Savinase

Further Reading

Maxatase k204

Alkalase k205

Code: k206
Latin name: Bacillus spp
Common names: Savinase
Savinase is an enzyme produced by Bacillus amyloliquefaciens.
 
Occupational
An occupational allergen, which may frequently induce hayfever, asthma and conjunctivitis in sensitised individuals.

Allergen Exposure

Geographical distribution
Microbial alkaline proteases lead the worldwide enzyme market and are enormously important to the detergent industry. Although protease production is an inherent property of all organisms, only those microbes that produce a substantial amount of extracellular protease have been exploited commercially. Of these, strains of Bacillus dominate the industrial sector.
 
Subtilisins are extracellular alkaline serine proteases, which catalyse the hydrolysis of proteins and peptide amides. Savinase is one of these enzymes; Alcalase, Esperase and Maxatase are others. (These are all trade names).
 
These enzymes are all produced using species of Bacillus. Maxatase and Alcalase (which have sometimes been considered virtually interchangeable) come from B. licheniformis, Esperase from an alkalophilic strain of a B. licheniformis, and Savinase from an alkalophilic strain of B. amyloliquefaciens (a mistaken attribution is often to B. subtilis, sometimes to B. lentus) (1). Alcalase and Maxatase are usually found in washing detergents recommended for use at 10 to 65°C and pH 7 to 10.5. Savinase and Esperase may be used at up to pH 11 and 12, respectively. The structure of Savinase is very similar to those of homologous Bacillus subtilisins (1).
 
Other enzymes used in washing detergents are the a-amylase enzymes, e.g., Termamyl, an enzyme also derived from B. licheniformis. This enzyme is also used in the production of glucose syrups. The a-amylase enzymes are particularly useful in dishwashing and de-starching detergents.
 
Mutations have led to newer protease preparations with improved catalytic efficiency and better stability against temperature, oxidizing agents and other changing wash conditions. Many newer preparations, such as Durazym, Maxapem and Purafect, have been produced (2).
 
B. amyloliquefaciens is used in the commercial biosynthesis of an a-amylase that is more thermally stable than the wild-type enzyme. B. lentus which is also used in the commercial biosynthesis of a subtilisin protease. The microorganisms are removed from the final enzyme products, which, again, are usually used in detergents.
 
All of these enzymes are high-molecular-weight antigens and have been implicated in allergic rhinitis and asthma.
 
Environment
Savinase is used in washing powders and dishwasher powders and removes stains that are protein-based, such as egg, gravy, blood and grass. These stains are often difficult to remove, for they are insoluble in water and stick strongly to clothes and crockery. Savinase, an enzyme, hydrolyses the proteins to peptides which are soluble in water.
 
Early attempts to use proteases foundered because of producers and users developing hypersensitivity. This was combated by developing dust-free granulates (about 0.5 mm in diameter) in which the enzyme is encased in an inner core; the granulates contain inorganic salts (e.g., NaCI) and sugars as preservatives, and are bound with reinforcing fibres of carboxymethyl cellulose or a similar protective colloid. This core is coated with inert waxy materials such as paraffin oil or polyethylene glycol, plus various hydrophilic binders, which later disperse in the wash. This combination of materials both prevents dust formation and protects the enzymes against damage by other detergent components during storage. In addition to the granulated forms of enzymes intended for use in detergent powders, liquid preparations in water solution and slurries of enzyme in a non-ionic surfactant are available as active ingredients of liquid stain-removing concentrates, used for removing stubborn stains. Some preparations contain both Termamyl and Alcalase (3).
 
Secretion of subtilisin is associated with onset of sporulation, and many mutations which block sporulation at early stages affect expression levels of subtilisin. However, subtilisin is not necessary for normal sporulation.
 
Unexpected exposure
See under Geographical distribution and Environment.
 
Allergens
No allergenic epitopes from this substance have yet been characterised.
 
Antigenic and allergenic characteristics of the enzymes Alcalase and Savinase and their potential cross-reactivity were evaluated using crossed immunoelectrophoresis and crossed radioimmuno-electrophoresis. Alcalase exhibited 2 distinct antigens, 1 electropositive and 1 electronegative. Savinase exhibited 1 electropositive and 2 electronegative antigens. The antigens of the 2 enzymes were clearly different from each other, the 3 Savinase antigens exhibiting greater electrophoretic mobility than the 2 Alcalase antigens. In crossed radioimmunoelectrophoresis studies, only the electropositive antigen of Alcalase, its retrograde complex, and the electropositive antigen of Savinase bound IgE from the sera of individuals who were skin test-positive to one or both enzymes (4).

Potential Cross-Reactivity

In a group of detergent enzyme workers with known exposure to Alcalase/Maxatase, sensitisation to Savinase, to which there was no previous occupational exposure, was demonstrated by skin-specific IgE. The researchers attributed this to either cross-reactivity between these enzymes or to foreign enzyme contaminants contained in the Savinase antigen (5).
 
No evidence of cross-reactivity was observed between Alcalase and Savinase in heterologous and tandem crossed immunoelectrophoresis studies and heterologous microimmunodiffusion reactions (4).

Clinical Experience

IgE-mediated reactions
Since the 1960s an increasing number of occupational allergies against natural and modified recombinant enzymes has been reported. Subtilisins have been shown to cause allergic reactions, in particular airway sensitisation. Symptoms, results of skin prick tests, detection of specific IgE antibodies, and results of specific bronchoprovocation tests have confirmed an immunologic mechanism for these high-molecular-weight antigens (6-15). Those affected are mainly workers in the detergent industry rather than consumers (16-20).
 
Occupational allergy to enzyme detergents has almost disappeared due to the use of preventive measures, both individually and in industries. Nonetheless, incomplete application of preventive measures within a factory may result in new instances of sensitisation and allergic disease (14).
 
A close relationship has been described between bronchial hyperresponsiveness and the provocation dose of an enzyme. Researchers have reported that bronchopulmonary testing with enzyme solutions is highly sensitive and specific in confirming the diagnosis of occupational asthma due to these proteolytic enzymes (15). Specific IgE tests have also been reported to be very useful in the diagnosis of sensitisation to these enzymes (16).
 
Not all the enzymes are equipotent in inducing allergic sensitisation. In guinea pig models, Termamyl was about 10-fold more potent than Alcalase/Maxatase, and the protease subtilisin B was shown to be less potent than these. Savinase was shown to be equivalent in potency to Alcalase/Maxatase. Prospective evaluation of skin-specific IgE tests of factory workers showed that sensitisations to Termamyl and Savinase were similar to sensitisations to Alcalase. The sensitisations to subtilisin B were weaker than those to Alcalase (21).
 
Sixty-five workers in a detergent industry were divided into 4 groups according to their prick test results, as follows: those positive for at least 1 enzyme (Lipolase, Savinase) (group I, n = 15); enzyme-negative cases (group II, n = 50); and from among these, enzyme-positive non-smoking cases (group Ia, n = 11); and enzyme-negative non-smoking cases (group IIa, n = 32). The observation that the FVC and FEV(1) of the workers were much lower than expected, especially among those sensitive to the enzymes, independently of occupational asthma and cigarette smoking, led to the conclusion that the cause might be sensitivity to the enzymes (20).
 
Eight new cases of asthma in a detergent factory led to a prospective clinical and aetiological enquiry. Sensitisation to the enzymes used (Savinase, Maxatase and Biozym P 300 S) was shown by skin tests (8 positive to all the enzymes), by the level of specific IgE to Rast (8 times greater than class 3 for Savinase and Maxitase), and by HBDT for Maxatase and Biozym P 300 S (greater than 50% in 8 patients). The incomplete application of preventive measures within the factory did not seem to completely explain the origin of this sensitisation. The study of the morphology of the enzyme grains that had been introduced into the factory helped to explain the mechanism. The capsules of the grain were fissured, allowing allergen contact. The replacement of these grains with others whose envelope was intact led to the disappearance of the symptoms and confirmed the hypothesis (14).
 
Other reactions
A 53-year-old woman developed respiratory symptoms while working with a cleaner containing subtilisins. Her symptoms intensified in the work environment and improved away from work. Computed tomography demonstrated alveolar and interstitial infiltrates with subsequent scarring. Pulmonary function showed a restrictive pattern with diminished diffusion capacity. Bronchoalveolar lavage showed lymphocytosis, and all cultures were negative. Precipitating antibodies to the enzyme were found in the patient's serum. These findings supported a diagnosis of extrinsic allergic alveolitis from the enzyme contained in the cleaner (22).
 
Compiled by Dr Harris Steinman, harris@zingsolutions.com

References

  1. Betzel C, Klupsch S, Papendorf G, Hastrup S, Branner S, Wilson KS. Crystal structure of the alkaline proteinase Savinase from Bacillus lentus at 1.4 A resolution. J Mol Biol 1992 Jan 20;223(2):427-45.
  2. Gupta R, Beg QK, Lorenz P. Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol. 2002;59(1):15-32.
  3. Chaplin M. The use of enzymes in detergents. London South Bank University. www.lsbu.ac.uk/biology/enztech/detergent.html 2004: December
  4. Arlian LG, Vyszenski-Moher DL, Merski JA, Ritz HL, Nusair TL, Wilson ER. Antigenic and allergenic characterization of the enzymes alcalase and savinase by crossed immunoelectrophoresis and crossed radioimmunoelectrophoresis. Int Arch Allergy Appl Immunol 1990;91(3):278-84.
  5. Bernstein DI, Bernstein IL, Gaines WG Jr, Stauder T, Wilson ER. Characterization of skin prick testing responses for detecting sensitization to detergent enzymes at extreme dilutions: inability of the RAST to detect lightly sensitized individuals. J Allergy Clin Immunol. 1994;94(3 Pt 1):498-507
  6. Reinheimer W, Utz G. Allergic asthma due to the detergent additive maxatase. [German] Dtsch Med Wochenschr. 1971 Feb 5;96(6):246-7

 

As in all diagnostic testing, the diagnosis is made by the physican based on both test results and the patient history.