TY - JOUR
AU1 - Białkowska, Aneta M
AU2 - Morawski, Krzysztof
AU3 - Florczak, Tomasz
AB - Abstract The objective of this review is to outline the crucial role that peptides play in various sectors, including medicine. Different ways of producing these compounds are discussed with an emphasis on the benefits offered by industrial enzyme biotechnology. This paper describes mechanisms of peptide bond formation using a range of proteases with different active site structures. Importantly, these enzymes may be further improved chemically and/or genetically to make them better suited for their various applications and process conditions. The focus is on extremophilic proteases, whose potential does not seem to have been fully appreciated to date. The structure of these proteins is somewhat different from that of the common commercially available enzymes, making them effective at high salinity and high or low temperatures, which are often favorable to peptide synthesis. Examples of such enzymes include halophilic, thermophilic, and psychrophilic proteases; this paper also mentions some promising catalytic proteins which require further study in this respect. Introduction Linear and cyclic peptides play an important role in numerous intracellular reactions. Under in vivo conditions, peptides are involved in the regulation of repair and metabolic processes, and may act as hormones (e.g., oxytocin, adrenocorticotropic hormone, and calcitonin), neurotransmitters, growth factors, and ion channel ligands. Many peptides produced by single cell bacteria and filamentous Streptomyces, such as gramicidins, polymyxins, bacitracins, and glycopeptides, reveal antimicrobial activity. Under in vitro conditions, they are typically used as biologically active substances in pharmaceuticals or their precursors. They may also confer specific benefits to foodstuffs, feeds, and cosmetic products. As they offer high safety and tolerability, the demand for these highly selective and efficacious substances has been on the rise over the years. According to forecasts for the pharmaceutical industry alone, in 2018, the value of the global peptide market is predicted to reach $ 25.4 billion, which represents a 1.8-fold increase on 2011 ($ 14.1 billion) [39]. While peptides are usually generated by complicated and laborious chemical synthesis, multi-step chemical methods are gradually being superseded by enzymatic technologies. Indeed, methods based on proteolytic biocatalysts are becoming increasingly reliable and economical. In addition, the replacement of the widely available and well-studied mesophilic proteases with their extremophilic counterparts significantly increases the possibilities of process optimization, as the latter due to their unique kinetic and structural adaptations remain active under extreme environmental conditions, such as very high salinity and temperatures higher than 60 °C or lower than 20 °C, which often favor peptide synthesis. This review shows the importance of peptides as products with significant value added in medicine and many industrial processes. It describes several methods of peptide synthesis, indicating their strengths and weaknesses. A special focus is placed on protease-based catalysis, which has recently been explored as an efficient tool for peptide bond formation. Indeed, modern biotechnology aimed at clean and cost-effective manufacturing should be continuously improved not only by chemical or genetic modifications of biocatalysts or by altering the reaction environment, but also by the replacement of conventional proteolytic enzymes with their counterparts retaining high activity under extreme environmental conditions, and often maximizing production efficiency. The application potential of peptides Peptides are considered important target molecules for pharmaceutical, nutritional, and cosmetic applications. Special attention has been given to their considerable therapeutic potential. Their natural properties and the important functions they fulfil in vivo make these molecules particularly well-suited for the treatment of numerous pathological conditions, including metabolic diseases and tumors (Table 1). Moreover, given their attractive pharmacological profile and intrinsic properties, peptides represent an excellent starting point for the design of novel therapeutics and their specificity has been seen to translate into excellent safety, tolerability, and efficacy profiles in humans. This aspect is the primary factor differentiating peptides from the traditional small molecules. Furthermore, peptide drugs are typically associated with lower production complexity compared to protein-based biopharmaceuticals, and therefore, their production costs are also lower, generally approaching those of small molecules [66]. Examples of commercially available peptide drugs Peptide (commercial name) . Application in the treatment of . Action . Source . Carfilzomib (Kyprolis) Multiple myeloma Acts as a proteasome inhibitor [72] Degarelix (Firmagon) Prostate cancer Blocks GnRH receptors, thus lowering testosterone secretion [51] Enfuviritide (Fuzeon) HIV Prevents viral entry into cells [75] Exenatide (Byetta, Bydureon) Type 2 diabetes mellitus Facilitates control of glucose levels [10] Glatiramer (Copaxone) Multiple sclerosis Mechanism of action has not been elucidated [105] Goserelin (Zoladex) Prostate and breast cancer Inhibits the production of gonadotropins [87] Icatibant (Firazyr) Hereditary angioedema Antagonist of bradykinin B2 receptors [30] Lanreotide (Somatuline, Angiopeptin) Neuroendocrine tumors and acromegaly Acts similar to somatostatin [18] Linaclotide (Linzess, Constella) Chronic idiopathic constipation Agonist of guanylate cyclase 2C [76] Lucinactant (Surfaxin) Respiratory insufficiency in children and neonates Mimics human surfactant protein-B (SP-B) [29] Mifamurtide (Mepakt) Bone cancer Activates monocytes [40] Nesiritide (Natrecor) Decompensated heart failure Causes vasodilation, affecting heart function [32] Octreotide (Sandostatin) Neuroendocrine tumors and acromegaly Acts similar to somatostatin [98] Pasireotide (Signifor) Cushing disease Blocks growth hormone secretion [114] Pramlintide (Symlin) Type 1 and 2 diabetes mellitus Helps maintain optimum blood glucose concentration [5] Romiplostim (Nplate) Idiopathic thrombocytopenic purpura Activates thrombopoietin receptors and stimulates Janus kinase 2 (JAK2) and signal transducers and activators of transcription 5 (STAT5) pathways [19] Teduglutide (Revestive, Gattex) Short bowel syndrome Acts as a GLP-2 receptor agonist [58] Teriparatide (Forteo) Some types of osteoporosis Due to its similarity to parathyroid hormone (PTH) increasing plasma calcium concentration, it activates osteoblasts [110] Ziconotide (Prialt) Pain Blocks neurotransmission [71, 84] Peptide (commercial name) . Application in the treatment of . Action . Source . Carfilzomib (Kyprolis) Multiple myeloma Acts as a proteasome inhibitor [72] Degarelix (Firmagon) Prostate cancer Blocks GnRH receptors, thus lowering testosterone secretion [51] Enfuviritide (Fuzeon) HIV Prevents viral entry into cells [75] Exenatide (Byetta, Bydureon) Type 2 diabetes mellitus Facilitates control of glucose levels [10] Glatiramer (Copaxone) Multiple sclerosis Mechanism of action has not been elucidated [105] Goserelin (Zoladex) Prostate and breast cancer Inhibits the production of gonadotropins [87] Icatibant (Firazyr) Hereditary angioedema Antagonist of bradykinin B2 receptors [30] Lanreotide (Somatuline, Angiopeptin) Neuroendocrine tumors and acromegaly Acts similar to somatostatin [18] Linaclotide (Linzess, Constella) Chronic idiopathic constipation Agonist of guanylate cyclase 2C [76] Lucinactant (Surfaxin) Respiratory insufficiency in children and neonates Mimics human surfactant protein-B (SP-B) [29] Mifamurtide (Mepakt) Bone cancer Activates monocytes [40] Nesiritide (Natrecor) Decompensated heart failure Causes vasodilation, affecting heart function [32] Octreotide (Sandostatin) Neuroendocrine tumors and acromegaly Acts similar to somatostatin [98] Pasireotide (Signifor) Cushing disease Blocks growth hormone secretion [114] Pramlintide (Symlin) Type 1 and 2 diabetes mellitus Helps maintain optimum blood glucose concentration [5] Romiplostim (Nplate) Idiopathic thrombocytopenic purpura Activates thrombopoietin receptors and stimulates Janus kinase 2 (JAK2) and signal transducers and activators of transcription 5 (STAT5) pathways [19] Teduglutide (Revestive, Gattex) Short bowel syndrome Acts as a GLP-2 receptor agonist [58] Teriparatide (Forteo) Some types of osteoporosis Due to its similarity to parathyroid hormone (PTH) increasing plasma calcium concentration, it activates osteoblasts [110] Ziconotide (Prialt) Pain Blocks neurotransmission [71, 84] Open in new tab Examples of commercially available peptide drugs Peptide (commercial name) . Application in the treatment of . Action . Source . Carfilzomib (Kyprolis) Multiple myeloma Acts as a proteasome inhibitor [72] Degarelix (Firmagon) Prostate cancer Blocks GnRH receptors, thus lowering testosterone secretion [51] Enfuviritide (Fuzeon) HIV Prevents viral entry into cells [75] Exenatide (Byetta, Bydureon) Type 2 diabetes mellitus Facilitates control of glucose levels [10] Glatiramer (Copaxone) Multiple sclerosis Mechanism of action has not been elucidated [105] Goserelin (Zoladex) Prostate and breast cancer Inhibits the production of gonadotropins [87] Icatibant (Firazyr) Hereditary angioedema Antagonist of bradykinin B2 receptors [30] Lanreotide (Somatuline, Angiopeptin) Neuroendocrine tumors and acromegaly Acts similar to somatostatin [18] Linaclotide (Linzess, Constella) Chronic idiopathic constipation Agonist of guanylate cyclase 2C [76] Lucinactant (Surfaxin) Respiratory insufficiency in children and neonates Mimics human surfactant protein-B (SP-B) [29] Mifamurtide (Mepakt) Bone cancer Activates monocytes [40] Nesiritide (Natrecor) Decompensated heart failure Causes vasodilation, affecting heart function [32] Octreotide (Sandostatin) Neuroendocrine tumors and acromegaly Acts similar to somatostatin [98] Pasireotide (Signifor) Cushing disease Blocks growth hormone secretion [114] Pramlintide (Symlin) Type 1 and 2 diabetes mellitus Helps maintain optimum blood glucose concentration [5] Romiplostim (Nplate) Idiopathic thrombocytopenic purpura Activates thrombopoietin receptors and stimulates Janus kinase 2 (JAK2) and signal transducers and activators of transcription 5 (STAT5) pathways [19] Teduglutide (Revestive, Gattex) Short bowel syndrome Acts as a GLP-2 receptor agonist [58] Teriparatide (Forteo) Some types of osteoporosis Due to its similarity to parathyroid hormone (PTH) increasing plasma calcium concentration, it activates osteoblasts [110] Ziconotide (Prialt) Pain Blocks neurotransmission [71, 84] Peptide (commercial name) . Application in the treatment of . Action . Source . Carfilzomib (Kyprolis) Multiple myeloma Acts as a proteasome inhibitor [72] Degarelix (Firmagon) Prostate cancer Blocks GnRH receptors, thus lowering testosterone secretion [51] Enfuviritide (Fuzeon) HIV Prevents viral entry into cells [75] Exenatide (Byetta, Bydureon) Type 2 diabetes mellitus Facilitates control of glucose levels [10] Glatiramer (Copaxone) Multiple sclerosis Mechanism of action has not been elucidated [105] Goserelin (Zoladex) Prostate and breast cancer Inhibits the production of gonadotropins [87] Icatibant (Firazyr) Hereditary angioedema Antagonist of bradykinin B2 receptors [30] Lanreotide (Somatuline, Angiopeptin) Neuroendocrine tumors and acromegaly Acts similar to somatostatin [18] Linaclotide (Linzess, Constella) Chronic idiopathic constipation Agonist of guanylate cyclase 2C [76] Lucinactant (Surfaxin) Respiratory insufficiency in children and neonates Mimics human surfactant protein-B (SP-B) [29] Mifamurtide (Mepakt) Bone cancer Activates monocytes [40] Nesiritide (Natrecor) Decompensated heart failure Causes vasodilation, affecting heart function [32] Octreotide (Sandostatin) Neuroendocrine tumors and acromegaly Acts similar to somatostatin [98] Pasireotide (Signifor) Cushing disease Blocks growth hormone secretion [114] Pramlintide (Symlin) Type 1 and 2 diabetes mellitus Helps maintain optimum blood glucose concentration [5] Romiplostim (Nplate) Idiopathic thrombocytopenic purpura Activates thrombopoietin receptors and stimulates Janus kinase 2 (JAK2) and signal transducers and activators of transcription 5 (STAT5) pathways [19] Teduglutide (Revestive, Gattex) Short bowel syndrome Acts as a GLP-2 receptor agonist [58] Teriparatide (Forteo) Some types of osteoporosis Due to its similarity to parathyroid hormone (PTH) increasing plasma calcium concentration, it activates osteoblasts [110] Ziconotide (Prialt) Pain Blocks neurotransmission [71, 84] Open in new tab Peptides are also widely used in the cosmetic industry, and especially in skin care products, such as anti-aging and anti-wrinkle preparations. The peptides they contain stimulate skin regeneration and collagen production. Furthermore, some peptides are used to combat age spots and promote hair growth (Table 2). Examples of commercial peptides in the cosmetic industry Peptide (commercial name) . Application . Action . Source . Copper tripeptide-1 Affects collagen synthesis as well as antioxidant and anti-inflammatory response Anti-wrinkle, anti-aging, moisturizing, hair growth stimulating [52] Pal-GHK Stimulates collagen and glycosaminoglycans production Anti-wrinkle, anti-aging, moisturizing, firming, protecting against UV [6] Argireline Inhibits the formation of the SNARE complex and catecholamine release Anti-wrinkle, moisturizing, firming, skin color modifying [12] Pal-KTTKS Stimulates the production of collagen type I, III, and VI, elastin, fibronectin, and glycosaminoglycans Anti-aging, anti-wrinkle [78] Tripeptide-10 citrulline Regulates collagen synthesis and affects the diameter and distribution of collagen fibers Anti-aging, firming [6] Peptide (commercial name) . Application . Action . Source . Copper tripeptide-1 Affects collagen synthesis as well as antioxidant and anti-inflammatory response Anti-wrinkle, anti-aging, moisturizing, hair growth stimulating [52] Pal-GHK Stimulates collagen and glycosaminoglycans production Anti-wrinkle, anti-aging, moisturizing, firming, protecting against UV [6] Argireline Inhibits the formation of the SNARE complex and catecholamine release Anti-wrinkle, moisturizing, firming, skin color modifying [12] Pal-KTTKS Stimulates the production of collagen type I, III, and VI, elastin, fibronectin, and glycosaminoglycans Anti-aging, anti-wrinkle [78] Tripeptide-10 citrulline Regulates collagen synthesis and affects the diameter and distribution of collagen fibers Anti-aging, firming [6] Open in new tab Examples of commercial peptides in the cosmetic industry Peptide (commercial name) . Application . Action . Source . Copper tripeptide-1 Affects collagen synthesis as well as antioxidant and anti-inflammatory response Anti-wrinkle, anti-aging, moisturizing, hair growth stimulating [52] Pal-GHK Stimulates collagen and glycosaminoglycans production Anti-wrinkle, anti-aging, moisturizing, firming, protecting against UV [6] Argireline Inhibits the formation of the SNARE complex and catecholamine release Anti-wrinkle, moisturizing, firming, skin color modifying [12] Pal-KTTKS Stimulates the production of collagen type I, III, and VI, elastin, fibronectin, and glycosaminoglycans Anti-aging, anti-wrinkle [78] Tripeptide-10 citrulline Regulates collagen synthesis and affects the diameter and distribution of collagen fibers Anti-aging, firming [6] Peptide (commercial name) . Application . Action . Source . Copper tripeptide-1 Affects collagen synthesis as well as antioxidant and anti-inflammatory response Anti-wrinkle, anti-aging, moisturizing, hair growth stimulating [52] Pal-GHK Stimulates collagen and glycosaminoglycans production Anti-wrinkle, anti-aging, moisturizing, firming, protecting against UV [6] Argireline Inhibits the formation of the SNARE complex and catecholamine release Anti-wrinkle, moisturizing, firming, skin color modifying [12] Pal-KTTKS Stimulates the production of collagen type I, III, and VI, elastin, fibronectin, and glycosaminoglycans Anti-aging, anti-wrinkle [78] Tripeptide-10 citrulline Regulates collagen synthesis and affects the diameter and distribution of collagen fibers Anti-aging, firming [6] Open in new tab In the food industry, peptides play an important role as stabilizers and agents increasing the microbiological safety of foodstuffs (e.g., nisin, a polycyclic antibacterial peptide produced by the bacterium Lactococcus lactis). In addition to their protective and antibiotic functions, peptides are also used as quality-enhancing food additives. Of particular note is aspartame (Asp-Phe-OMe), a common dipeptide sweetener. Some peptides, which have been found to confer health benefits, serve as functional additives. For instance, the dipeptides Ile-Tyr, Lys-Trp, Val-Tyr, and Ile-Trp have been proven to lower blood pressure [136]. In some countries, such as Japan, food supplemented with these substances has been given nutraceutical status [136]. Moreover, much attention has been devoted to antimicrobial peptides in agriculture. Genes encoding desirable peptides have been introduced to the cells of cultivable plants (Table 3) to prevent diseases and boost productivity. Examples of commercial peptides in agriculture Peptide . Properties . Application . Source . Cecropin B Antibacterial, antiviral Antibacterial factor in transgenic rice Antiviral factor controlling fish pathogens [25, 118] MSI-99 Antibacterial, antifungal Antibacterial and antifungal factor in transgenic bananas and tobacco [20] Lactoferricin Anti-inflammatory Prevention of udder inflammation in goats [141] Polyoxin B Antifungal, insecticidal Control of mildews in apple and nectarine trees and grapevines [107] alfAFP Antifungal Antifungal effects in transgenic potatoes [43] Peptide . Properties . Application . Source . Cecropin B Antibacterial, antiviral Antibacterial factor in transgenic rice Antiviral factor controlling fish pathogens [25, 118] MSI-99 Antibacterial, antifungal Antibacterial and antifungal factor in transgenic bananas and tobacco [20] Lactoferricin Anti-inflammatory Prevention of udder inflammation in goats [141] Polyoxin B Antifungal, insecticidal Control of mildews in apple and nectarine trees and grapevines [107] alfAFP Antifungal Antifungal effects in transgenic potatoes [43] Open in new tab Examples of commercial peptides in agriculture Peptide . Properties . Application . Source . Cecropin B Antibacterial, antiviral Antibacterial factor in transgenic rice Antiviral factor controlling fish pathogens [25, 118] MSI-99 Antibacterial, antifungal Antibacterial and antifungal factor in transgenic bananas and tobacco [20] Lactoferricin Anti-inflammatory Prevention of udder inflammation in goats [141] Polyoxin B Antifungal, insecticidal Control of mildews in apple and nectarine trees and grapevines [107] alfAFP Antifungal Antifungal effects in transgenic potatoes [43] Peptide . Properties . Application . Source . Cecropin B Antibacterial, antiviral Antibacterial factor in transgenic rice Antiviral factor controlling fish pathogens [25, 118] MSI-99 Antibacterial, antifungal Antibacterial and antifungal factor in transgenic bananas and tobacco [20] Lactoferricin Anti-inflammatory Prevention of udder inflammation in goats [141] Polyoxin B Antifungal, insecticidal Control of mildews in apple and nectarine trees and grapevines [107] alfAFP Antifungal Antifungal effects in transgenic potatoes [43] Open in new tab Some peptides, including lipopeptides and peptide amphiphiles, exhibit the ability to reduce surface tension and offer an interesting alternative to the conventional surfactants. In addition, many of these substances also have antibiotic properties, and their biocompatibility and renewability may play a major role in some applications [28]. A well-known molecule with such properties is surfactin, a cyclic lipopeptide produced by Bacillus subtilis, consisting of seven amino acids bonded to the carboxyl and hydroxyl groups of a 14-carbon fatty acid. At 20 mM, the compound decreases the surface tension of water from 72 to 27 mN/m [140]. Another interesting application of peptides is the production of liquid crystal phases. Such systems exhibit the properties of liquids, but their molecules are organized in a crystal-like manner. Peptide liquid crystal phases have attracted considerable interest due to their potential uses in monitoring enzymatic activity and controlling cellular behavior [56, 102]. Peptide production methods Current technologies of peptide production include extraction from natural sources, genetic engineering, cell-free expression systems, transgenic animals and plants, and chemical and enzymatic synthesis [47]. Each of these methods has some strengths and weaknesses, with the most important ones listed in Table 4. For instance, peptide isolation from natural sources is often tedious, idiosyncratic, and impractical. However, this method facilitates obtaining of long and cyclic peptides. There are commercial technologies of microbial synthesis of such peptides, particularly those exhibiting antimicrobial and antibiotic activities, despite relatively high costs. One of the examples is nisin—a natural antimicrobial peptide produced via fermentation of fluid milk or whey by strains of Lactococcus lactis subsp. lactis. This bacteriocin is synthesized ribosomally and used as a safe food preservative in more than 50 countries and a potential agent in pharmaceutical, veterinary, and health care products. A major limitation to the practical application of nisin is that commercial nisin is very expensive, because it is produced in a costly fermentation medium and the yield of nisin derived from the fermentation is low. Most of the nisin-producing bacterial strains have poor growth and cause acidification of milk [7]. The similar problems, caused by the lack of a relatively inexpensive fermentation technology, are related to production of bacitracin, which is a metal dependent, branched cyclic polypeptide produced by Bacillus licheniformis and Bacillus subtilis. It is synthesized non-ribosomally by the large multienzyme complex BacABC and consists of a mixture of structurally similar polypeptides, containing 12 amino acid residues. Bacitracin is one of the most important antibiotics used in human medicine as a topical medication and applied after surgical operations. This antibiotic is also commonly used in animal and poultry feed additives which increase feed efficiency and reduce infectious diseases [1]. In addition, other peptide antibiotics produced by species of the genus Bacillus, such as polymyxin, gramicidin, and tyrothricin, are commercially available, despite their high price. Polymyxin B is an antibiotic primarily used for resistant Gram-negative infections. It is derived from the bacterium Bacillus polymyxa. Tyrothricin is a cyclic polypeptide antibiotic mixture from Bacillus brevis. Gramicidin is a heterogeneous mixture of six antibiotic compounds, gramicidins A, B, and C, making up 80, 6, and 14% respectively, all of which are obtained from the soil bacterial species Bacillus brevis and called collectively gramicidin [100]. The necessity of production of novel peptides of that kind causes that the alternative and less expensive production technologies, based on renewable substrates from industrial wastes and/or involving improved, recombinant microbial strains, have been intensively sought after. In contrast to peptide isolation from natural sources, genetic engineering enables the production of much greater quantities of peptides, and selected amino acids can be replaced with ones desirable for a given application. In this case, however, the major limitation is peptide aggregation at high levels of expression of the gene encoding the final product. For those reasons, the prevalent commercial methods of peptide production are of chemical nature, with the most widespread one being solid-phase peptide synthesis (SPPS) [21]. Crucially, it offers the possibility to isolate and purify intermediate reaction products, effectively deprotect functional groups, and recombine peptides to obtain larger molecules with desirable amino acid sequences. The drawbacks include complicated and laborious protection–deprotection procedures, harsh reaction conditions, and toxic reagents. A much cleaner and milder peptide technology production is enzymatic synthesis involving biological catalysts. Peptide bonds can be formed, often in a stereoselective manner, by proteases, which are well-characterized and widely used in the industry. Their high catalytic activity under mild conditions and high specificity is economically beneficial, enabling a reduction in the number of process steps, the amount of energy consumed, and the quantity and toxicity of waste products. The disadvantage of enzymatic methods is the need to optimize most process parameters, such as temperature, organic solvent concentration, biocatalytic activity, substrate concentration, etc. Nevertheless, enzymatic peptide synthesis is gaining in importance as a technology consistent with the concept of sustainable development. Increasingly stringent health and safety regulations and the growing demand for biologically active peptides have prompted extensive efforts to find biotechnological alternatives to chemical synthesis of peptides relevant in the medical and nutritional areas [47]. So far, small peptides (typically di- and tripeptides) have been obtained in enzymatic processes involving mostly papain, α-chymotrypsin, and thermolysin, and successfully used in foodstuffs, animal feeds, pharmaceuticals, and agrochemicals. Some of them have already found commercial applications, including the non-caloric sweetener aspartame, a lysine-containing sweet peptide, kyotorphin, angiotensin, enkephalin, and dynorphin, as well the tripeptide Arg–Gly–Asp, a novel drug for the treatment of heavy burns and dermal ulcers [47]. The synthesis of longer peptides requires several proteases with different substrate specificities. Such an approach has been deployed by Kimura et al., who produced Z-l-Tyr-Gly-Gly-l-Phe-l-Leu-OEt. In the first step, they obtained Z-Gly-Gly-OBut and Z-l-Tyr-Gly-Gly-OBut using papain and α-chymotrypsin, then Z-l-Phe-l-LeuOEt, and finally the target peptide using thermolysin [70]. In turn, Kullmann synthesized cholecystokinin by means of chemical condensation of peptides obtained using several enzymes: papain, α-chymotrypsin, arylsulfatase (EC 3.1.6.1; desulfation of tyrosine-O-sulfate), thermolysin, and aminopeptidase M [73]. Comparison of peptide synthesis methods (modified table from [44]) Crucial aspects . Chemical synthesis (SPPS) . Enzymatic synthesis . Genetic engineering . Final peptide length Medium to long Short Long Limitations of amino acid sequence None Addition of proline and hydroxyproline residues may be problematic None Strategy of protection for functional groups involved in the peptide bond Global Partial to minimal Not required Racemization Some Absent Absent Reaction conditions Hazardous Mild Mild Substrate/reagent costs Very expensive Relatively inexpensive Inexpensive Need for process optimization Partial Yes Yes Yield of the desired sequence Very high Medium to high Low to medium Process scale mg to tens of g g to kg to ton g to kg to ton Industrial application Widely used in research and industrial production Some specific industrial uses and increasingly used in research Widely used in research and industrial production Crucial aspects . Chemical synthesis (SPPS) . Enzymatic synthesis . Genetic engineering . Final peptide length Medium to long Short Long Limitations of amino acid sequence None Addition of proline and hydroxyproline residues may be problematic None Strategy of protection for functional groups involved in the peptide bond Global Partial to minimal Not required Racemization Some Absent Absent Reaction conditions Hazardous Mild Mild Substrate/reagent costs Very expensive Relatively inexpensive Inexpensive Need for process optimization Partial Yes Yes Yield of the desired sequence Very high Medium to high Low to medium Process scale mg to tens of g g to kg to ton g to kg to ton Industrial application Widely used in research and industrial production Some specific industrial uses and increasingly used in research Widely used in research and industrial production Open in new tab Comparison of peptide synthesis methods (modified table from [44]) Crucial aspects . Chemical synthesis (SPPS) . Enzymatic synthesis . Genetic engineering . Final peptide length Medium to long Short Long Limitations of amino acid sequence None Addition of proline and hydroxyproline residues may be problematic None Strategy of protection for functional groups involved in the peptide bond Global Partial to minimal Not required Racemization Some Absent Absent Reaction conditions Hazardous Mild Mild Substrate/reagent costs Very expensive Relatively inexpensive Inexpensive Need for process optimization Partial Yes Yes Yield of the desired sequence Very high Medium to high Low to medium Process scale mg to tens of g g to kg to ton g to kg to ton Industrial application Widely used in research and industrial production Some specific industrial uses and increasingly used in research Widely used in research and industrial production Crucial aspects . Chemical synthesis (SPPS) . Enzymatic synthesis . Genetic engineering . Final peptide length Medium to long Short Long Limitations of amino acid sequence None Addition of proline and hydroxyproline residues may be problematic None Strategy of protection for functional groups involved in the peptide bond Global Partial to minimal Not required Racemization Some Absent Absent Reaction conditions Hazardous Mild Mild Substrate/reagent costs Very expensive Relatively inexpensive Inexpensive Need for process optimization Partial Yes Yes Yield of the desired sequence Very high Medium to high Low to medium Process scale mg to tens of g g to kg to ton g to kg to ton Industrial application Widely used in research and industrial production Some specific industrial uses and increasingly used in research Widely used in research and industrial production Open in new tab Protease-catalyzed synthesis of peptide bonds may be either kinetically or thermodynamically controlled (Fig. 1). In neither case, is it necessary to protect amino or carboxyl groups on the substrate amino acids, as the sequence of the target peptide is determined by the specificity of the enzyme used [138]. Fig. 1 Open in new tabDownload slide Protease-catalyzed peptide synthesis in kinetically (I + II + III) and thermodynamically (III) controlled processes. In these reactions, proteases may act as transferases (I) or hydrolases (II, III): k t and k h, apparent second-order transferase and hydrolase rate constants Kinetically controlled synthesis of peptides Kinetically controlled peptide synthesis (KCS) may be conducted using serine and cysteine proteases, which in this case act as transferases and catalyze the transfer of an acyl group from a donor to an amino acid nucleophile through the formation of an acyl–enzyme intermediate (Fig. 2). The substrates are acyl donors activated to increase their affinity for the active sites of the enzymes. Activation is needed for the reaction to proceed until complete exhaustion of the substrate regardless of the reaction equilibrium for peptide bond synthesis/hydrolysis. Only then, it is possible to obtain equilibrium concentrations of the substrates and products (a peptide synthesis/hydrolysis equilibrium). In both groups of enzymes, following the formation of an enzyme–substrate complex, the acyl group may be transferred to a water molecule (in a hydrolysis reaction) or to another nucleophile, such as the amino group of another amino acid (in a transferase reaction). The key issue is to select appropriate process conditions for the peptide synthesis reaction to prevail, or to maximize the ratio of the transferase to hydrolase rate constants (k t/k h)app for a given enzyme. Synthesis efficiency depends on many factors, the most important ones being molar excess of nucleophilic groups competing with water for the acyl. In this respect, an important parameter is the affinity of the nucleophile for the S1′ pocket of the enzyme, which is affected by the substrate specificity of the biocatalyst and the ionic strength of the medium. While designing the process, one should also take into account other physical factors, such as reaction medium pH. High pH resulting from an increased concentration of OH ions favors hydrolysis, while bringing pH below p I of the nucleophile produces more charged nucleophilic groups, which is undesirable as only uncharged groups (“free bases”) can react with acyl–enzyme intermediates. Furthermore, the higher the temperature, the lower the (k t/k h)app, which compromises the efficiency of peptide bond formation. This observation has led to the development of frozen aqueous systems for the purposes of synthesis [14, 22, 47, 65, 138]. Fig. 2 Open in new tabDownload slide Mechanism of peptide bond synthesis involving serine proteases (a) and substrate (acyl) binding by cysteine protease (b) Thermodynamically controlled synthesis of peptides Thermodynamically controlled peptide synthesis (TCS) may be conducted using all types of proteases, irrespective of their mechanism of action, and acyl donors with a free (non-activated) carboxyl group may be applied as substrates (Fig. 3). However, intermediate formation from a non-activated substrate proceeds slowly and requires large amounts of the biocatalyst. Therefore, such reactions are characterized by very low efficiency, which is the main drawback of the method. Fig. 3 Open in new tabDownload slide Thermodynamically controlled peptide synthesis In the simplest of terms, in this kind of synthesis, the reaction rate constant (K app) is manipulated to avoid reaching an equilibrium to drive the reaction in the desired direction (see equation below). [AN] =Kapp[AON][NH][H2O] $$[ {\text{AN] = }}\frac{{K_{\text{app}} [ {\text{AON][NH]}}}}{{ [ {\text{H}}_{ 2} {\text{O]}}}}$$ where K app is the reaction rate constant, [AN] is the target peptide concentration, [AOH] is the acyl donor concentration; [NH] is the nucleophile concentration; and [H2O] is the water concentration. This objective may be achieved by adjusting reagent concentrations and the physical parameters of the process. The former can be done by the removal of the generated product from the reaction medium or by reducing water content in the medium (using unconventional media, such as ionic liquids, supercritical liquids, emulsions, or organic solvents). The physical parameters of the medium that may shift the reaction rate constant towards synthesis include pH, ionic strength, and temperature. These factors directly affect the ionization (K ion) of substrates used in synthesis reactions (Fig. 2). According to the literature data, for optimum peptide synthesis, efficiency medium pH should be adjusted to minimize the difference between p K of the acyl group donor and that of the nucleophile. In the case of ionic strength, it has been found that an increase in this parameter leads to a reduction in the reaction rate constant and product concentration due to the higher stability of the ionized reagents present in the solution. In contrast, K app and synthesis efficiency rise with temperature. However, it should be borne in mind that all of the above parameters also affect the activity and specificity of the enzymes, which should be taken into consideration during process design [14, 22, 47, 65, 138]. Improvement of proteases with a view to their use in synthesis reactions Along with lipases, microbiological proteases are currently deemed to present the greatest biotechnological potential. Their industrial output is qualitatively diverse and quantitatively the largest among enzymes, while studies on the relationship between their properties and structure enable rational engineering of conventional proteases to make them better suited to the process conditions. Enzyme engineering includes techniques ranging from deliberate chemical modification to genetic remodeling of wild-type enzymes. These modifications may consist of replacement of a single amino acid or group of amino acids, or entail changes within the entire protein molecule. They are typically aimed at (1) increasing enzyme stability in media containing organic solvents; (2) reducing the rate of competitive hydrolysis of the acyl donor ester, especially when water is used as the reaction medium; (3) limiting the usually undesirable proteolytic activity of proteases to prevent competitive peptide cleavages during the synthesis reaction; and (4) extending or altering the specificity or enantioselectivity of the native enzyme [14]. Modifications often affect more than one parameter of the biocatalyst, further increasing the efficiency of peptide bond synthesis. It should be noted that both chemical and genetic modifications lead to biocatalysts exhibiting some desired functions absent from the parent enzyme. Chemical enzyme modification Chemical modification can be considered the original method of improving enzyme properties, with the main advantages including: (1) wide applicability; (2) cost-effectiveness and ease of implementation, also on a large scale; and (3) the possibility to incorporate non-coded amino acid moieties, leading to enzyme species that cannot be generated by genetic engineering. Nakatsuka et al. chemically modified a protease to enhance its esterolytic activity while minimizing amidolysis [91]. They obtained a chemical derivative of subtilisin with the serine hydroxyl in the active site replaced by a thiol group. The resulting thiolsubtilisin is deemed a promising catalyst for both small and large peptides, being a rudimentary peptide ligase by virtue of the preference of the thiol–acyl intermediate to react with α-amines rather than to hydrolyze [91]. Using a similar approach, Wu and Hilvert converted the serine in the subtilisin active site (Ser221) into selenocysteine, producing an enzyme with strong peptide synthesis activity [135]. Subtilisin was also successfully modified by methylation of the active site histidine [142]. Chemical modifications of proteases have also been used to make them more stable and preserve their activity in organic solvent media, which are often required in peptide bond synthesis. An example of modifications stabilizing protein tertiary structure is crosslinking by means of bifunctional reagents as their two reactive groups can be used to form both inter- and intramolecular bridges [49]. In the case of proteases, such reagents include glutaraldehyde, bisimidoesters, adipimidates, dianhydrides, suberimidates, and succinimide esters, as confirmed by Tafertshofer et al. [46] and Gleich et al. [123]. According to the former, the half-life of α-chymotrypsin crosslinked with dianhydrides and N-hydroxysuccinimide esters (NHS) of carbonic acid increased from 1 h to 22.2 h at 50 °C, with its pH optimum shifting to a higher value and its isoelectric point to the acid region [123]. The latter research team reported that trypsin crosslinked with NHS of dicarboxylic acids, dianhydrides, and bisimidoesters showed enhanced stability while retaining sufficiently high activity [46]. Protease stability was improved in a similar way by Manonmani and Joseph, who crosslinked an extracellular alkaline protease from Trichoderma koningii with glutaraldehyde [82]. Similar effects were obtained by Yandri et al. for a protease from Bacillus subtilis ITBCCB148 using dimethyl adipimidate (DMA), a bifunctional reagent [137]. They were able to modify 69, 75, and 76% of the lysine residues in the protein and increase its thermal stability relative to its native form by 9.2, 14.4, and 34.9%, respectively (as determined at 60 °C after 300 min) [137]. Additional advantages of crosslinking, such as resistance to proteolysis, were observed by St. Clair and Navia [120] and Persichetti [101], who treated thermolysin crystals with glutaraldehyde. The resulting protein retained high activity under harsh conditions including high temperature, extreme pH, and organic solvents. The same crosslinking factor was used by Sangeetha and Abraham following prior subtilisin aggregation using ammonium sulfate and polyethylene glycol (PEG) with surfactants such as Triton X-100 and Tween 20 [113]. As compared to the native protein, immobilized crosslinked enzyme aggregates (CLEA) exhibited a higher optimum temperature (70 vs. 55 °C) and pH (9.0 vs. 7.0) as well as enhanced stability (by approx. 50% during 5 h incubation at 40–70 °C). Moreover, subtilisin CLEA has good stability in nonpolar organic solvents, such as hexane and cyclohexane, and can, therefore, be used as a catalyst for the biotransformation of compounds which are not soluble in aqueous media [113]. Another approach to increasing enzyme stability in organic media was presented by Cao et al. [16], who immobilized papain on magnetic nanocrystalline cellulose (MNCC), a novel bio-based nanocomposite. In addition to higher stability in organic solvents, the studied protease (PA@MNCC) was characterized by a much higher optimum temperature and pH as compared to the free enzyme. The chemical modification applied by the authors affected some of the properties of the enzyme, turning it into an efficient catalyst for the synthesis of the dipeptide N-(benzyloxycarbonyl)–alanyl–glutamine (Z–Ala–Gln) in a deep eutectic solvent (choline chloride:urea, 1:2), with a yield of approx. 71.5% [16]. Another type of chemical modification stabilizing some enzymes with minimal loss of activity is the covalent attachment of large molecular weight polyhydroxy moieties, such as PEG, or carbohydrates to proteins. PEG coupling has been used to dissolve enzymes in non-aqueous solvents, opening up a new area of enzyme chemistry. For instance, Murphy and Fagain modified bovine trypsin with ethylene glycol bis(succinic acid N-hydroxysuccinimide ester). The resulting protein exhibited a decreased rate of autolysis and retained higher activity in aqueous mixtures of 1,4-dioxane, dimethylformamide, dimethylsulfoxide, and acetonitrile. Moreover, the modified trypsin gave higher yields and reaction rates than the native enzyme in kinetically controlled synthesis of benzoyl argininyl–leucinamide in acetonitrile and in t-butanol [88]. Interesting results were also reported by Nakashima et al. [90], who investigated an enzyme covalently modified by comb-shaped poly(ethylene glycol). While the native form was not active in the tested ionic liquids, the modified biocatalyst exhibited high activity both in those liquids and in toluene. It was the most efficient in ionic liquid [emim][Tf2N] (1-ethyl-3-methylimidazolium tetrafluoroborate), where 65% yield of N-Ac-l-Phe-OEt transesterification with 1-butanol was obtained [90]. A covalent attachment of Carlsberg subtilisin to different forms of chitosan was described by Macquarrie and Bacheva [80] with a 25–92% increase in activity as compared to the free enzyme. That enabled a successful application of the modified subtilisin in the synthesis of Z–Ala–Ala–Leu–Phe–pNA in a 6:4 DMF–acetonitrile mixture, with a yield of 100% after only 40 min [80]. High peptide synthesis yields were also reported by Stolarow et al. [122], who covalently immobilized trypsin on magnetic particles in dioxane. The modified enzyme showed higher specific activity and coupling yield in peptide synthesis reaction. According to the authors, those results indicate a substantial potential of their organic protease immobilization method for the preservation of enzymatic activity during the coupling step. However, the results also revealed that the organic method suffers from enzyme solubility limitations under those conditions [122]. Protein engineering An example of genetic modifications of protein sequences to increase catalytic activity in organic reaction media was described by Ogino et al. [97]. To enhance the activity of protease PST-01 from Pseudomonas aeruginosa in aspartame synthesis while retaining its substrate specificity, the authors substituted Tyr114Phe near the active site. In this way, they made this fragment of the enzyme similar to the homologous region in thermolysin, an enzyme produced by Bacillus thermoproteolyticus, which is by nature a much more efficient catalyst in organic media. The modification did significantly increase PST-01 activity in the tested media without affecting its stability [97]. In turn, Zhong et al. introduced five point mutations (Asn218Ser, Gly169Ala, Met50Phe, Gln206Cys, and Asn76Asp) to subtilisin. The modified enzyme had a half-life of 350 h in DMF and 1600 h in aqueous solutions at pH 8.4 and 2 °C, as compared to 20 min and 15 h, respectively, for the wild type [142]. The enzyme was also studied by Chen and Arnold, who generated a library of different variants of the gene encoding it using error prone PCR. Based on screening, they selected two muteins: Asp60Asn, Gln103Arg, Met218Ser and Asp60Asn, Asp97Gly, Gln103Arg, Gly131Asp, Glu156Gly, Asn181Ser, Ser188Pro, Met218Ser, and Thr255Ala. In the synthesis of poly(l-methionine), the activity of the former enzyme increased 38-fold in 85% DMF, while the latter 256-fold in 60% DMF, relative to the native form [23, 24]. Directed evolution was used by Martinez et al. [83] to modify an alkaline psychrophilic protease from Bacillus gibsonii. They obtained an MF1 hybrid (Ile21Val, Ser39Glu, Asn74Asp, Asp87Glu, Met122Leu, and Asn253Asp), whose specific activity at 15 °C was 1.5 times higher and whose half-life at 60 °C was more than 100 times longer than that of the wild type (224 min compared to 2 min) [83]. Genetic engineering can be used not only to introduce point mutations, but modifications of entire protein domains. For instance, Fang et al. [36] reported replacement of N- and C-terminal domains in the keratinases KerSMF and KerSMD from Stenotrophomonas maltophilia BBE11-1. In addition to higher activity, the authors obtained increased thermal stability for three out of the six developed muteins [36]. Proteases from extremophilic microorganisms as efficient tools in peptide synthesis Few proteolytic enzymes have been applied in peptide bond synthesis to date. Studies in this field have mostly focused on proteases isolated from plants (bromelain, papain), animals (trypsin, chymotrypsin, pepsin), and microorganisms (subtilisin, an alkaline protease from Bacillus, and thermolysin, a thermostable enzyme from Bacillus thermoproteolyticus) (Table 5). The application of other proteases for peptide synthesis has been marginal. Researchers have not provided a rationale for the selection of these catalysts; it seems that the main reason is their easy accessibility in the form of commercial preparations rather than their specific structural properties. At the same time, most studies concern peptide synthesis in non-conventional reaction media (mostly hydrophobic). Such environments are favorable from the point of view of process efficiency, but may adversely affect biocatalysts, leading to a loss of activity or stability, or to altered substrate specificity. The section “Improvement of proteases” presents methods of enzyme modification that may impart desirable synthesis-promoting properties not present in their native forms. However, such methods are laborious and difficult to design. A good alternative would be to expand the range of proteases used in peptide synthesis to include those produced by extremophilic microorganisms, whose proteins are evolutionarily adapted to retain activity under extreme conditions. The properties of some of them (especially halophilic enzymes) appear to be particularly useful in reactions conducted in media with reduced water content. Catalysis in hydrophobic media may also be facilitated by enzymes isolated from microorganisms adapted to extreme temperatures, whether high (thermozymes) or low (psychrozymes). Both types of extremozymes differ from mesophilic proteins in terms of active structure stabilization and water activity in the catalytic process. Most common proteolytic enzymes currently used in peptide synthesis Protease . Substrates . Synthesis conditions . Yield (%) . References . Acyl donor . Nucleophile . Reaction medium . pH . T (°C) . Bromelain l-Lys-OEt (l-Lys)n-OEt Aqueous 7.6 40 80 [104] Bromelain Cbz-Gly l-Leu-OMe Ethyl acetate (+triethylamine) – 37 86 [124] α-Chymotrypsin N-Ac-l-Phe-OEt l-Ala-OMe Hexane with iron oxide nanoparticles – 25 >97 [68] α-Chymotrypsin l-Cys-OEt (l-Cys)n-OEt Frozen aqueous solution, Na-phosphate buffer 8.0 −20 85 [93] Papain l-Ala-OEt (l-Ala)n-OEt Phosphate buffer 7.0 40 67 [9] Papain (1) l-Ala-OEt (2) l-Lys-OEt (1) (l-Ala)n-OEt (2) (l-Lys)n-OEt 1 M PBS 7.6 40 (1) >99 (2) 80 [35] Papain l-Glu(OEt)2 Methyl 4-aminobutyrate hydrochloride Phosphate buffer 8.0 40 53 [139] Papain l-Ala-Gly-OEt (l-Ala-Gly-OEt)n – 9.0 40 48 [103] Papain Tyr-OEt (Tyr)n-OEt Phosphate buffer (containing DTT) – 30 83 [85] Papain Tyr-OEt (Tyr)n-OEt Phosphate buffer 7.5 25 81 [94] Subtilisin A Ac-Thr(t-Bu)-Ser(t-Bu)-Asp(O-t-Bu)-Leu-Ser(t-Bu)-Lys(Boc)-Gln-OCam (7-mer) H-Met-Glu(O-t-Bu)-Glu(O-t-Bu)-Glu(O-t-Bu)-Ala-NH2 (5-mer) (1) DMF/MTBE (2) CH2Cl2 – 37 (1) 62 (2) 98 [96] Subtilisin H-Phe-NH2 Z-Phe-OCam THF (anhydrous) – 25 <48 [134] Subtilisin (immobilized on poly(vinyl alcohol)-cryogel) Z-Ala-Ala-Leu-OMe Phe-pNA MeCN:DMF 2:3 – 20 80–95 [8] Trypsin Bz-Arg-OEt Bz-Arg-NH2 80% ethanol, 20% tris buffer 9.0 – 90 [122] Trypsin Cbz-Phe-Arg-OMe Leu-PA Acetonitrile + DMF – 20 60–70 [79] Trypsin N-α-benzoyl-l-Arg-OEt Phe-NH2 Borate buffer 9.0 10 70 [38] Protease . Substrates . Synthesis conditions . Yield (%) . References . Acyl donor . Nucleophile . Reaction medium . pH . T (°C) . Bromelain l-Lys-OEt (l-Lys)n-OEt Aqueous 7.6 40 80 [104] Bromelain Cbz-Gly l-Leu-OMe Ethyl acetate (+triethylamine) – 37 86 [124] α-Chymotrypsin N-Ac-l-Phe-OEt l-Ala-OMe Hexane with iron oxide nanoparticles – 25 >97 [68] α-Chymotrypsin l-Cys-OEt (l-Cys)n-OEt Frozen aqueous solution, Na-phosphate buffer 8.0 −20 85 [93] Papain l-Ala-OEt (l-Ala)n-OEt Phosphate buffer 7.0 40 67 [9] Papain (1) l-Ala-OEt (2) l-Lys-OEt (1) (l-Ala)n-OEt (2) (l-Lys)n-OEt 1 M PBS 7.6 40 (1) >99 (2) 80 [35] Papain l-Glu(OEt)2 Methyl 4-aminobutyrate hydrochloride Phosphate buffer 8.0 40 53 [139] Papain l-Ala-Gly-OEt (l-Ala-Gly-OEt)n – 9.0 40 48 [103] Papain Tyr-OEt (Tyr)n-OEt Phosphate buffer (containing DTT) – 30 83 [85] Papain Tyr-OEt (Tyr)n-OEt Phosphate buffer 7.5 25 81 [94] Subtilisin A Ac-Thr(t-Bu)-Ser(t-Bu)-Asp(O-t-Bu)-Leu-Ser(t-Bu)-Lys(Boc)-Gln-OCam (7-mer) H-Met-Glu(O-t-Bu)-Glu(O-t-Bu)-Glu(O-t-Bu)-Ala-NH2 (5-mer) (1) DMF/MTBE (2) CH2Cl2 – 37 (1) 62 (2) 98 [96] Subtilisin H-Phe-NH2 Z-Phe-OCam THF (anhydrous) – 25 <48 [134] Subtilisin (immobilized on poly(vinyl alcohol)-cryogel) Z-Ala-Ala-Leu-OMe Phe-pNA MeCN:DMF 2:3 – 20 80–95 [8] Trypsin Bz-Arg-OEt Bz-Arg-NH2 80% ethanol, 20% tris buffer 9.0 – 90 [122] Trypsin Cbz-Phe-Arg-OMe Leu-PA Acetonitrile + DMF – 20 60–70 [79] Trypsin N-α-benzoyl-l-Arg-OEt Phe-NH2 Borate buffer 9.0 10 70 [38] Open in new tab Most common proteolytic enzymes currently used in peptide synthesis Protease . Substrates . Synthesis conditions . Yield (%) . References . Acyl donor . Nucleophile . Reaction medium . pH . T (°C) . Bromelain l-Lys-OEt (l-Lys)n-OEt Aqueous 7.6 40 80 [104] Bromelain Cbz-Gly l-Leu-OMe Ethyl acetate (+triethylamine) – 37 86 [124] α-Chymotrypsin N-Ac-l-Phe-OEt l-Ala-OMe Hexane with iron oxide nanoparticles – 25 >97 [68] α-Chymotrypsin l-Cys-OEt (l-Cys)n-OEt Frozen aqueous solution, Na-phosphate buffer 8.0 −20 85 [93] Papain l-Ala-OEt (l-Ala)n-OEt Phosphate buffer 7.0 40 67 [9] Papain (1) l-Ala-OEt (2) l-Lys-OEt (1) (l-Ala)n-OEt (2) (l-Lys)n-OEt 1 M PBS 7.6 40 (1) >99 (2) 80 [35] Papain l-Glu(OEt)2 Methyl 4-aminobutyrate hydrochloride Phosphate buffer 8.0 40 53 [139] Papain l-Ala-Gly-OEt (l-Ala-Gly-OEt)n – 9.0 40 48 [103] Papain Tyr-OEt (Tyr)n-OEt Phosphate buffer (containing DTT) – 30 83 [85] Papain Tyr-OEt (Tyr)n-OEt Phosphate buffer 7.5 25 81 [94] Subtilisin A Ac-Thr(t-Bu)-Ser(t-Bu)-Asp(O-t-Bu)-Leu-Ser(t-Bu)-Lys(Boc)-Gln-OCam (7-mer) H-Met-Glu(O-t-Bu)-Glu(O-t-Bu)-Glu(O-t-Bu)-Ala-NH2 (5-mer) (1) DMF/MTBE (2) CH2Cl2 – 37 (1) 62 (2) 98 [96] Subtilisin H-Phe-NH2 Z-Phe-OCam THF (anhydrous) – 25 <48 [134] Subtilisin (immobilized on poly(vinyl alcohol)-cryogel) Z-Ala-Ala-Leu-OMe Phe-pNA MeCN:DMF 2:3 – 20 80–95 [8] Trypsin Bz-Arg-OEt Bz-Arg-NH2 80% ethanol, 20% tris buffer 9.0 – 90 [122] Trypsin Cbz-Phe-Arg-OMe Leu-PA Acetonitrile + DMF – 20 60–70 [79] Trypsin N-α-benzoyl-l-Arg-OEt Phe-NH2 Borate buffer 9.0 10 70 [38] Protease . Substrates . Synthesis conditions . Yield (%) . References . Acyl donor . Nucleophile . Reaction medium . pH . T (°C) . Bromelain l-Lys-OEt (l-Lys)n-OEt Aqueous 7.6 40 80 [104] Bromelain Cbz-Gly l-Leu-OMe Ethyl acetate (+triethylamine) – 37 86 [124] α-Chymotrypsin N-Ac-l-Phe-OEt l-Ala-OMe Hexane with iron oxide nanoparticles – 25 >97 [68] α-Chymotrypsin l-Cys-OEt (l-Cys)n-OEt Frozen aqueous solution, Na-phosphate buffer 8.0 −20 85 [93] Papain l-Ala-OEt (l-Ala)n-OEt Phosphate buffer 7.0 40 67 [9] Papain (1) l-Ala-OEt (2) l-Lys-OEt (1) (l-Ala)n-OEt (2) (l-Lys)n-OEt 1 M PBS 7.6 40 (1) >99 (2) 80 [35] Papain l-Glu(OEt)2 Methyl 4-aminobutyrate hydrochloride Phosphate buffer 8.0 40 53 [139] Papain l-Ala-Gly-OEt (l-Ala-Gly-OEt)n – 9.0 40 48 [103] Papain Tyr-OEt (Tyr)n-OEt Phosphate buffer (containing DTT) – 30 83 [85] Papain Tyr-OEt (Tyr)n-OEt Phosphate buffer 7.5 25 81 [94] Subtilisin A Ac-Thr(t-Bu)-Ser(t-Bu)-Asp(O-t-Bu)-Leu-Ser(t-Bu)-Lys(Boc)-Gln-OCam (7-mer) H-Met-Glu(O-t-Bu)-Glu(O-t-Bu)-Glu(O-t-Bu)-Ala-NH2 (5-mer) (1) DMF/MTBE (2) CH2Cl2 – 37 (1) 62 (2) 98 [96] Subtilisin H-Phe-NH2 Z-Phe-OCam THF (anhydrous) – 25 <48 [134] Subtilisin (immobilized on poly(vinyl alcohol)-cryogel) Z-Ala-Ala-Leu-OMe Phe-pNA MeCN:DMF 2:3 – 20 80–95 [8] Trypsin Bz-Arg-OEt Bz-Arg-NH2 80% ethanol, 20% tris buffer 9.0 – 90 [122] Trypsin Cbz-Phe-Arg-OMe Leu-PA Acetonitrile + DMF – 20 60–70 [79] Trypsin N-α-benzoyl-l-Arg-OEt Phe-NH2 Borate buffer 9.0 10 70 [38] Open in new tab Halophilic proteases Halophilic proteases are adapted for catalysis in reaction media with high concentrations of salt, whose hygroscopic properties significantly lower water activity. In the natural environments of halophilic organisms, salinity is similar to that of organic solvent media. For this reason, halophilic enzymes seem to be particularly well-suited, and are regarded by some as “perfect candidates,” for reactions conducted in aqueous-organic and organic solvents [133]. Importantly, the number of known extremozymes of this kind increases year by year, most of them being serine proteases, which are already well-characterized (Table 5). Importantly, some halophilic enzymes are highly stable in organic solvents promoting peptide bond synthesis (Table 6). One of the most interesting examples of halotolerant biocatalysts resistant to hydrophobic media is a serine protease obtained from a γ-Proteobacterium strain (the species was not exactly identified). The enzyme preserves approx. 80% of its initial activity following 18 h of incubation in 35% NaCl solution. Moreover, aqueous-organic solvent systems (including ethylene glycol, ethanol, butanol, etc.) lead to a more than twofold increase in the activity and stability of the enzyme as compared to the control media containing only water [112]. Selected halophilic proteases and their characteristics (table modified from Białkowska et al. [11]) Microorganism/producer . NaCl stability . Optimum hydrolysis conditions . References . Opt. NaCl (M) . Opt. pH . Opt. T (°C) . Haloferax lucentensis VKMM 007a NA 4.3–5.13 8.0 60 [81] Natrialba asiatica 172 P1 Stable in 4 M NaCl at 4 °C 5.1 10.7 70 [60, 61, 63] Chromohalobacter sp. TVSP101a Stable in 4.5 M NaCl at 60 and 70 °C for 2 h Stable in 4.5 M NaCl at room temp. for 20 days and at −4 °C for 45 days 4.5 8.0 75 [133] Haloferax mediterranei 1538 Stable in 4.5 M NaCl 4.5 8.0–8.5 55 [121] Halobacillus sp. SR5-3 Stable in 3.4–6.0 M NaCl at 37 °C for 24 h 4.3 10.0 50 [92] Haloferax mediterranei R4 (AATC 33500) Stable in 3 M NaCl at 5 °C for 20 h 4.3 NA NA [62] Halogeometricum borinquense TSS101 Stable in 3.4 M NaCl at 60 °C for 2 h and 30 days at 4 °C 3.4–4.3 10.0 60 [132] Filobacillus sp. RF2-5 Stable in 4.3 M NaCl at 30 °C for 24 h 2.6–4.3 10.0–11.0 60 [50] Halobacterium halobium NA 4 8.0–9.0 NA [57] Halobacterium halobium ATCC 43214 NA 4 8.0–11.0 NA [26, 69, 109] Halobacterium halobium S9 NA 4 8.7 40 [17] Virgibacillus sp SK33a Stable in 0–4.3 M NaCl at 55 °C for 60 min 1.7–3.4 7.5 55 [119] Halobacterium salinarium IM NA 3 8.0 NA [95] Natrialba magadiia NA 1.5 8.0–10.0 NA [45] Microorganism/producer . NaCl stability . Optimum hydrolysis conditions . References . Opt. NaCl (M) . Opt. pH . Opt. T (°C) . Haloferax lucentensis VKMM 007a NA 4.3–5.13 8.0 60 [81] Natrialba asiatica 172 P1 Stable in 4 M NaCl at 4 °C 5.1 10.7 70 [60, 61, 63] Chromohalobacter sp. TVSP101a Stable in 4.5 M NaCl at 60 and 70 °C for 2 h Stable in 4.5 M NaCl at room temp. for 20 days and at −4 °C for 45 days 4.5 8.0 75 [133] Haloferax mediterranei 1538 Stable in 4.5 M NaCl 4.5 8.0–8.5 55 [121] Halobacillus sp. SR5-3 Stable in 3.4–6.0 M NaCl at 37 °C for 24 h 4.3 10.0 50 [92] Haloferax mediterranei R4 (AATC 33500) Stable in 3 M NaCl at 5 °C for 20 h 4.3 NA NA [62] Halogeometricum borinquense TSS101 Stable in 3.4 M NaCl at 60 °C for 2 h and 30 days at 4 °C 3.4–4.3 10.0 60 [132] Filobacillus sp. RF2-5 Stable in 4.3 M NaCl at 30 °C for 24 h 2.6–4.3 10.0–11.0 60 [50] Halobacterium halobium NA 4 8.0–9.0 NA [57] Halobacterium halobium ATCC 43214 NA 4 8.0–11.0 NA [26, 69, 109] Halobacterium halobium S9 NA 4 8.7 40 [17] Virgibacillus sp SK33a Stable in 0–4.3 M NaCl at 55 °C for 60 min 1.7–3.4 7.5 55 [119] Halobacterium salinarium IM NA 3 8.0 NA [95] Natrialba magadiia NA 1.5 8.0–10.0 NA [45] aHalophilic enzymes studied and characterized in terms of their stability in selected organic solvents Open in new tab Selected halophilic proteases and their characteristics (table modified from Białkowska et al. [11]) Microorganism/producer . NaCl stability . Optimum hydrolysis conditions . References . Opt. NaCl (M) . Opt. pH . Opt. T (°C) . Haloferax lucentensis VKMM 007a NA 4.3–5.13 8.0 60 [81] Natrialba asiatica 172 P1 Stable in 4 M NaCl at 4 °C 5.1 10.7 70 [60, 61, 63] Chromohalobacter sp. TVSP101a Stable in 4.5 M NaCl at 60 and 70 °C for 2 h Stable in 4.5 M NaCl at room temp. for 20 days and at −4 °C for 45 days 4.5 8.0 75 [133] Haloferax mediterranei 1538 Stable in 4.5 M NaCl 4.5 8.0–8.5 55 [121] Halobacillus sp. SR5-3 Stable in 3.4–6.0 M NaCl at 37 °C for 24 h 4.3 10.0 50 [92] Haloferax mediterranei R4 (AATC 33500) Stable in 3 M NaCl at 5 °C for 20 h 4.3 NA NA [62] Halogeometricum borinquense TSS101 Stable in 3.4 M NaCl at 60 °C for 2 h and 30 days at 4 °C 3.4–4.3 10.0 60 [132] Filobacillus sp. RF2-5 Stable in 4.3 M NaCl at 30 °C for 24 h 2.6–4.3 10.0–11.0 60 [50] Halobacterium halobium NA 4 8.0–9.0 NA [57] Halobacterium halobium ATCC 43214 NA 4 8.0–11.0 NA [26, 69, 109] Halobacterium halobium S9 NA 4 8.7 40 [17] Virgibacillus sp SK33a Stable in 0–4.3 M NaCl at 55 °C for 60 min 1.7–3.4 7.5 55 [119] Halobacterium salinarium IM NA 3 8.0 NA [95] Natrialba magadiia NA 1.5 8.0–10.0 NA [45] Microorganism/producer . NaCl stability . Optimum hydrolysis conditions . References . Opt. NaCl (M) . Opt. pH . Opt. T (°C) . Haloferax lucentensis VKMM 007a NA 4.3–5.13 8.0 60 [81] Natrialba asiatica 172 P1 Stable in 4 M NaCl at 4 °C 5.1 10.7 70 [60, 61, 63] Chromohalobacter sp. TVSP101a Stable in 4.5 M NaCl at 60 and 70 °C for 2 h Stable in 4.5 M NaCl at room temp. for 20 days and at −4 °C for 45 days 4.5 8.0 75 [133] Haloferax mediterranei 1538 Stable in 4.5 M NaCl 4.5 8.0–8.5 55 [121] Halobacillus sp. SR5-3 Stable in 3.4–6.0 M NaCl at 37 °C for 24 h 4.3 10.0 50 [92] Haloferax mediterranei R4 (AATC 33500) Stable in 3 M NaCl at 5 °C for 20 h 4.3 NA NA [62] Halogeometricum borinquense TSS101 Stable in 3.4 M NaCl at 60 °C for 2 h and 30 days at 4 °C 3.4–4.3 10.0 60 [132] Filobacillus sp. RF2-5 Stable in 4.3 M NaCl at 30 °C for 24 h 2.6–4.3 10.0–11.0 60 [50] Halobacterium halobium NA 4 8.0–9.0 NA [57] Halobacterium halobium ATCC 43214 NA 4 8.0–11.0 NA [26, 69, 109] Halobacterium halobium S9 NA 4 8.7 40 [17] Virgibacillus sp SK33a Stable in 0–4.3 M NaCl at 55 °C for 60 min 1.7–3.4 7.5 55 [119] Halobacterium salinarium IM NA 3 8.0 NA [95] Natrialba magadiia NA 1.5 8.0–10.0 NA [45] aHalophilic enzymes studied and characterized in terms of their stability in selected organic solvents Open in new tab The biological activity exhibited by halophilic enzymes at reduced water activity results from numerous structural adaptations, and especially altered amino acid composition, as compared to homologous enzymes from other groups of microorganisms. Halophilic proteins contain up to 20% more acidic amino acid residues (Asp and Glu), clustered on the surface, and a smaller number of basic Lys residues. The amount of small hydrophobic residues (Ala, Gly, and Val) is increased at the expense of aromatic residues (Phe, Trp, and Tyr). Due to their greater negative charge, halophilic proteins bind more hydrogen ions, reducing surface hydrophobicity, and thus limiting the tendency to aggregate at high salinity. This is the basic adaptation of halophilic proteins to media with a decreased content of water, which is bound by the salts present in the environment, typically at high concentrations [11]. Despite the fact that halophilic enzymes exhibit many structural properties enabling efficient activity in reaction media favoring peptide synthesis, there is a dearth of reports on their actual application. Exceptions include the work of Ryu et al. [109], who synthesized peptides using a protease form the extreme halophile Halobacterium halobium (ATCC 43214). The obtained halophilic enzyme was characterized by a broad acyl donor specificity for amino acid derivatives and much greater nucleophilic acceptor specificity. Synthesis was conducted using the substrates N-Ac-l-Phe-OEt and Gly–NH2 in the presence DMF at pH 10 and 2.8 M NaCl and afforded a yield of 70% after 15 h [109]. The application of another halophilic protease stable in an aqueous-organic medium containing 1.5 M NaCl and 15–30% organic solvents (including DMSO), obtained from the haloalkalophilic archaeon Natrialba magadii, was described by Ruiz et al. [108]. Synthesis was carried out using Ac–Phe–OEt and Gly–Phe–NH2 in a medium containing 30% DMSO and 1.5 M NaCl; the obtained yield was 58% after 24 h [108]. In turn, Z-l-Ala-l-Leu-NH2 was synthesized using the halophilic protease PST-01 isolated from Pseudomonas aeruginosa PST-01 [13]. The maximum reaction rate was observed at pH 8.0 and 37 °C. Higher reaction rates were obtained in the presence of DMSO, glycerol, methanol, and ethylene glycol. The equilibrium yield was the highest in the presence of DMSO. The equilibrium yield of Cbz–Asp–Phe–OMe using PST-01 was 83% in the presence of 50% (v/v) DMSO [128]. Thermophilic proteases Thermophilic proteases retain their catalytic activity at temperatures exceeding 60 °C. Many enzymes in this category also exhibit high resistance to organic solvents, extreme pH, and other denaturing factors. Toplak et al. [126] described a serine protease from Coprothermobacter proteolyticus which is not only thermostable and resistant to detergents, but also exhibits optimum activity in basic environments. In turn, Cannio et al. presented a Sulfolobus solfataricus protease with maximum activity at 70–90 °C, pH 2 and a half-life of 20 days at 80 °C [15]. Thermophilic proteases combine the beneficial characteristics of halophilic enzymes, that is, high stability in atypical media with low water content, with thermal stability. Thus, catalysis may be conducted at higher temperatures enabling superior substrate and product solubility and better mass transfer. Similarly as in the case of halophilic enzymes, the specific properties of thermozymes result from structural adaptations. These proteins are densely packed and more rigid than their mesophilic counterparts as they contain more stabilizing elements, including hydrogen and ionic bonds, disulfide bridges, and hydrophobic interactions. In a study of a Bacillus stearothermophilus protease, the introduction of only one additional hydrogen bond (by substituting Ser for Ala) led to a 0.7 °C increase in thermal stability [31]. As compared to the homologous mesophilic enzymes, thermozyme sequences contain more Glu, Lys, and Arg residues, which participate in the formation of salt bridges. The introduction of these amino acids at appropriate positions to a mesophilic protein molecule increases its thermal stability [48]. Such enzymes are also characterized by a larger number of hydrophobic interactions. An estimated average increase in stability of 1.3 ± 0.5 kcal/mol is attained with each additional methyl group buried in the molecule during protein folding [99]. Thermal stability is also affected by metal ions (e.g., Ca2+) bound to thermozyme molecules [67]. The other factors found to influence this property include: reduction in entropy of unfolding, increased content of α-helices in the structure, fewer hydrophilic regions on the surface of the protein, fewer residues susceptible to deamination and oxidation, and shortening of the loops present in the molecule [77]. However, there is no universal mechanism ensuring stability at high temperatures. The thermophilic proteinase most useful in enzymatic peptide synthesis is undoubtedly thermolysin, a metalloproteinase isolated as early as in 1961 from B. thermoproteolyticus. This protein is very well characterized and has been commercially available for many years. It cleaves peptide bonds on the N-terminus side of hydrophobic amino acid residues including alanine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine. The first report on its application in peptide synthesis comes from 1977 [55]. Thermolysin may be treated as a model enzyme, which for many years has been used to study the ways in which peptide bond synthesis is influenced by different factors, such as reaction media (typically organic [27], aqueous-organic systems [3, 4, 54], solid-to-solid systems [34], micro-aqueous systems [53], and ionic liquids [33]), enzyme modification [74], as well as methods of enzyme addition to the reaction medium [129]. Due to the low specificity of this protease, research has involved a wide spectrum of substrates, with the most frequent configuration being Leu or Phe in position P1′. Table 7 shows examples of synthesis yields obtained for thermolysin-catalyzed reactions in different systems. Importantly, the enzyme has found an industrial application in peptide synthesis, and in particular in the production of aspartame [2, 86], widely used as a sweetener. Furthermore, thermolysin is also used in one of the steps of synthesizing cholecystokinin, a potential therapeutic agent for the control of gastrointestinal function and the treatment of epilepsy, obesity, and inflammatory diseases [111] as well as in the synthesis of the dipeptide Ala–Phe, a bitter-tasting food additive [127, 130]. All of these processes are centered on thermolysin resistance to organic solvents, but not its high thermal stability, and so they do not to take advantage of the full potential of this protein. The opportunities afforded by the application of high temperatures in peptide production were convincingly shown in experiments conducted by Toplak et al. [125], who studied two thermophilic proteases: TaqSbt from Thermus aquaticus and DgSbt from Deinococcus geothermalis. Both purified enzymes were characterized by high thermal stability and synthesized peptide bonds in neat acetonitrile at 60 and 80 °C with excellent conversion (>90%). The enzymes tolerated high levels of N,N-dimethylformamide (DMF) as a cosolvent (40–50% v/v), which improved substrate solubility and gave good conversion in 5 + 3 peptide condensation reactions. Moreover, it was shown that for both thermozymes higher conversion rates were obtained in synthesis conducted at 60 °C rather than 37 °C [125]. Examples of reaction yields for peptides catalyzed by thermolysin in different media Synthesized product . Reaction medium . Reaction conditions . Catalyst form . Yield (%) . References . Z-Ala-Phe-OMe Acetate buffer 50 °C, pH 6 Free enzyme 66 [130] Fua-Phe-Leu-NH2 Water-acetonitrile (1:3) 37 °C, pH 7 Immobilized and arylated (Ac–Phe, Ac–Ala) 73–77 [74] l-Asp-Phe-OMe BP6 (ionic liquid); 5% water 37 °C Free enzyme 95 [33] Z-l-Asp-l-Phe-OEt/OMe Toluene, a w = 0.73–0.78 30 °C Immobilized on celite R-640 96–99 [27] Fmoc-Asp-Tyr-OMe Water RT, pH 6 Free enzyme 57 [111] Z-Asp-Leu-NH2 Acetonitrile, 4% water 25 °C Immobilized 92 [106] Synthesized product . Reaction medium . Reaction conditions . Catalyst form . Yield (%) . References . Z-Ala-Phe-OMe Acetate buffer 50 °C, pH 6 Free enzyme 66 [130] Fua-Phe-Leu-NH2 Water-acetonitrile (1:3) 37 °C, pH 7 Immobilized and arylated (Ac–Phe, Ac–Ala) 73–77 [74] l-Asp-Phe-OMe BP6 (ionic liquid); 5% water 37 °C Free enzyme 95 [33] Z-l-Asp-l-Phe-OEt/OMe Toluene, a w = 0.73–0.78 30 °C Immobilized on celite R-640 96–99 [27] Fmoc-Asp-Tyr-OMe Water RT, pH 6 Free enzyme 57 [111] Z-Asp-Leu-NH2 Acetonitrile, 4% water 25 °C Immobilized 92 [106] Open in new tab Examples of reaction yields for peptides catalyzed by thermolysin in different media Synthesized product . Reaction medium . Reaction conditions . Catalyst form . Yield (%) . References . Z-Ala-Phe-OMe Acetate buffer 50 °C, pH 6 Free enzyme 66 [130] Fua-Phe-Leu-NH2 Water-acetonitrile (1:3) 37 °C, pH 7 Immobilized and arylated (Ac–Phe, Ac–Ala) 73–77 [74] l-Asp-Phe-OMe BP6 (ionic liquid); 5% water 37 °C Free enzyme 95 [33] Z-l-Asp-l-Phe-OEt/OMe Toluene, a w = 0.73–0.78 30 °C Immobilized on celite R-640 96–99 [27] Fmoc-Asp-Tyr-OMe Water RT, pH 6 Free enzyme 57 [111] Z-Asp-Leu-NH2 Acetonitrile, 4% water 25 °C Immobilized 92 [106] Synthesized product . Reaction medium . Reaction conditions . Catalyst form . Yield (%) . References . Z-Ala-Phe-OMe Acetate buffer 50 °C, pH 6 Free enzyme 66 [130] Fua-Phe-Leu-NH2 Water-acetonitrile (1:3) 37 °C, pH 7 Immobilized and arylated (Ac–Phe, Ac–Ala) 73–77 [74] l-Asp-Phe-OMe BP6 (ionic liquid); 5% water 37 °C Free enzyme 95 [33] Z-l-Asp-l-Phe-OEt/OMe Toluene, a w = 0.73–0.78 30 °C Immobilized on celite R-640 96–99 [27] Fmoc-Asp-Tyr-OMe Water RT, pH 6 Free enzyme 57 [111] Z-Asp-Leu-NH2 Acetonitrile, 4% water 25 °C Immobilized 92 [106] Open in new tab To the best of our knowledge, one more thermophilic enzyme has been studied in terms of peptide synthesis. It constitutes a special case as it was obtained via genetic modification. Site-directed mutagenesis of catalytic Ser491 into Cys in an Acidothermus cellulolyticus aminopeptidase led to an enzyme with interesting catalytic properties and reduced hydrolytic activity [131]. The engineered enzyme, known as aminolysin-A, can produce linear homo-oligopeptides, hetero-dipeptides, and cyclic dipeptides using cost-effective substrates in a one-pot reaction. Aminolysin-A can recognize several C-terminal-modified amino acids, including their l- and d-forms, as acyl donors as well as free amines, including amino acids and puromycin aminonucleoside, as acyl acceptors. The absence of amino acid esters prevents the formation of peptides; therefore, the reaction mechanism involves aminolysis rather than the reverse reaction of hydrolysis. The authors observed that the aminolysin system may be a useful tool for facile preparation of structurally diverse peptide mimetics [131]. Psychrophilic proteases Peptide bond synthesis may be promoted not only by higher temperatures, as it was described above, but also by colder conditions causing the water present in the reaction medium to nucleate. Then, water, which is a nucleophile, becomes less competitive in accepting the acyl group, which shifts the reaction equilibrium towards peptide bond synthesis. The literature provides many examples of the application of such systems in peptide synthesis, but most authors have conducted catalysis with commercially available mesophilic enzymes, such as chymotrypsin and trypsin, which are not particularly suited to cold environments. Efficient catalysis at low temperatures may be conducted by psychrophilic enzymes obtained from cold-adapted microorganisms which grow below 20 °C (the most resilient microbe found to date is the bacteria Planococcus halocryophilus, which remains metabolically active at −25 °C in permafrost microcosms [89]). These molecules are also highly resistant to hydrophobic solvents due to an increased number of ion pairs limiting the influence of hydrophobic media on protein folds and higher structural flexibility of these proteins [117]. This translates into the possibility to use psychrophilic proteases in media with low water content. The metabolic activity exhibited by psychrophilic enzymes at low temperatures is enabled by structural adaptations. Such enzymes are characterized by greater structural flexibility as compared to their mesophilic counterparts due to fewer and weaker intramolecular interactions (especially hydrophobic), due to which the core of the molecule has a greater volume and the whole molecule is more loosely packed. On the other hand, such enzymes exhibit more hydrophilic interactions with solvents due to an increased number of polar amino acid residues. Thus, the active sites of these enzymes are usually more readily accessible to compensate for lower substrate diffusion at low temperatures. In terms of their amino acid composition, as compared to their mesophilic counterparts, psychrophilic proteins contain fewer proline residues (especially in the loops) and basic amino acid residues, which may make the molecule more rigid due to the formation of hydrogen bonds. On the other hand, psychrophilic proteins have more glycine residues, which often occur in clusters [37, 59, 64]. Despite the aforementioned suitability of psychrozymes for peptide synthesis, the literature gives very few examples of psychrophilic proteases actually used for those purposes. However, these described proteases were isolated from organisms living in the cold environment. A notable exception is Fuchise et al. [41, 42], who studied trypsin from Pacific Cod (Gadus macrocephalus). While that enzyme is characterized by relatively high optimum temperature (50 °C), it is highly thermolabile (unstable over 40 °C), which sets it apart from most mammalian trypsins. This enzyme is active in the synthesis of N-Boc-d-Ala-l-Ala-pNA and N-Boc-l-Ala-l-Ala-pNA conducted at 25 °C in 40% DMSO with chemically modified (inverse) substrates [41, 42]. The potential of cold-adapted enzymes was also appreciated by Sekizaki et al. [115, 116], who reported similar properties for trypsin from chum salmon. In this case, peptide synthesis was conducted at 0 °C due to the low stability of the acyl donors, which were amino acids with a general formula of N-Boc-d/l-AA-OAm (where: AA = Gly, l,d-β-Ala, l,d-Leu, l,d-Phe, l,d-Pro; Am = amidinophenyl); l-Ala-pNA served as a nucleophile. The reaction was conducted in 50% DMSO and offered a yield of up to 90% over 120 h [115, 116]. Summary In protease-catalyzed peptide bond synthesis of critical importance is reaction medium engineering aimed at limiting hydrolysis. Favorable reaction conditions are often extreme due to hydrophobicity, high or low temperature, and salt concentration. Indeed, analyzing previous research it is surprising that peptide synthesis has been mostly carried out using enzymes requiring moderate temperatures and salinity, which are suboptimal in such applications. The use of such enzymes seems to be primarily motivated by their availability as well as the ability to compare one’s own study results with those of other workers examining the same enzyme under different conditions and with different substrates. From the molecular and thermodynamic point of view, it seems that in peptide synthesis enzymes naturally characterized by high tolerance of, or preference for, hydrophobic environments, would be a more suitable choice and proteolytic extremozymes from halophilic, thermophilic, and psychrophilic microorganisms could be applied on a much wider scale. The isolation and study of these proteins no longer represent serious problems (e.g., in terms of available analytical and preparative techniques), and an increasing number of such molecules are well-characterized. The range of available proteases has also been expanded by metagenomic research of extreme environments that enabled identification of the genes of some enzymes with very interesting properties. Without neglecting conventional ways of exploring proteolytic microorganisms, metagenomic and metatranscriptomic studies should be intensified as they offer greater access to the protease-encoding genes of those microbes which as of now cannot be cultured in laboratory conditions. Further development may be achieved through examination of subtle molecular adaptations of extremophilic proteases to facilitate rational engineering of known proteases and, in the future, design new protease variants tailored to different applications and process conditions. References 1. 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TI - Extremophilic proteases as novel and efficient tools in short peptide synthesis
JF - Journal of Industrial Microbiology and Biotechnology
DO - 10.1007/s10295-017-1961-9
DA - 2017-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/extremophilic-proteases-as-novel-and-efficient-tools-in-short-peptide-v2x7OQJuml
SP - 1325
EP - 1342
VL - 44
IS - 9
DP - DeepDyve
ER -