Site Directed Mutagenesis of Subtilisin

Site Directed Mutagenesis of Subtilisin Introduction 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 f rom 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 ag ents. 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 ÃŽ ± 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. Fermentation: 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 37ËÅ ¡C. 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