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(genetically unmodified protein) are the safest choice [57, 97, 98].

      2.3.3 Engineered Proteins

      2.3.3.1 Technical Enzymes: e.g. Proteases and Lipases

      The demand for technical enzymes corresponded to a market size equal to 1 billion USD in 1999 [100]. Some of these enzymes are the thermostable enzymes, which are well represented in different industrial processes and constitute more than 65% of the worldwide market [101]. Enzymes were implemented in many important industrial products and applications such as in the paper industry, detergents, drugs, degradation of different wastes, textiles, food, pharmaceuticals, leather, degumming of silk goods, manufacture of liquid glue, cosmetics, meat tenderization, cheese production, growth promoters, etc. Enzymes used with detergent are the most important and profitable applications with a market size equal to 0.6 billion USD in 2000 (Novozymes data) [100]. The first use of enzymes in detergents occurred in 1913 when Röhm and Haas introduced crude trypsin into their detergent Burnus based on a German patent issued to Otto Röhm (1913) [100]. Enzymes used with detergent must be stable and function well in the presence of a variety of potentially unfriendly detergent ingredients (e.g., anionic/ non-ionic/cationic surfactants, chelators (e.g. EDTA), builders, polymers, bleaches) and in various forms of detergent products (i.e., liquids and powders) [100]. Thermostable enzymes are active and stable at temperatures higher than optimal growth of their producer strains. Bacilli strains isolated from diverse sources with diverse properties have made these organisms the focus of attention in biotechnology. Thermostable enzymes can be produced by both thermophilic and mesophilic microbes. The use of high temperature has many significant applications due to solubility and reducing viscosity [102, 103].

       2.3.3.1.1 Proteases

      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

      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

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