactions at 37 °C. The effects of different concentrations of lipase powder (LP), substrate (high oleic sunflower oil, O) and surfactant (gum arabic, A) on lipase activity (R) were assessed using experimental designs. Considered variable bounds were: 0.05–0.15 g_LP, 0.07–0.20 oil:aqueous phase (w/w) and 0–0.025 g gum arabic/mL. All variables had significant effects on the transformed response, R ^1/2. The most important result was the negative effect of gum arabic in lipase activity, even when high oil concentrations were used. Experimental lipase activities involved in this work were within 0.32–16.90 mmol_FFA/g_oil·g_LP·h. Using 0.05 g_LP and 0.20 oil:aqueous phase (w/w) without gum arabic, the activity of 20.47 ± 7.19 mmol_FFA/g_oil·g_LP·h was reached.     

Extraction,purification and application of thermostable and halostable alkaline protease from Bacillus alveayuensis CAS 5 using marine wastes

A marine bacterium Bacillus alveayuensis CAS 5 produced protease when grown at 55 °C for 60 h in 100 ml of basal medium containing 1% SSP (w/v) and purified to 7.77 fold with specific activity of 518.78 U/mg. The optimum temperature, pH and NaCl for enzyme activity were 50 °C, 9 and 35% respectively. The enzyme was highly stable even at 80 °C, pH 12, 35% NaCl and presence of ionic, non-ionic and commercial detergents. The protease was investigated for its application as cleansing additive in blood stain removal. The preset study emphasized that marine wastes can be utilized to generate high value-added products and hidden potential in the production of functional foods.     

Development of an Environmentally Acceptable Detergent Formulation for Fatty Soils Based on the Lipase from the Indigenous Extremophile Pseudomonas aeruginosa Strain

A lipase derived from an indigenous extremophile Pseudomonas aeruginosa strain isolated from rancid metalworking fluid was evaluated as a detergent additive. Applicability of the obtained enzyme as an additive in detergent formulations was confirmed by its implementation in the formulations of several new products differing in surfactant type and concentrations, demonstrating satisfactory performance in terms of degreasing efficiency and composition of the washing wastewater. The degreasing efficiency of different enzyme‐containing detergent formulations was studied on cotton fabric samples stained with triolein and compared to that of formulations containing only surfactant. The highest efficiency of the fatty soil removal in formulations with a low content of surfactants (0.4 %) was noted in the enzyme formulation containing Lutensol^® XP‐80 (degreasing efficiency >80 %) and Triton^® X‐100 (degreasing efficiency >60 %). An attempt was then made to optimize the composition of the enzyme formulation on the basis of one or both of these surfactants using statistically planned experiments and response surface methodology (RSM). Taking into consideration the environmental aspects and the shown detergency, it appeared that rather high degreasing effects were achieved in formulations based on a low quantities of Lutensol^® XP‐80 (0.4 %) at all pH values. However, pH seemed to have a notable effect since the degreasing efficiency significantly increased with increasing pH and the amount of the enzyme. Formulations having a moderate alkaline pH profile and higher amount of enzyme exhibited a high cleaning performance of fatty soil even at a low concentration of the surfactant.     

Lipid distribution on textiles in relation to washing with lipases

Effectiveness of lipase in detergency was studied using three test soils (lard, artificial sebum, and olive oil) on a woven cotton fabric. Distribution of oily soil on fabrics was determined for three different treatments (unwashed, washed with detergent without lipase, and washed with detergent plus lipase). Osmium tetroxide was used to label lipid soil for analysis in the scanning electron microscope. Both longitudinal and cross-sectional backscattered electron images for unwashed samples showed that soil was present on surfaces of the cotton fibers and in interfiber spaces of the yarn bundle. Lard soiled samples had large deposits on the fabric surfaces, while artificial sebum and olive oil appeared more uniformly distributed throughout the textile. Oil was deposited in the interfiber capillaries of the yarn bundle and in the crenulation, secondary walls, and lumen of the fibers. Energy dispersive X-ray microanalysis was used to determine relative concentrations of oil at selected morphological locations within the fiber structure and at the fiber surface. Soil distributions within the fibrous structures differed with type of soil and laundry treatment. Backscattered electron images dramatically demonstrated the effect of lipase on cleaning. After washing with detergent plus lipase, yarn surfaces had much less residual soil; residual soil that remained was in the irregularities of the cotton fiber surfaces. Concentrations of oil in the secondary walls, crenulations, lumen, and the fiber surfaces were lower after lipase treatment for all three soils. While washing with detergent removed soil from the yarn and fiber surfaces and the crenulation of the cotton fiber, only the samples washed with detergent plus lipase had lower concentrations of soils within the secondary wall and lumen of the cotton fibers. Fabrics soiled with olive oil and washed with detergent plus lipase had the lowest concentrations of residual soil across the textile structure; the residual soil observed was mainly in the irregularities on the fiber surfaces.