(genetically unmodified protein) are the safest choice [57, 97, 98].
There are many concepts controlling the uses of protein in medicinal applications such as is purity. In technical applications (e.g., technical enzyme) the boundaries of purity are different. Regarding activity and stability, the protein must match perfectly the purpose of its use. The importance of protein engineering in industry continues to grow as the number of applications of proteins expands, and the technology used to discover proteins efficiently with useful properties is better able to address industrially relevant problems. Recent advances in directed evolution are implemented in many established industrial laboratories as well as in start-up companies, augmenting the rational design approach. Additionally, organisms from extreme environments are becoming an important source of new backbones for engineering proteins with significantly different properties. The successfully engineered protein generally requires a proper combination of properties. For example, a detergent protease would require, at minimum, stability in the presence of detergent and activity against certain protein stains. Nevertheless, the control of a few basic properties is a recurring theme in many applications. Properties such as sufficient stability, high activity (in the case of enzymes), and the ability to interact correctly with surfaces are necessary for a variety of industrially important proteins [99].
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
Up to the 1980s, proteases were considered the only commercially relevant enzymes. Today, many laundry-detergent products contain at least a protease, and many contain cocktails of enzymes including proteases, amylases, cellulases, and lipases [100]. The major source of proteases 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