Subtilisin is a monomeric protein serine protease, which is produced by Gram positive bacteria and fungi.(1) Serine proteases are a subgroup of carbonyl hydrolase. These carbonyl hydrolases can be naturally occurring or recombinant. Naturally occurring carbonyl hydrolases consists of subtilisin matalloproteases, serine carboxypeptidase and thiol proteinase. In case of recombinant carbonyl hydrolase, the sequence coding for the wild type enzyme is altered to produce a mutant either by insertion, substitution or deletion of amino acid. Proteases are a diverse class of enzymes having several biological functions and specificities. The catalytic machinery of these enzymes is attributed to subtilisin and mammalian chymotrypsin related bacterial serine protease.(2) They are responsible for cleaving peptide bonds found in proteins. The proteases play an important role in cell wall turnover and are maximally expressed in the stationary phase. Serine proteases have molecular weights ranging from 25,000-30,000. They are inhibited by diidopropylfluorophosphate but are resistant to EDTA ( ethylene diamaine tetra acetic acid.) (3)
The amino acid sequences of several subtilisins like subtilisin Carlsberg, subtilisin BPN, and subtilisin DY are known. Subtilisin BPN is a 275 amino acid serine protease, which is secreted by Bacillus amyloliquefaciens. This enzyme is industrially important and its gene has been cloned and expressed in Bacillus subtilis. This molecule has two enzyme binding sites- one of the site binds weakly to cations and the other one binds calcium with high affinity. These calcium binding sites are common characteristics of microbial proteases since they contribute towards kinetic and thermodynamic stability. Subtilisin is majorly used in industries wherein the environment contains high levels of metal chelators which remove the calcium from subtilisin , thus reducing its stability. It would be highly beneficial to create a stable subtilisin which would be independent of calcium. These mutated subtilisin enzymes will have an enhanced stability even in the presence of detergents and chaotropic agents. X-ray crystallographic analysis of mutants revealed that the conformational changes due to the mutations are localized, with very less distortion of the backbone structure. Thus a large increase in stability can be obtained with very minor changes in the protein structure. Mutants can be created by deletion, addition or substitution of amino acids at positions 75-83. The deletion of amino acids 75-83 has shown to eliminate the calcium binding site while still retaining its enzymatic activity. However, calcium free subtilisins are susceptible to proteolysis. This can be overcome by converting the active site serine 221 to cysteine (S221C). This allows the protein to fold without any auto-degradation by proteolysis. A recombinant DNA was created which encoded an active subtilisin protein without the ability to bind to calcium. The protein had no codons which specifies amino acids 75-83 and had certain additional stabilizing mutations at the N-terminal amino acids 1-8 or the ? helix amino acids 70-74 or the helix turn amino acids 84-89. Suitable host cells were transformed with an expression vector containing this DNA segment. Upon culturing the host cells the enzymatically active subtilisin mutant was expressed and recovered.(1)
Fabric cleaning compositions containing subtilisin BPN mutants
The ability to hydrolyze proteins can be exploited by incorporating these proteases as an additive to laundry detergents. Most of the stains on clothes are proteinaceous and these enzymes can efficiently aid in their removal. The amino acid sequence of the protease is responsible for its characteristics. The subtilisin BPN variants have modifications at 2-3 amino acid positions. This variant has an increased hydrolytic power and decreased adsorption to an insoluble substrate as compared to the original subtilisin. A decreased adsorption to the substrate results in a better cleaning performance.
In subtilisin BPN, the amino acids ranging from position 199-220 form a large exterior loop on the molecule. This loop plays an important role in mediating the adsorption of enzyme to the surface bound peptide and mutations in this region will affect the adsorption. The substituting amino acids in case of a mutation are either neutral or negatively charged. The substituting amino acid for position 199 is mostly His, Ala, Cys, Pro, Glu, Asp, Gly, Asn or Gln and for position 207 is Glu or Asp. The cleaning compositions comprise of 0.001-1% of one or more enzyme variants. The enzyme variants can be used along with other conventional ingredients to produce efficient fabric laundry composition. These fabric cleaning compositions also contain various zwitterionic or anionic surfactants, hydrotopes, dyes or pigments, primary or secondary alcohols, chelating agents and brighteners.
The Bacillus subtilis cells having the subtilisin mutant are allowed to grow till mid log phase in LB glucose broth and is then inoculated into a fermentor.
The cells are grown overnight at 37EsC. Chloramphenicol is added for the antibiotic selection of mutagenized plasmid.
The cells are removed from the broth by centrifugation
The enzyme is purified by affinity adsorption or cation exchange chromatography.
The active enzyme concentration is determined by the pNA assay. ( para nitro anilide) This assay measures the rate at which pNA is released as the enzyme hydrolyses the substrate. The rate at which the yellow colour is produced is measured at 410nm with the help of a spectrophotometer and is directly proportional to the enzyme concentration. The total protein concentration can also be estimated by determining the absorbance at 280nm. (4)
Production of subtilisin variants
Techniques used in microbiology, molecular biology, protein purification and recombinant DNA technology are all used in developing a subtilisin variant, which is a part of dish washing and fabric cleaning compositions.
Cassette mutagenesis method for production of protease variants
The gene encoding the protease is sequenced
The sequence is screened for a point at which desired mutation can be made
The sequences flanking this region are checked for the presence of restriction enzyme site so as to replace a short segment of the gene with an oligonucleotide which will then encode various mutants.
The gene is mutated by primer extension
The oligonucleotides are synthesized to have the same restriction sites, eliminating the need for synthetic linkers to create the restriction site. The genes coding for serine proteases of B.amyloliquefaciens, B.subtilis and B.licheniformis can be used as targets for mutagenesis. The gene can be inserted into a suitable vector and introduced into a host strain ( Bacillus PB92) for expression and production of mutant proteases.
These mutations / substitutions enhance the performance and stability of subtilisin in detergent compositions. These serine proteases can be used in the form of granules and liquid composition both in laundry dishwashing and cosmetic applications. These enzymes are often in the form of encapsulated particles in order to protect it from the other components. Encapsulation also improves the enzyme performance and helps in regulating its availability. The encapsulating material can be derived from carbohydrates, silicates, polyvinyl alcohol, borates, PEG or paraffin waxes.
Fabric cleaning performance by Blood Milk Ink microswatch assay. (BWI)
This assay is performed on a microtitre plate. Samples of the subtilisin variants and reference subtilisin are obtained from filtered culture broths. 10ul samples of the enzyme are added to the BWI swatch plate along with 90ul of working detergent solution. The plates are incubated for 30 minutes. 100ul of the supernatant is transferred into a new microtiter plate and its absorbance is measured. Control wells contain the detergent solution without the protease sample. The measurement at 450nm tracks pigment removal and at 600nm tracks turbidity and cleaning. The performance index (PI) of the variant is calculated. PI compares the performance of variant and reference enzyme at the same protein concentration.
PI>1 the variant is better than the wild type.
PI=1 variant and standard have the same performance
PI<1 the standard performs better than the variant (5)
Producing mutations in subtilisin genes
Cloning the subtilisin gene
The gene for subtilisin is cloned from any fungus or Gram positive bacteria. The subtilisin producing clones can be identified by introducing fragments of genomic DNA into an expression vector. They are then used to transform protease negative bacteria. The cells are plated on a media containing skim milk ( substrate for subtilisin.) The colonies producing subtilisin will be surrounded by clear agar due to the digestion of the skim milk by the secreted subtilisin.
Generation of random mutations
Mutations can be introduced into the gene by nitrous acid, sodium bisulphite or by replicating in a mutator strain of E.coli. In mutagenesis by nitrous acid, the coding region of subtilisin gene is cloned in M13 phage. A single stranded phage DNA is prepared and made to react with 1M nitrous acid ( pH 4.3 ) for around 15-60 minutes at 23EsC or 1-5 minutes with 2.4 M formic acid at 23EsC. After mutagenesis, a primer is annealed to the DNA and the double stranded molecule is synthesized.
Expression of the mutants
The mutated gene can be expressed in an expression vector.
Screening of mutants
The transformed B.subtilis are cultivated in the presence of a nitrocellulose filter paper. The secreted enzyme binds to it and can be identified by subjecting it to certain conditions which will help in differentiating the expression of the mutant product from the wild type product. For instance, the product can be treated under conditions that would inactivate the wild type enzyme. Therefore a preserved enzyme activity after this treatment indicates that the mutation conferred an enhanced stability on the enzyme. (3)
Mutant type subtilisin YaB and its applications
The subtilisin YaB mutanats are created by substituting the glycine residues at positions 124, 151 and 159 with other amino acids by site directed mutagenesis. The mutants have a higher casein hydrolyzing activity and an improved substrate specificity. They can be used for quality improvement of meat and other foodstuff. The ale gene encoding subtilisin YaB is carried by an expression vector like pUC, pBR or pET. Site directed mutagenesis of this sequence is carried out by PCR technique. The initial codon of ale was mutated from TTG to ATG. The plasmid with the wild type and mutant ale genes are then used to transform suitable host cells like Bacillus, E. coli or S. cerevisiae and expressed. The protein is isolated and purified by acetone precipitation or histamine affinity chromatography.
Elastin is a protein which is present in the arteris and dermis of vertebrates. It is insoluble in water and is difficult to hydrolyze by ordinary proteases. Subtilisin YaB, originally known as alkaline elastase, is an extracellular protease secreted by Bacillus YaB. Bacillus YaB is an aerobic, rod shaped Gram positive bacterium. It is highly hydrolytic for collagen and elastin. At a pH lower than its isoelectric point (10.6) , it has high binding affinity to elastin. The enzyme has substrate specificity for small amino acids like glycine and alanine which constitute 50-60% of the elastin protein. The optimal reaction pH of the enzyme is 11.75, whereas that of the other subtilisins is around 9-10.5. Meat like pork, beef, chicken consists of connective tissues containing collagen and elastin. Meat tenderizers which are commercially available contain plant proteases like papain. However the substrate specificity of papain is very low and proteins like myosin and actin can also get hydrolyzed which will detoriate the taste of the meat. Subtilisin YaB mutants efficiently hydrolyze elastin and collagen and retains the good taste. (6)
Other applications of mutant proteases
Mutant proteases have important features that make them suitable for several applications. Enhanced oxidative stability, thermal stability and chelating stability are certain properties which make them ideal for use in cleaning compositions. They are also used for cleaning contact lenses. They are utilized for waste treatment, peptide hydrolysis, cleaning of medical devices, several textile applications and in cleaning compositions for oral care, hair and skin care. Proteases are part of the composition used for treatment of wool or silk. The wool fibres or animal hair are subjected to pre-treatment with these proteolytic enzymes for enhancing their overall properties which is then followed by the plasma treatment. The mutant proteases are used for processing skin or hides into leather. Hides and skins are brought to the tanneries in the form of dry or salted hides. The processing involves several steps like soaking, unhairing and bating. The enzymatic treatment with protease can be done anytime during these steps. (7)