which form a system of scaffolding that maintains cell shape. Other proteins are important in cell signaling, immune responses, cell adhesion, and the cell cycle [57].
Proteins are natural chains of amino acids joined by amide linkages. They are degraded by enzymes (proteases). For thousands of years people used natural proteins such as wool [58], silk [45] and hair (keratin) for clothes, decoration or to display their wealth. After understanding their composition, they were reformulated and their properties changed to match certain demands. Numerous proteins were studied for developing natural bio-based materials such as keratin, collagen, albumin, gelatin, and fibroin [59]. They are degraded by enzymes. Nowaday some old techniques are still used to produce products from some types protein–polymer such as wool [60], silk [61–64], and hair [65–67]. However, some modern applications have been invented such as the use of Keratin [68–70] in hydrogel. Silk is used in tissue engineering, in drug delivery for musculoskeletal therapeutics. The first industrial applications of protein as polymer were in the early 1930s and 1940s with casein and with soy protein. Protein biopolymers can be classified with animal proteins (e.g. casein [71–75], whey [76–79], keratin [58, 68, 80, 81], collagen [82–85] and gelatine [86], polyglutamic) and in plant proteins (wheat, corn, soy, pea and potato proteins) and microbial protein such as polyglutamic [87–89], cyanophycin [90–93] protein biopolymers remained present in some niche markets such as encapsulates (pharmaceutical), coatings (food industry), adhesives or surfactants. They are used in the packaging industry for breweries, wineries and essential oil composite film for refrigerated products; microcapsules based on biodegradable polymers [72, 90, 93]. Protein biopolymers remain in some niche markets such as encapsulates (pharmaceutical), coatings (food industry), adhesives or surfactants. They are used in the packaging industry for breweries, wineries and refrigerated products essential oil composite film, microcapsules based on biodegradable polymers [57, 72].
Mammalians are not able to produce all 20 amino acids (essential amino acids). They obtain those essential amino acids through their diet. The first protein to be fully sequenced was insulin, by Frederick Sanger, who won the Nobel Prize for this achievement in 1958. It is important to highlight that Sanger succeed in solving the insulin sequence using insulin itself rather using the related genes. It was a complicated process especially in the presence of the disulfide bond between A and B fragments. Sanger discovered early the importance of the 3D structure of the protein. The first protein structures to be solved using data obtained from x-ray diffraction analysis included hemoglobin and myoglobin, by Max Perutz and Sir John Cowdery Kendrew, respectively, in 1958 [94, 95]. The 3D structures of both proteins were determined; Perutz and Kendrew shared the 1962 Nobel Prize in Chemistry for these discoveries. Rapid advances in site-directed mutagenesis and total gene synthesis combined with new expression systems in prokaryotic and eukaryotic cells have provided the molecular biologist with tools for modification of existing proteins to improve catalytic activity, stability and selectivity, for construction of chimeric molecules and for synthesis of completely novel molecules that may be endowed with some useful activity. The results are used in the improvement of the design by using knowledge-based procedures that exploit facts, rules and observations about proteins of known 3D structure [96].
2.3.2 The Biology of the Protein
Most of organic compounds have applications that were successfully synthesized chemically after solving their structures. Even though the basic concept of the protein structure and composition was solved, proteins resist chemical synthesis, this is due mainly to its long variant polymeric chain. Proteins cannot be synthesized by organic chemists in large quantities and they cannot be manipulated in vitro to modify single amino acids in a protein and leave all other amino acids of that variety unchanged. However, the majority of proteins can be manipulated using in vivo genetic engineering. Once a gene coding for a protein has been cloned from the original wild-type genome into a vector it can be manipulated by using synthetic oligonucleotides to produce site specific mutations in the cloned material. This is specific and can alter any side chain of a particular amino acid to any other of the 20 naturally occurring amino acids. The technique of site-directed mutagenesis can alter any number of amino acids in a protein and can be used to build proteins from scratch. The position of the amino acid is decided by inspection of the tertiary structure of the primary structure (using conserved amino acids and site directed mutagenesis experiments) and the tertiary structure of the protein (using protein modeling), and the interaction of the amino acid with the substrate or with other parts of the main protein is evaluated mathematically. Conserved amino acids can be determined from the protein primary structure using alignment. Conserved amino acids are usually responsible for important functions. It is, of course, necessary to have a reasonable idea of what property one is trying to enhance in the target protein. Wrong manipulation of protein could lead to fatal problems. Proteins produced through biological systems (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].
