Protease and Amylase Stability in the Presence of Chelators Used in Laundry Detergent Applications: Correlation Between Chelator Properties and Enzyme Stability in Liquid Detergents

Chelators are a common ingredient in most laundry detergents. They have a number of different functions such as reducing water hardness, assisting in keeping particulate soil in suspension and the removal of certain stains, thus complementing the action of the anionic surfactants. Another important group of components in a modern liquid detergent is enzymes, mainly proteases and amylases. As the most commonly used enzymes within the detergent industry are dependent on bound calcium ions to maintain conformational stability and function, the presence of both chelators and enzymes in a liquid detergent presents a challenge. The three commonly used Ca²⁺ chelators: citrate, DTPA (diethylene triamine pentaacetic acid) and HEDP (1-hydroxyethane-1,1-diyl)bis(phosphonic acid), were studied with regard to their impact on protease and amylase stability in buffer and in a model liquid detergent. Enzyme stability was characterized by differential scanning calorimetry (DSC) and activity studies, and correlated to the chelator-Ca²⁺ interaction properties. The results show that a chelator’s ability to reduce water hardness and its Ca²⁺ affinity are in reality two separate aspects in the context of their use in liquid detergents. In the presence of DTPA, stoichiometric surplus of free Ca²⁺ is required to maintain sufficient amylase and protease stability. In the presence of the weaker chelators, HEDP and citrate, the total Ca²⁺ concentration is more important to protein stability than stoichiometric balancing between chelator and Ca²⁺. Thus, for these chelators their total concentration only has a minor impact on the Ca²⁺ concentration required to maintain or improve enzyme storage stability. The results underline the importance of Ca²⁺ in liquid detergent formulations, and suggest how proper balancing of chelators and Ca²⁺ can be used to improve overall enzyme stability.     

Calcium Chelating Sugar-Based Surfactants for Hard-Water Detergency

Hard water can decrease the detergency efficiency of surfactants due to a significant concentration of divalent cations as Ca²⁺ or Mg²⁺. The formulation of a cleaning detergent must be modified and it is usual to add more surfactants or a huge quantity of sequestrating agents. This supplementary addition can have deleterious effects on the environment and increases the price of the formulation. A surfactant, presenting both detergency capacities in the presence of Ca²⁺ and Ca²⁺ chelating properties, would be of great interest. In this paper, we report on Ca²⁺ extraction with sugar-based chelating surfactants using an experimental device, namely a flotation column, as both chelating properties and foaming properties in the presence of Ca²⁺ are important in the flotation process as well as in detergency. Among all the sugar-based surfactants tested, a few of them were able to extract calcium and thus demonstrated the expected characteristics.     

Natural Mutations Affect Structure and Function of gC1q Domain of Otolin-1

Otolin-1 is a scaffold protein of otoliths and otoconia, calcium carbonate biominerals from the inner ear. It contains a gC1q domain responsible for trimerization and binding of Ca²⁺. Knowledge of a structure–function relationship of gC1q domain of otolin-1 is crucial for understanding the biology of balance sensing. Here, we show how natural variants alter the structure of gC1q otolin-1 and how Ca²⁺ are able to revert some effects of the mutations. We discovered that natural substitutions: R339S, R342W and R402P negatively affect the stability of apo-gC1q otolin-1, and that Q426R has a stabilizing effect. In the presence of Ca²⁺, R342W and Q426R were stabilized at higher Ca²⁺ concentrations than the wild-type form, and R402P was completely insensitive to Ca²⁺. The mutations affected the self-association of gC1q otolin-1 by inducing detrimental aggregation (R342W) or disabling the trimerization (R402P) of the protein. Our results indicate that the natural variants of gC1q otolin-1 may have a potential to cause pathological changes in otoconia and otoconial membrane, which could affect sensing of balance and increase the probability of occurrence of benign paroxysmal positional vertigo (BPPV).     

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.     

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.     

A Novel GUCA1A Variant Associated with Cone Dystrophy Alters cGMP Signaling in Photoreceptors by Strongly Interacting with and Hyperactivating Retinal Guanylate Cyclase

Guanylate cyclase-activating protein 1 (GCAP1), encoded by the GUCA1A gene, is a neuronal calcium sensor protein involved in shaping the photoresponse kinetics in cones and rods. GCAP1 accelerates or slows the cGMP synthesis operated by retinal guanylate cyclase (GC) based on the light-dependent levels of intracellular Ca²⁺, thereby ensuring a timely regulation of the phototransduction cascade. We found a novel variant of GUCA1A in a patient affected by autosomal dominant cone dystrophy (adCOD), leading to the Asn104His (N104H) amino acid substitution at the protein level. While biochemical analysis of the recombinant protein showed impaired Ca²⁺ sensitivity of the variant, structural properties investigated by circular dichroism and limited proteolysis excluded major structural rearrangements induced by the mutation. Analytical gel filtration profiles and dynamic light scattering were compatible with a dimeric protein both in the presence of Mg²⁺ alone and Mg²⁺ and Ca²⁺. Enzymatic assays showed that N104H-GCAP1 strongly interacts with the GC, with an affinity that doubles that of the WT. The doubled IC₅₀ value of the novel variant (520 nM for N104H vs. 260 nM for the WT) is compatible with a constitutive activity of GC at physiological levels of Ca²⁺. The structural region at the interface with the GC may acquire enhanced flexibility under high Ca²⁺ conditions, as suggested by 2 μs molecular dynamics simulations. The altered interaction with GC would cause hyper-activity of the enzyme at both low and high Ca²⁺ levels, which would ultimately lead to toxic accumulation of cGMP and Ca²⁺ in the photoreceptor outer segment, thus triggering cell death.     

Erythroid Differentiation Regulator 1 Strengthens TCR Signaling by Enhancing PLCγ1 Signal Transduction Pathway

Erythroid differentiation regulator 1 (Erdr1) has previously been reported to control thymocyte selection via TCR signal regulation, but the effect of Erdr1 as a TCR signaling modulator was not studied in peripheral T cells. In this report, it was determined whether Erdr1 affected TCR signaling strength in CD4 T cells. Results revealed that Erdr1 significantly enhanced the anti-TCR antibody-mediated activation and proliferation of T cells while failing to activate T cells in the absence of TCR stimulation. In addition, Erdr1 amplified Ca²⁺ influx and the phosphorylation of PLCγ1 in CD4 T cells with the TCR stimuli. Furthermore, NFAT1 translocation into nuclei in CD4 T cells was also significantly promoted by Erdr1 in the presence of TCR stimulation. Taken together, our results indicate that Erdr1 positively modulates TCR signaling strength via enhancing the PLCγ1/Ca²⁺/NFAT1 signal transduction pathway.     

Detergent-hard water interactions in machine dishwashing

Insufficient detergent concentration has recently been recognized as a major contributor to film formation on glassware in machine dishwashing. The film is shown to consist largely of calcium tripolyphosphate, Ca₅(P₃O₁₀)₂; evidence is cited for the presence of a silicate, probably calcium metasilicate, as well. Systematic variation of detergent concentration and of water hardness allows construction of a response surface describing conditions of maximum and minimum rates of film formation. The extent of film formation is minimized when the ionic ratio of Ca or Mg or both to tripolyphosphate is 1:1 and maximized when the ratio is 5:2. These ratios correspond to the 1:1 chelate and the uncharged precipitate, Ca₅(P₃O₁₀)₂, respectively. The implications of this treatment to other detergency processes are also considered. Presented at the AOCS Meeting, San Francisco, April 1969.     

Production and Characteristics of an Enantioselective Lipase from Burkholderia sp. GXU56

The lipase production of Burkholderia sp. GXU56 was influenced by carbon and nitrogen sources, inorganic salts, initial pH of the medium and cultivation temperature. The maximum lipase production was 580.52 U/mL and reached 5 times the level of the basic medium in the optimum medium at pH 8.0, 32 °C, 200 rpm and 40–48 h. The lipase was purified 53.6 fold to homogeneity and the molecular weight was 35 KDa on SDS‐PAGE. The optimum pH and temperature of the lipase were 8.0 and 40 °C, respectively, and it was stable in the range of pH 7–8.5 and at temperatures below 45 °C. The lipase activity was strongly inhibited by Zn²⁺, Cu²⁺, Co²⁺, Fe²⁺, Fe³⁺ ions and SDS, while it was stimulated by Li⁺ and Ca²⁺ ions and in presence of 0.1 % CTAB, 0.1 % Triton X‐100 and 10 % DMSO. K_m and V_max of the lipase were calculated to be 0.038 mmol/L, and 0.029 mmol/L min^–1, respectively, with PNPB as the substrate. The GXU56 lipase showed enantioselective hydrolysis of (R,S)‐methyl mandelate to (R)‐mandelic acid, which is an important intermediate in the pharmaceutical industry.     

Clusters of Phospholipid Vesicles as Platforms for Glucose Oxidase‐Catalyzed Reaction in a Bubble‐Column Bioreactor

Clusters of negatively charged liposomes encapsulated with glucose oxidase were prepared in the presence of Ca²⁺ and used to catalyze the oxidation of glucose in an external loop airlift bubble column. The clusters exhibited higher catalytic activity compared to nonclustered glucose oxidase‐containing liposomes (GOLs) when the liposome membranes were incorporated with cholesterol. The clusters were structurally altered in shear flow to give sufficient interfacial area accessible to glucose. The reactivity of GOL clusters could be modulated on the basis of lipid composition of the membranes which affected the mode of interaction among liposomes through Ca²⁺. Part of GOL clusters could be separated from the reaction mixture by centrifugation, which would be advantageous for reusing liposomal catalysts. The liposome clusters can be the platforms to regulate the catalytic performance of glucose oxidase in the airlift.     

Kinetics of the thermal inactivation of Bacillus subtilis α‐amylase and its application on the desizing of cotton fabrics

The thermal inactivation of Bacillus subtilis α‐amylase was studied in the presence and in the absence of Ca²⁺ at various temperatures. Inactivation rate constant (k), half‐life time (t_1/2), and activation energy (E_a) were determined to characterize the inactivation of the enzyme. Results obtained showed that the thermal inactivation of Bacillus subtilis α‐amylase followed a first‐order kinetics. The addition of Ca²⁺ had a good thermostabilizing effect on the enzyme. The stabilizing effect of Ca²⁺ is reflected by the increased values of the activation energy, which is about two times higher in the presence than that in the absence of 20 mM Ca²⁺, and the decreased values of the inactivation rate constants. The desizing of the cotton fabrics was performed through steaming at 100°C with Bacillus subtilis α‐amylase. The desizing efficiency seemed to be dependent on the concentration and pH value of the enzyme solution. It was found that through the steaming process with α‐amylase, the desizing ratio of the cotton fabrics could be beyond 98% and little damage happened to the fibers of the fabrics. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Phospholipid liposomes: Preparation,characterization, and uses

Rhodopsin and cyclic guanosine monophosphat (cGMP)-dependent channel proteins are isolated from the rod outer segment disk membranes of dark-adopted bovine retinae and incorporated in liposomes, prepared by the method of detergent removal dialysis. The ion channel does not lose its transport function (release of Ca²⁺ ions by injection of cGMP) when incorporated in a liposome. Its activity depends on the degree of protein solubilization and the kind of detergent used. The highest activity is obtained by use of the detergent CHAPS. Shape, size, and size distribution of the liposomes are deduced from elastic and quasi-elastic light scattering, the liposome number density by viscometry, and the photopigment or Ca²⁺ content by optical absorbance. The liposomes are heterogeneous with respect to size and shape. Small unilamellar liposomes (R_h = 80 nm) and a narrow size distribution (U_D = 0.16) are obtained by using the detergent CHAPS. With increasing rhodopsin content per liposome, the hydrodynamic radius R_h increases and at the same time the shape of a liposome converts from a sphere to a prolate ellipsoid. The amount of entrapped Ca²⁺ per liposome reaches its maximum value when the Rhodopsin nearest-neighbor distance approaches its minimum value. This suggests an intermembrane protein-lipid-protein lattice, which serves as barriere for Ca²⁺. The influence of temperature or total used Ca²⁺ content is less profound. Increasing temperature yields slightly smaller liposomes.     

Methyl Ester Sulfonate: A High-Performance Surfactant Capable of Reducing Builders Dosage in Detergents

The detergency performance of α-sulfo fatty-methyl ester sulfonate (α-MES) under different water hardness conditions was compared against the dominant workhorse in Home Care products, linear alkylbenzene sulfonate (LAS). Results demonstrate that α-MES has a higher soil removal index and its detergency performance is not drastically affected by water hardness, compared to that of LAS. The addition of α-MES to LAS also shows an improved cleaning performance and better water hardness resistance, due to the structural characteristics of α-MES, which allow the molecules to be relatively insensitive toward polyvalent ions such as Ca²⁺ and Mg²⁺. The washing performance of α-MES, α-MES/LAS, and LAS with different builders at various dosages was studied, and the results indicated that the dosage of builders in the detergent product could be reduced up to 33% with the application of α-MES, while the detergency is not sacrificed.     

Purification and biochemical characterization of an intracellular lipase by Rhizopus chinensis under solid‐state fermentation and its potential application in the production of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)

BACKGROUND: Purification and characterization of an intracellular lipase produced by Rhizopus chinenesis cultured in solid‐state fermentation was investigated. The potential application in concentrating eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil by the pure enzyme was also studied. RESULTS: Through four successive purification steps, the enzyme was purified to homogeneity with an apparent molecular mass of 36 kDa. The lipase was active for pH between 7.0 and 9.0 and temperatures 20–45 °C. Lipase activity was slightly increased in the presence of Ca²⁺ and Mg²⁺, but strongly inhibited by Hg²⁺ and SDS. The pure enzyme was most active on medium chain p‐nitrophenol esters, with the highest activity towards pNP‐caprylate (C8). The enzyme is a non‐specific lipase, because it cleaved not only the 1,3‐positioned ester bonds but also the 2‐positioned bond in triolein. High EPA (17.6%) and DHA (32.9%) contents were achieved using the pure lipase (100 U) within 10 h. CONCLUSION: The enzymatic activity of the lipase on a wide variety of substrates and its stability in the presence of some organic solvents suggest that the lipase should be investigated for a range of commercial applications. The pure lipase was proved to possess potential ability for the production and concentration of EPA and DHA from fish oil. Copyright © 2008 Society of Chemical Industry     

Cationic–electrokinetic improvement of an expansive soil

(Cationic–electrokinetic) soil improvement was shown to be an effective technique for delivering, homogeneously, stabilizing agents into soils. The results of the electrokinetic-stabilization procedure using the K⁺ and Ca²⁺ ions as stabilizing agents varied according to the type of the stabilizing ions. The plasticity index of the natural soil (PI = 40) was reduced to 32 for the Ca²⁺-stabilized soil, and to merely 8 for the K⁺-stabilized soil. The free swell value was reduced from 14% for the natural soil to 3.1% for the Ca²⁺-treated soil, and to only 0.4% for the K⁺-treated soil under identical conditions of dry density and initial moisture content. The friction angle was increased from ϕ = 24° for the natural soil to 30.9° for the Ca²⁺-treated soil, and to 36° for the K⁺-treated soil under identical conditions.     

Functional and Molecular Properties of DYT-SGCE Myoclonus-Dystonia Patient-Derived Striatal Medium Spiny Neurons

Myoclonus-dystonia (DYT-SGCE, formerly DYT11) is characterized by alcohol-sensitive, myoclonic-like appearance of fast dystonic movements. It is caused by mutations in the SGCE gene encoding ε-sarcoglycan leading to a dysfunction of this transmembrane protein, alterations in the cerebello-thalamic pathway and impaired striatal plasticity. To elucidate underlying pathogenic mechanisms, we investigated induced pluripotent stem cell (iPSC)-derived striatal medium spiny neurons (MSNs) from two myoclonus-dystonia patients carrying a heterozygous mutation in the SGCE gene (c.298T>G and c.304C>T with protein changes W100G and R102X) in comparison to two matched healthy control lines. Calcium imaging showed significantly elevated basal intracellular Ca²⁺ content and lower frequency of spontaneous Ca²⁺ signals in SGCE MSNs. Blocking of voltage-gated Ca²⁺ channels by verapamil was less efficient in suppressing KCl-induced Ca²⁺ peaks of SGCE MSNs. Ca²⁺ amplitudes upon glycine and acetylcholine applications were increased in SGCE MSNs, but not after GABA or glutamate applications. Expression of voltage-gated Ca²⁺ channels and most ionotropic receptor subunits was not altered. SGCE MSNs showed significantly reduced GABAergic synaptic density. Whole-cell patch-clamp recordings displayed elevated amplitudes of miniature postsynaptic currents and action potentials in SGCE MSNs. Our data contribute to a better understanding of the pathophysiology and the development of novel therapeutic strategies for myoclonus-dystonia.     

Partial purification and characterization of a Ca2+-stimulated lipoxygenase from soybean seeds

A highly purified Ca²⁺-stimulated lipoxygenase was isolated from the Hill variety of soybean seeds. Separation of Ca²⁺-stimulated lipoxygenase from lipoxygenase active in the absence of Ca²⁺ (lipoxygenase-1) was readily obtained using a DEAE-cellulose column. Sample size applied to the ion exchange column was found to be critical. Both enzymes were bound to the column, although some highly active Ca²⁺-stimulated lipoxygenase eluted with buffer in the presence of bound lipoxygenase-1. Ca²⁺-stimulated lipoxygenase bound to DAAE-cellulose required the use of a NaCl gradient for elution. Ca²⁺-stimulated lipoxygenase showed an apparent isoelectric point at pH 5.90 and optimum activity at pH 7.5 and at 1.1 mM calcium. Lipoxygenase-1 was inhibited over 95% in the presence of 60 μM methyl mercuric chloride, while Ca²⁺-stimulated lipoxygenase showed a maximum of only 20% inhibition under the same conditions. Paper No. 4993 Mississippi Agricultural and Forestry Experiment Station.     

Type 1 Diabetes Impairs Cardiomyocyte Contractility in the Left and Right Ventricular Free Walls but Preserves It in the Interventricular Septum

Type 1 diabetes (T1D) leads to ischemic heart disease and diabetic cardiomyopathy. We tested the hypothesis that T1D differently affects the contractile function of the left and right ventricular free walls (LV, RV) and the interventricular septum (IS) using a rat model of alloxan-induced T1D. Single-myocyte mechanics and cytosolic Ca²⁺ concentration transients were studied on cardiomyocytes (CM) from LV, RV, and IS in the absence and presence of mechanical load. In addition, we analyzed the phosphorylation level of sarcomeric proteins and the characteristics of the actin-myosin interaction. T1D similarly affected the characteristics of actin-myosin interaction in all studied regions, decreasing the sliding velocity of native thin filaments over myosin in an in vitro motility assay and its Ca²⁺ sensitivity. A decrease in the thin-filament velocity was associated with increased expression of β-myosin heavy-chain isoform. However, changes in the mechanical function of single ventricular CM induced by T1D were different. T1D depressed the contractility of CM from LV and RV; it decreased the auxotonic tension amplitude and the slope of the active tension–length relationship. Nevertheless, the contractile function of CM from IS was principally preserved.     

Identification and Characterization of a Novel Thermophilic,Organic Solvent Stable Lipase of <Emphasis Type="Italic">Bacillus</Emphasis> from a Hot Spring

A novel lipase gene lip256 was cloned and identified from the genomic library of hot spring strain Bacillus sp. HT19. The deduced amino acid sequence of lip256 has less than 32% identity to a predicted esterase (Cog1752) from Photobacterium leiognathi lrivu.4.1 and contains a novel motif (GTSAG) that differs from other clusters in the lipase superfamily. Following purification, a single band was obtained with a molecular mass of 33 kDa by SDS-PAGE, and the optimal temperature and pH for lipolytic activity of Lip25 were 70 °C and 9.0, respectively. Lip256 exhibited high activity at high temperatures, with 40% maximum activity at 80 °C and good stability at temperatures ranges between 50 and 80 °C. Additionally, the enzyme was highly stable in the presence of butyl-alcohol, glycerol, acetonitrile, pyridine, and urea. However, the presence of acetone, methanol, trichloromethane, petroleum ether, hexane, tert-butanol, isopropanol, dithiothreitol, ethylenediaminetetraacetic acid, polyhexamethylene biguanide, dimethyl sulfoxide, benzene, Triton X-100, Tween-20, Tween-80, and sodium dodecyl sulfate suppressed or absolutely inhibited enzyme activity. Furthermore, Ca²⁺, Mg²⁺, and Cu²⁺ suppressed enzyme activity, whereas Na⁺, Fe³⁺, K⁺, Fe²⁺, and Sr²⁺ enhanced enzyme activity. The unique characteristics of novel lipase Lip256, including its thermo-alkaliphilic performance, high tolerance toward metal ions, inhibitors, and detergents, and high stability in organic solvents, implied that this enzyme might be an interesting candidate for industrial processes.     

Some Biochemical Properties of Lipase from Bay Laurel (<Emphasis Type="Italic">Laurus nobilis</Emphasis> L.) Seeds

Lipase was isolated from bay laurel (Laurus nobilis L.) seeds, some biochemical properties were determined. The bay laurel oil was used as the substrate in all experiments. The pH optimum was found to be 8.0 in the presence of this substrate. The temperature optimum was 50 °C. The specific activity of the lipase was found to be 296 U mg protein⁻¹ in optimal conditions. The enzyme activity is quite stable in the range of pH 7.0–10. The enzyme was stable for 1 h at its optimum temperature, and retained about 68% of activity at 60 °C during this time. K _m and V _max values were determined as 0.975 g and 1.298 U mg protein⁻¹, respectively. Also, storage stability and metal effect on lipolytic activity were investigated. Enzyme activity was maintained for 9, 12, and 42 days at room temperature, 4 and −20 °C, respectively. Ca²⁺, Co²⁺, Cu²⁺, Fe²⁺, and Mg²⁺ lightly enhanced bay laurel lipase activity.