Detergent Enzymes Research Articles

ENDOSCOPE CONTAMINATION FROM HBV‐ AND HCV‐POSITIVE PATIENTS AND EVALUATION OF A CLEANING/DISINFECTING METHOD USING STRONGLY ACIDIC ELECTROLYZED WATER

Background: It is well known that strongly acidic electrolyzed water (SAEW) has a potent bactericidal effect. We examined residual viruses on endoscopes that were used in hepatitis B virus (HBV)‐positive and hepatitis C virus (HCV)‐positive patients and evaluated the effectiveness of SAEW in cleaning/disinfecting the endoscopes.Methods: A random sample of endoscopes used in 109 endoscopies on HBV‐positive patients and 107 endoscopies on HCV‐positive patients, who underwent upper gastrointestinal endoscopy for various reasons was taken to determine the degree of HBV and HCV contamination. Samples were taken using 10 mL of physiological saline injected through the forceps channel of each endoscope and collected at the distal end to be assayed using polymerase chain reaction (PCR). After examination, each endoscope was treated with air aspiration, then 200 mL of tap water that contained an enzyme detergent was absorbed, and SAEW was aspirated after cleaning with a brush. After each procedure, PCR was used for comparison and to identify any residual viruses.Results: In saline collected after air aspiration, viruses were detected in 39/109 endoscopes used in HBV patients and in 20/107 endoscopes used in HCV patients. In the saline aspirated with tap water containing an enzyme detergent, HBV was detected in 12/109 endoscopes and HCV was detected in 6/107 endoscopes. However, neither HBV nor HCV was detected after the endoscopes were cleaned manually with a brush and disinfected with SAEW.Conclusion: Endoscopes contaminated with HBV and HCV are effectively cleaned and disinfected by SAEW.

Carbohydrate-Active Enzymes from Alkaliphiles

The market in industrial enzymes for detergents, starch, food, fabric, and so on has been estimated at more than 1600 million US dollars in 1999. The usage of detergent enzymes, such as protease, cellulase, aamylase, and lipase, forms 35% of the world market. The enzymes now are incorporated into more than 80% of heavy-duty laundry detergents in the world. Furthermore, alkaline protease and alkaline aamylase have been frequently used in automatic dishwashing detergents especially in the US and Europe. We have exploited large-scale production of alkaline cellulases, alkaline proteases, and an alkaline α-amylase, and the enzymes have been incorporated into heavy-duty compact detergents and/or bleaches. The problems are that traditional alkaline proteases and alkaline α-amylases are seriously inactivated by chemical oxidants and chelating reagents and are thermally unstable, especially when the enzymes are used in automatic dishwashers. Here, we describe the improvement of thermostability and oxidative stability of our alkaline liquefying α-amylase designated LAMY. Furthermore, we show a novel amylase that is extremely resistant to chemical oxidants and chelating reagents.

Effects of drying methods and additives on the structure, function, and storage stability of subtilisin: role of protein conformation and molecular mobility

Proteins and enzymes for commercial use are often dried in order to extend their shelf lives. This study examined the effects of disaccharides (sucrose and trehalose), polymers (dextran and maltodextrin) and disaccharide–polymer mixtures on the stability of subtilisin, a common industrial laundry detergent enzyme, during drying and during subsequent storage in dried solids containing perborate, a laundry detergent additive that hydrolyzes to form hydrogen peroxide in the presence of water. Subtilisin is susceptible to oxidation by hydrogen peroxide at a partially buried methionine residue, which results in loss of catalytic activity. Two drying methods were compared: spray coating and lyophilization. During both drying methods, the presence of a disaccharide, which can hydrogen bond to subtilisin in the place of lost water, was necessary to inhibit dehydration-induced unfolding of subtilisin. Dehydration-induced unfolding was assessed by second-derivative IR spectroscopy. However, even in the samples in which the protein’s secondary structure was perturbed, the protein refolded to the native conformation and exhibited full activity upon rehydration. None of the additives protected Met222 from oxidation during storage of the initially dry protein formulations at 37 °C and 20 or 75% relative humidity under oxidizing conditions. In contrast, the two additional methionine residues in the enzyme, Met119 and Met175, which are deeply buried in the interior of the protein, remained unmodified during storage of the dried formulations and became oxidized only if the enzyme was completely unfolded in aqueous solution.

Protease Adsorption and Reaction on an Immobilized Substrate Surface

Enzymatic reactions with surface-bound substrates present an interesting problem in biomolecular surface science, as they require us to consider traditional enzyme kinetics in the context of protein adsorption. These reactions are important in such applications as detergent enzyme additives, food processing, and contact lens cleaning. We study the interaction of a serine protease (subtilisin) with an immobilized substrate (bovine serum albumin) surface through the simultaneous use of surface plasmon resonance and surface plasmon enhanced fluorescence techniques. We measure adsorbed enzyme concentrations and substrate cleavage rates in situ and compare the reactivities with those in solution. By varying the ionic strength of the reaction environment and studying several single point mutations of subtilisin, we find the adsorption behavior of the enzyme is strongly influenced by its electrostatic interactions with the charged bovine serum albumin surface. The surface reactivity of each of the mutants is cou...

Occupational asthma and allergy in the detergent industry: new developments.

This review highlights the latest developments in the control of enzyme-induced occupational asthma and allergy (rhinitis and conjunctivitis) in the detergent industry. The industry has developed guidelines for the safe handling of enzymes in order to reduce the risk of occupational allergy and asthma. Those manufacturing facilities that follow all of the guidelines enjoy very low or no cases of asthma and allergy among workers exposed to enzymes. The key to the success of the management of enzyme-induced allergy and asthma is prospective surveillance for the development of enzyme-specific IgE antibody before the onset of allergic symptoms. This allows for continuing interventions to reduce exposures, so as to minimize or eliminate those associated with symptoms. Workers with IgE to enzymes can still continue to work in the industry symptom-free for their entire career. This indicates that exposures needed to induce sensitization are different and probably lower than exposures needed to elicit enzyme allergic symptoms. The experience of the detergent enzyme industry in controlling occupational allergens can be applied to other industries. The detergent enzyme story can be viewed as a model for the control of type 1 protein allergens in the workplace.

Allergic contact dermatitis to detergents: A multicenter study to assess prevalence

Background: Allergic contact dermatitis (ACD) to optical brighteners and enzymes in laundry detergents was the focus of numerous reports in the early 1970s. Subsequently, there has been little published on the incidence of allergic reactions to chemicals in laundry detergents. Nonetheless, consumers and physicians continue to ascribe allergic contact reactions to laundry detergents. Objective: This article reports the findings of a multicenter study on the prevalence of patch test reactions to a liquid and a granular laundry detergent provided by Procter & Gamble Company (Cincinnati, Ohio). Methods: Patients referred to members of the North American Contact Dermatitis Group for evaluation of potential ACD were invited to participate in the study, which involved the placement of 2 patch tests (a 0.1% aqueous dilution of a granular laundry detergent and a 0.1% aqueous dilution of a liquid laundry detergent). Whether the patients had atopic dermatitis and whether they or their physicians felt that their dermatitis might be related to laundry detergents were noted. Reactions to the laundry detergents were correlated with allergic reactions to the following screening chemicals: fragrances, nickel, and potassium dichromate. Patients who experienced a reaction to at least one of the laundry detergents could enter phase II of the study, which involved testing to varying dilutions of the laundry detergents, to 0.1% sodium lauryl sulfate (as an irritant control), and to laundered patches of cotton. Patients positive in phase II could enter phase III, which involved wearing a garment laundered with the detergent. Phases II and III were double blinded. Results: Of the 3120 patients seen by members of the North American Contact Dermatitis Group during the 2 years of this study, 738 patients volunteered to enroll. Enrollment was not statistically randomized. Of these 738, 5 (0.7%) had positive patch test reactions to granular laundry detergent (0.1%, aqueous); 3 of these 5 also had positive reactions to the liquid laundry detergent (0.1%, aqueous). In 4 of the 5 patients, the reaction to detergent was thought to have present relevance to their dermatitis; in 1 of the 5, the reaction was deemed to have past relevance. One of these 5 patients had allergy to fragrances. None of the patients was positive to nickel or chromate. Two of the 5 entered phases II and III. Of these 2 patients, 1 had essentially negative repeat dilutional patch testing and “use testing” suggesting that the earlier reaction patterns may have been irritant. The remaining patient had positive dilutional reactions to both the liquid and granular laundry detergent; however, she also had a positive reaction to sodium lauryl sulfate and to a swatch from a T-shirt laundered without detergent. Upon “use testing” in phase III, this latter patient experienced diffuse dermatitis under both the half of the T-shirt laundered with detergent and that laundered without detergent. Conclusion: Laundry detergents appear to be a rare cause of ACD. Among 738 patients with dermatitis, 5 (0.7%) reacted to a 0.1% aqueous dilution of a laundry detergent. Only 2 of these 5 patients could be evaluated in greater detail to differentiate allergic from irritant patch test reactions to detergents. Upon further testing in 2 patients, the reaction in 1 of 2 could not be reduplicated and the reaction in the other was invoked both by the detergents and the controls. Thus, whether our study patients were truly allergic and, if so, what the allergenic material(s) in detergents might be, remains unknown. Therefore the reported incidence rate for detergent-induced allergy of 0.7% in dermatitic patients may be too high, possibly because of false-positive irritant reactions. (J Am Acad Dermatol 2002;46:200-6.)

Application of infrared spectroscopy (attenuated total reflection) for monitoring enzymatic activity on substrate films

Infrared film analysis, a method based on infrared spectroscopy in the mode of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), is demonstrated as a novel analytical method for monitoring enzymatic activity on surface-attached substrate films in the mid infrared range (400–4000 cm −1). The ATR-FTIR technique is sensitive to molecules within a distance of approximately 1 μm from the ATR sampling unit surface (a 7 cm 2 hydrophobic ZnSe crystal). Applying a 0.2–0.3 μm thick film on the ATR unit surface, any chemical changes within this film as well as at the interface can be continuously monitored, even having an aqueous phase on top of the film. Infrared film analysis is considered especially useful for studying detergent enzymes, which act on surface bound films consisting of food component like vegetable oils (triacylglycerols) and carbohydrates (e.g. starch). Experimental data are presented for hydrolysis of a triacylglycerol film (triolein) by use of a triacylglycerol lipase (cutinase), and starch film degradation by use of an α-amylase.

A new hydrogen peroxide–based medical-device detergent with germicidal properties: Comparison with enzymatic cleaners

Background: The objective of this study was to evaluate the efficacy of the cleaning and bacterial killing ability of a new non–enzyme-based formulation (killing detergent solution [KDS]) compared with commercially available enzymatic detergents that included Metrizyme (Metrex Research Division of Sybron Canada Ltd. Morrisburg, Ontario) and Gzyme (Germiphene Corp, Brantford, Ontario). KDS is a hydrogen peroxide–based detergent formulation that combines cleaning efficacy with the ability to kill microorganisms. The KDS formulation helps ensure the protection of the health care worker from infectious risk during the soaking and cleaning stages of medical device reprocessing and reduces the bioburden on devices before sterilization/disinfection. Methods: Test organisms that included Enterococcus faecalis , Salmonella choleraesuis , Staphylococcus aureus , and Pseudomonas aeruginosa were suspended in artificial test soil (ATS-B; patent submitted), inoculated at 106 colonyforming units per carrier and dried overnight before detergent exposure. The ATS-B mimics the blood, protein, carbohydrate, and endotoxin levels of patient-used medical devices. Plastic lumen carriers and a flexible colonoscope were used for surface and simulated-use testing, respectively. Results: The results for the microbial challenge dried onto polyvinyl chloride (PVC) carriers demonstrated that the ability of KDS to remove protein, blood, carbohydrate, and endotoxin from surface test carriers was as effective as the enzyme detergents that were evaluated. Furthermore, KDS was able to effect approximately a 5-Log10 reduction in microbial loads with a 3-minute exposure at room temperature, whereas none of the other detergents were as effective. In simulated-use testing of a soiled colonoscope, KDS was significantly better at ensuring microbial killing compared with Gzyme and Metrizyme and was equivalent to the enzymatic detergents in cleaning ability. Conclusions: In summary the KDS has excellent microbial-killing ability in 3-minute exposures at room temperature and cleans as well as the existing enzymatic detergent formulations that were tested. (Am J Infect Control 2001;29:168-77)

Can medical surveillance measures improve the outcome of occupational asthma?

Occupational asthma (OA) has been reported to account for 3% to 20% of all asthma cases in studies that have included populations in Europe, North America, Australia, New Zealand, and Japan.1 Even after the diagnosis has been confirmed and the worker removed from ongoing exposure to the causative work agent, it can still be associated with ongoing asthma, significant morbidity,2 and even mortality.3 For some workplace sensitizers the risk of OA in the workplace can be reduced by primary prevention, particularly control of workplace respiratory exposure. It has been shown for several workplace sensitizers4-6 that the population risk of sensitization relates to the airborne exposure level. Reduced inhalation exposure levels have been achieved as a result of process or product changes (eg, for detergent enzymes by encapsulation of the enzymes7 and for natural rubber latex by powder-free lowprotein glove use8). Other measures may include use of alternative nonsensitizing agents (elimination/substitution), occupational hygiene measures of ventilation, robots in high-exposure areas, and appropriate respirators. Regulations to control and monitor airborne concentrations of common sensitizers such as diisocyanates may enhance the likelihood of reducing work exposures to safer levels. An additional method for primary prevention of OA would be to identify workers at risk of sensitization to specific workplace agents at the time of pre-employment and exclude their exposure to these agents. Unfortunately, at present those at risk cannot be predicted with sufficient sensitivity and specificity for this to be practical: although risk markers for those exposed to sensitizers, such as genetic markers, atopy, and smoking history9

Safe Use of Detergent Enzymes in the Workplace

Safe Use of Detergent Enzymes in the Workplace

Chemically modified papain for applications in detergent formulations

Papain was modified using succinic anhydride, and the modified papain so obtained was compared with the native papain for its activity and stability in detergents. This study was done using commercial enzyme detergents as references. It was found that modified papain retained activity comparable to the commercial enzyme detergents. Chemically modified papain may prove to be an inexpensive alternative to alkaline proteases that are used in detergents.

Safe use of detergent enzymes in the workplace.

This article gives a detailed description of a program to control workplace exposure to enzymes during manufacture of laundry detergents. We believe this same program could, in large part, be re-applied to control exposure to enzymes in other industries or to control exposure to other industrial allergens. We chose to publish at this time because we believe enzyme use will increase in the future. The information contained here could potentially help new users ensure safe deployment of enzyme technology. Also, we have recently acquired new knowledge to improve our management system: this knowledge could help current enzyme users. We offer two new approaches that greatly increase worker participation and also improve understanding of the production process. We also highlight limitations of traditional air sampling. The new approaches are semi-quantitative tools to detect, rank, and control sources of dust release and product spillage. Our main conclusions are that detergent enzymes can be handled safely in the workplace provided there is no visible dust or spillage. Finally, we highlight the value of prick testing as a safe, simple tool to objectively asses the effectiveness of a hygiene program and to provide an "early alert" in the event of outages in the management system.

The world wide problem of occupational asthma

The world wide problem of occupational asthma

Influence of MHC background on the antibody response to detergent enzymes in the mouse intranasal test.

The mouse intranasal test (MINT) was developed to assess the immunogenic potential of detergent enzymes. The BDF1 mouse (H-2(b/d)), a cross of C57Bl/6 (H-2(b)) x DBA/2 (H-2(d)) has been used for most of the development work. Preliminary data in the CB6F1(H-2(d/b)), a cross of Balb/c (H-2(d)) x C57Bl/6 showed that this strain was similar to the BDF1 in its response to enzymes. These data also showed that the parental strains responded differently to the enzymes. To understand better the influence the major histocompatibility complex (MHC) background has on immune responses to enzymes, 3 different enzymes were tested in 4 inbred strains (C57Bl/6, DBA/2, Balb/c, and C57Bl/10), 2 hybrid strains (BDF1 and CB6F1), and 2 congenic strains (Balb.B10 and B10.D2). BDF1 mice rank enzymes the same as the guinea pig, which in turn correlates with sensitization in occupationally exposed humans. The ranking is based upon the dose of enzyme needed to give one-half maximal IgG1 antibody response (ED(50)) where Termamyl is more potent than Alcalase, which is equipotent to Savinase. The H-2(d) strains ranked the enzymes the same as the BDF1 but generated ED(50)s for the proteases that were one order of magnitude greater than the BDF1 ED(50)s. The response to Termamyl was the same in the two F1 strains and the H-2(d) strains. The H-2(b) strains did not rank the enzymes the same as BDF1 but the ED(50)s for the proteases were similar to the ED(50)s in the F1 strains. The response to Termamyl in the H-2(b) strains was lower than the response in the F1 and H-2(d) strains. Initial data show that the inbred strains will make enzyme-specific IgE antibody to high doses of enzyme with DBA/2 > Balb/c > C57Bl/6 in terms of the robustness of the response. The IgG1 responses in the congenic strains were similar to the responses in the H-2 matched strains. In addition, the antibody response to enzymes was consistent regardless of the source of BDF1 mice. The responses to these enzymes are clearly MHC linked with a role for Class II I-E molecules indicated. The current data strongly support the use of the F1 hybrid as an appropriate strain for evaluating allergic responses to enzymes.

Lipase from Ralstonia pickettii as an additive in laundry detergent formulations

An ideal detergent enzyme should be stable at high pH and temperature, withstand oxidising and chelating agents, be effective at low concentrations and have a broad substrate specificity. Studies have been carried out to find the efficacy of removal of triglyceride soil in laundry by lipase from Ralstonia pickettii with or without detergents. Important parameters viz. lipase and detergent concentration, time and temperature have been studied. Under optimal conditions, lipase in the presence of detergent increased the removal of oil by 24–27% over treatment with detergent alone.

Ultrafiltration performance of heat-treated shamouti orange [Citrus sinensis (L.) Osbeck] juice.

During ultrafiltration of orange juice with inorganic membranes, heating of the juice prior to the filtration experiment resulted in a significant increase of the fouling indices. The effect of the irreversible fouling (Rif) was always high, whereas the reversible fouling (Rrf) depended on the treatment. It was clearly seen that fouling was reduced after pectin degradation, but the heat treatment applied to the juice before filtration still resulted in reduced fluxes. It is suggested that pectins and proteins that undergo flocculation/coagulation during the heat treatment tend to interact with the membrane-filtering layer and to cause reduction of permeation flux. To clean the membrane to restore its pure water flux, close to the initial one, a proteolitic enzyme detergent wash was needed.

The role of enzymes in modern detergency

AbstractEnzymes have effectively assisted the development and improvement of modern household and industrial detergents. The major classes of detergent enzymes—proteases, lipases, amylases, and cellulases—each provide specific benefits for application in laundry and automatic dishwashing. Historically, proteases were first to be used extensively in laundry detergents. In addition to raising the level of cleaning, they have also provided environmental benefits by reducing energy consumption through shorter washing times, lower washing temperatures, and reduced water consumption. Today proteases are joined by lipases and amylases in improving detergent efficacy especially for household laundering at lower temperatures and, in industrial cleaning operations, at lower pH levels. Cellulases contribute to overall fabric care by rejuvenating or maintaining the new appearance of washed garments. Enzymes are produced by fermentation technologies that utilize renewable resources.

Alkaline detergent enzymes from alkaliphiles: enzymatic properties, genetics, and structures.

The cleaning power of detergents seems to have peaked; all detergents contain similar ingredients and are based on similar detergency mechanisms. To improve detergency, modern types of heavy-duty powder detergents and automatic dishwasher detergents usually contain one or more enzymes, such as protease, amylase, cellulase, and lipase. Alkaliphilic Bacillus strains are often good sources of alkaline extracellular enzymes, the properties of which fulfil the essential requirements for enzymes to be used in detergents. We have isolated numbers of alkaliphilic Bacillus that produce such alkaline detergent enzymes, including cellulase (CMCase), protease, alpha-amylase, and debranching enzymes, and have succeeded in large-scale industrial production of some of these enzymes. Here, we describe the enzymatic properties, genetics, and structures of the detergent enzymes that we have developed.

Use of the Mouse Intranasal Test (MINT) to Determine the Allergenic Potency of Detergent Enzymes: Comparison to the Guinea Pig Intratracheal (GPIT) Test,

A mouse intranasal test (MINT) was developed to determine the relative allergenicity of detergent enzymes. In this simple method, various doses of the enzymes are administered in a detergent matrix, via intranasal instillation, on days 1, 3, and 10, with serum samples collected on day 15. The sera are then analyzed for enzyme specific IgG1 antibody by an antigen specific enzyme immunoassay. The protease enzyme Alcalase (protease Subtilisin Carlsberg) has been used as a benchmark enzyme for development and characterization of the model. The objective of the current studies was to obtain potency comparisons with other protease and nonprotease enzymes and to begin to assess the validity of the model by comparison with potency determinations obtained with the guinea pig intratracheal (GPIT) test. The range of potencies detected among several enzymes of different classes was approximately 60-fold (compared with Alcalase). Modification of the dosing regimen to permit slightly more extended dosing did not change the relative potency determination. Comparison of data from the MINT and GPIT methods indicate that both the mouse and the guinea pig recognize the bacterial amylase Termamyl and a fungal exocellulase as more potent than Alcalase, a serine protease (Subtilisin B) and a fungal alpha-amylase (Fungamyl) as less potent than Alcalase, and the serine protease, Savinase, as equivalent to Alcalase. Also, these data are in alignment with clinical data on the prevalence of occupational enzyme sensitization. Given the simplicity and low cost of the MINT method compared with the GPIT test, these results support continued development of the method as an alternative approach for assessing the allergenicity of enzymes.

Direct Evidence for the Involvement of Two Glucose 6-Phosphate-binding Sites in the Glucose-6-phosphatase Activity of Intact Liver Microsomes: CHARACTERIZATION OF T1, THE MICROSOMAL GLUCOSE 6-PHOSPHATE TRANSPORT PROTEIN BY A DIRECT BINDING ASSAY

S 5627 is a synthetic analogue of chlorogenic acid. S 5627 is a potent linear competitive inhibitor of glucose 6-phosphate (Glc-6-P) hydrolysis by intact microsomes (Ki = 41 nM) but is without effect on the enzyme in detergent- or NH4OH-disrupted microsomes. 3H-S 5627 was synthesized and used as a ligand in binding studies directed at characterizing T1, the Glc-6-P transporter. Binding was evaluated using Ca2+-aggregated microsomes, which can be sedimented at low g forces. Aside from a modest reduction in K values for both substrate and S 5627, Ca2+ aggregation had no effect on glucose-6-phosphatase (Glc-6-Pase). Scatchard plots of binding data are readily fit to a simple "two-site" model, with Kd = 21 nM for the high affinity site and Kd = 2 microM for the low affinity site. Binding to the high affinity site was competitively blocked by Glc-6-P (Ki = 9 microM), whereas binding was unaffected by mannose-6-phosphate, Pi, and PPi and only modestly depressed by 2-deoxy-D-glucose 6-phosphate, a poor substrate for Glc-6-Pase in intact microsomes. Thus the high affinity 3H-S 5627 binding site fits the criteria for T1. Permeabilization of the membrane with 0.3% (3-[(chloramidopropyl)-dimethylammonio]-1-propanesulfonate) activated Glc-6-Pase and broadened its substrate specificity, but it did not significantly alter the binding of 3H-S 5627 to the high affinity sites or the ability of Glc-6-P to block binding. These data demonstrate unequivocally that two independent Glc-6-P binding sites are involved in the hydrolysis of Glc-6-P by intact microsomes. The present findings are the strongest and most direct evidence to date against the notion that the substrate specificity and the intrinsic activity of Glc-6-Pase in native membranes are determined by specific conformational constraints imposed on the enzyme protein. These data constitute compelling evidence for the role of T1 in Glc-6-Pase activity.