is microorganisms while proteases from plant origin have not been well investigated. Based on their catalytic mechanisms, proteases can be classified into Ser, Cys, Aspartic and metalloproteases. In nature, proteases have valuable biochemical and physiological functions. They can be very specific, not only cleaving proteins into amino acids or short peptides but also can cleaved specifically to produce useful peptides. Bacillus licheniformis, Bacillus subtilis and Bacillus pumilus are the most well-known species used in industry for alkaline protease production [104]. Proteases were used widely in many industrial applications included detergent, wool quality improvement, meat tenderization, leather, etc. Ideally, proteases used in detergent formulations should have high activity and stability within a broad range of pH and temperatures, and should be compatible with various detergent components along with oxidizing and sequestering agents [105].
Protein engineering was used to improve the stability of BPN’ from Bacillus amyfoliquefaciens in the chelating environment of the detergent by deleting the strong calcium-binding site (residues 75–83) and re-stabilizing the enzyme through interactions not involving metal ion binding. Stability increases of greater than 1000-fold in EDTA were reported for this protease [106]. The surface properties of BPN’ have also been engineered. It was found that variants containing mutations that produce negative charges in the active site region of the molecule adsorbed less strongly and gave better laundry performance.
2.3.3.1.2 Lipases
Lipases were characterized by their ability to hydrolyze long chain triglycerides [107]. Lipase catalyzes the hydrolysis (or synthesis) of insoluble esters. The primary use of lipase is in cleaning applications, although its use in the chiral synthesis of high value chemicals is also important. A comparison of the experimental results of several site-directed variants with structural modeling has provided much insight into the catalytic mechanism of a fungal lipase from Rhizopus oryzae at the molecular level [108]. In order to understand lipase activity fully one must also take into account its ability to interact with a macroscopic substrate, such as a triglyceride surface. Most lipases are activated at the oil(substrate)–water interface by a conformational change to adapt the enzyme–substrate interaction [109]. Changes at Glu87 and Trp89 were reported to alter activity of the lipase from Humicola lanuginosa (Lipolase) [110]. Surfactant and calcium sequestering agents, such as sodium tripolyphosphate, reduce the activity of current lipases 100–1000-fold in laundry detergents [111, 112]. Some progress in designing variants that reduce this inhibition by creating favorable surfactant–enzyme interactions were reported to give improved laundry performance. The commercial applications of lipases include, detergents such as in dishwashing, clearing of drains clogged by lipids in food processing or domestic/industrial effluent treatment plants [96].
2.3.3.2 Pharmaceutical Applications
The estimate of about 350 biotechnology drugs currently undergoing development, including vaccines, gene therapy, antisense technology and antibodies derived from “humanized” transgenic mice. Protein engineering was used to produce therapeutic proteins with improved properties such as increased solubility and stability. Many of the early protein drugs derived from biotechnology failed because they were primary molecules with suboptimal affinity or poor half-life in vivo, leading to poor efficacy. In other cases, many of the original protein drug molecules were non-human and caused immune responses against the drug itself. Affinity, half-life and dosing are all interrelated and play a role in determining the clinical efficacy and financial viability of protein-based drugs. This increased understanding of the issues affecting success in drug development was paralleled by increased capabilities in protein engineering and selection/screening technologies. These were used to improve the effectiveness of a number of protein drug candidates.
2.3.3.3 Reducing the Immunogenicity of Protein Drug Molecules
Many early attempts at introducing protein therapeutic molecules failed because the protein drug molecules were recognized as non-human and led to an immune response against the drug itself. As a result, most proteins used in clinical trials now are primarily human or are humanized, even if the original “proof of concept” work was done with non-human proteins. For example, Pulmozyme (Genentech) is a drug based on human DNAse which was developed for use in managing cystic fibrosis, following successful “proof of principle” studies with bovine pancreatic DNAse I [113]. The immunogenicity of mouse antibodies in humans was one of the major reasons why early monoclonal antibodies did not deliver the anticipated therapeutic benefits. This led to the development of chimaeric antibodies, created by fusing mouse variable domains to human constant domains to retain binding specificity while reducing the proportion of mouse sequence. TNFα-neutralizing chimaeric monoclonal antibody, was approved for use in treating Crohn’s disease and rheumatoid arthritis [114]. The reduction in monoclonal antibody immunogenicity was taken a stage further by complementarity-determining region (CDR) grafting, where the 34 CDRs of mouse antibodies were grafted onto human frameworks to reduce the proportion of mouse sequences in the drug still further while retaining binding specificity [115].
2.3.3.3.1 Insulin
Insulin was engineered through mutagenesis to create monomeric forms that are fast acting (insulin lispro and insulin aspart). Conversely, another form (insulin glargine) was created by mutagenesis to precipitate upon injection and give a sustained release of insulin. More research was done on insulin. Whittingham et al. 1997 reported a crystal structure of a prolonged-acting insulin with albumin-binding properties [116].
2.3.3.3.2 Catalytic Antibody
A catalytic antibody is a variant of an antibody. Antibodies are proteins that normally bind to a specific molecule but do not alter the bound molecule in any way. A catalytic antibody is one which was changed by mutations to have a novel sequence that folds into a structure that catalyzes a specific reaction (such as amide bond formation, ester hydrolysis, and decarboxylation). Catalytic antibodies function like enzymes, and are created to catalyze reactions for which there are no naturally occurring enzymes. Fifty or more different reactions have been catalyzed by the action of catalytic antibodies that were obtained individually by methods of protein engineering [117].
2.3.3.3.3 Polyketide Synthases
Antibiotics such as erythromycin are made by large multidomain proteins called polyketide synthases. Site-directed mutagenesis was used to modify the substrate specificity of one polyketide synthase reaction so that the product contains a malonate unit, whereas the product of the original enzyme contained a methylmalonate unit. In addition to site-directed mutagenesis, the order of the polyketide synthase domains was shuffled to create proteins that could catalyze the synthesis of new antibiotics. An extension of site-directed mutagenesis allows non-natural amino acids to be incorporated into proteins. Non-natural amino acids are not naturally encoded by the genome, but instead include a wide variety of amino acids that are present in cells or produced by synthetic methods [117].
2.3.4 Traditional Protein
2.3.4.1 Casein
Casein is a natural polymer extracted from skimmed milk proteins. Casein protein is used in many industrial and technical applications [71, 118, 119], such the manufacture of adhesives and the packaging industry for breweries, wineries and refrigerated products and it can also be used as a plasticizer for concrete. Casein is also used as microcapsules and in synthetic peptides [120]. Caseins evolved from members of a group of secreted calcium (phosphate)-binding phosphoproteins. The first industrial applications of protein as polymer were in the early 1930s and 1940s with casein and with soy protein. Casein is also used as microcapsules and in synthetic peptides [75, 121].
