Chapter and techniques to utilize and improve what
Chapter 1. Application Scope of Proteins and Existing Challenges Since the first time human beings learned how to use fire, how to build a house, how to hunt with tools, we never stop exploiting and changing the nature. Advances of modern science and technology offer us more and more knowledge and techniques to utilize and improve what nature presents us. Enzymes, as great gifts from nature, are catalysts bearing excellent properties (high activity, selectivity and specificity) that modulate the most complex chemical processes under the most benign conditions (room temperature, aqueous solution). They participate in every biological process, mediating various functions of living organisms, ranging from metabolism to neurotransmission, from photosynthesis to DNA synthesis. As our knowledge on the structure and function of enzymes accumulates, enzymes have been found useful beyond their intrinsic biological roles. The very initial application of enzymes may dated back to thousands of years ago, when man used naturally occurring microorganisms and the enzyme they produce to make food, such as bread, cheese, beer and wine. Today, besides their applications in food processing, enzymes are used in an increasing range of applications. In chemical industry, especially fine chemical syntheses, enzymatic catalysis is considered as a promising field due to benign reaction conditions, high chemo- and stereo- selectivity, and tolerance to substrate functional groups. In therapeutic applications, therapeutic enzymes have been used to treat various diseases, especially the ones caused by enzyme deficiency. In addition, enzymes also play significant roles in biosensing, bioremidiation, and so on. In a technical perspective, recent advancement in biotechnology have paved the way for the applications of enzymes. Recombinant DNA techniques, in particular, make possible the production of most enzymes at a commercially acceptable cost1,2. However, wide applications of enzymes are still facing other challenges. 1 Poor stability of proteins, especially in non-physiological environment, is one of the most critical obstacles for their wide applications3,4. Due to the fast denaturation and inactivation, enzymes commonly associate with low-temperature storage, short shelf life, sensitivity to heat and spectator chemicals, and vulnerability to protease digestion. Enzyme’s poor stability, therefore, largely limits the use of enzymes in almost every field of applications, including industrial catalysis, biopharmaceutical industry, protein therapeutics, environmental decontamination, and biosensor fabrication. In addition, native proteins may appear inadequate in front of the designed task they are not empowered to do by nature. For example, therapeutic use of protein has been largely limited by its low pharmacokinetic accessibility to intracellular drug target5. Various other properties might need to be introduced or improved to meet the demand of specific applications, such as increased hydrodynamic radius for long circulation time in vivo6,7, heterogeneous form to be recycled and reused in industrial catalysis8-10, altered substrate specificity for biosensor fabrication11,12. Great endeavors have been made by biologists, chemists, and material scientists to fit enzymes into a wide spectrum of applications. However, in addition to enhancing protein stability, these task- oriented properties are too diverse to be introduced in a universal approach. 1.1. Protein Immobilization In industry, the poor stability of enzymes is addressed by enzyme immobilization. Basically, enzyme immobilization can be dived into three main categories: immobilization to a support, entrapment and cross-linking8. Immobilization to a support involves binding of enzymes to a preformed matrix support via non-covalent interactions, such as hydrophobic interaction, electrostatic interaction, or covalent bonding13-15. The second approach is enzyme entrapment, in which enzymes are included into a gel matrix during the formation of the matrix10,16,17. Although 2 enzymes can be physically entrapped, additional chemical conjugation is usually required to prevent any enzyme leakage. As a more recent approach, cross-linking of enzyme aggregates or crystals without support matrix has been demonstrated to exhibit various extraordinary characteristics, such as effective enzyme stabilization, concentrated bioactivity in the catalyst, and low production costs 18-20. These immobilization strategies can improve the enzyme stability with various extents of successes. Some protocols in multipoint immobilization could result in impressive stabilizing effect, in many cases with stabilization factor as high as 1000–10,000-fold9. The high stabilization factors can be attributed to rigidification of enzyme 3D structures, thus reducing any conformational change involved in enzyme inactivation 21-23. In addition to enhanced stability, enzyme immobilization offers other advantages: (1) Immobilization technique makes possible the fabrication of heterogeneous enzyme catalysts10,18,24, enabling the separation and recycling of enzymes and therefore eliminating protein contamination of the product and reducing manufacture costs. (2) In the enzyme catalysis in organic solvents, immobilization technique significantly increases the solvent accessible surface of catalyst compared with traditional lyophilized powders18,25. (3) Immobilization technique provides a platform to incorporate multienzyme or chemoenzymatic cascade catalysts to overcome inactivation issues associated with their mutual interactions in free solution26-29. However, as enzyme immobilization technique is developed to meet the qualification of industrial biocatalysis, transferring this technology to other applications appears to be challenging. For example, most of the immobilized enzymes could not be applied into therapeutic applications due to the biocompatibility issues. 3 1.2. Protein Engineering In contrast to the enzyme immobilization technique chemists and material scientists developed, biologists take the route nature evolution takes to endow polyaminoacids with designed functions suitable for specific applications30,31. Via technologies such as site-directed mutagenesis 32,33 and DNA-shuffling 34,35, protein engineers could mimic and accelerate natural evolution to provide mutant enzymes tailored for applications in research. In addition, the ever-increasing use of computational simulation greatly aids the understanding and design of protein structures and properties 36-39. As a consequence, protein can be engineered to acquire a variety of improved properties, such as greater specificity 40,41, higher stereoselectivity 42-45, and altered pH optimum 46-48. In addition, mutagenesis can also be used to remove undesired properties of native protein, such as ubiquitous binding and immune response 49-51. Inhibition site on enzyme can also be eliminated from native enzyme to create inhibitor-resistant mutant 52,53. Advances in protein engineering have also allowed the exploration of the relationship between protein structure and stability, and thus guide the rationally design of the protein with improved stability in addition to screening for stable mutants. It was found that, improving packing in hydrophobic core54, building intramolecular salt bridge 4,55or disulfide bond 56,57, and adding new metal binding sites 58 may cause beneficial effect in protein stability. As a special case for protein engineering, fusion protein technique creates chimeric proteins via the combination of multiple genes, which originally code for separate proteins or peptides 59,60. This technique opens an avenue for combining two or more desired functionalities into one single protein. With this technique, a protein with function of interest can be labeled with an affinity tag for convenient separation; targeting modules can be installed on a therapeutic protein; 4 two enzymes in a tandem reaction can be closely associated to deliver enhanced catalytic efficiency28.Furthermore, active-site redesign is shown able to create new enzyme functions, which do not originally exist in the native form or even in nature. The creation of a complete spectrum of fluorescent proteins is a convincing example. Mutation introduced in different sites in wild-type green fluorescent protein (wtGFP) creates not only more stable variant (EGFP), but also fluorescent protein with altered excitation and emission wavelength (Table 1)61. More amazingly, substitution of the active site amino acid may completely change the bioactivity of an enzyme. A single amino acid mutation may convert a cyclophilin into a protease62, or a 3?- hydroxysteroid dehydrogenase to a ?4-3-ketosteroid-5?-reductase (5?- reductase)63.