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Applications of Biotechnology: Biology Doing Chemistry
Published in Richard J. Sundberg, The Chemical Century, 2017
Aspartame is an artificial sweetener approved by the FDA in 1981. Worldwide consumption in 2000 was about 20,000 t (see Section 9.2.2.3). Aspartame is produced enzymatically from methyl phenylalanate and N-Cbz l-aspartic acid by the Tosoh process. Aspartame is also produced by a chemical route developed by the Nutrasweet Company. A main advantage of the enzymatic process is that it can use racemic methyl phenylalanate, whereas the chemical process requires the pure L-enantiomer. The enzyme that is used is a metalloprotease, thermolysin. This protease is more selective for amide bond formation over methyl ester hydrolysis than other industrial proteases. The catalyzed reaction is the reverse of the reaction usually catalyzed by proteases. The basis of this transformation is the selective precipitation of the coupled N-protected product as a complex with the unreacted methyl d-phenylalanate. The methyl d-phenylalanate is recovered, racemized by base, and recycled. Figure 9.2 gives a flow sheet for the process.
Proteases from Thermophiles and Their Industrial Importance
Published in Devarajan Thangadurai, Jeyabalan Sangeetha, Industrial Biotechnology, 2017
D. R. Majumder, Pradnya P. Kanekar
Several other proteases from Thermoactinomyces fusca (Desai et al., 1969), Thermoactinomyces albus (Goodfellow et al., 1988), Thermoactinomyces thalpophilus (Odibo et al., 1988), Thermoactinomyces sacchari (Georgieva et al., 2000), Thermoactinomyces sp. HS628 (Georgieva et al., 2000), Thermoactinomyces sp. 27a (Zabolotskaya et al., 2004), Thermoactinomyces sp. (Yallop et al., 1997), have been isolated. Thermolysin – the enzyme was first studied from Bacillus thermoproteolyticus Rokko by Matsubara (1970). Thermophilic actinomycete, Thermoactinomyces thalpophilus isolated from thermal spring in Maharashtra, India was found to produce thermolysin like protease (TLP). The enzyme has unique property of condensation of two amino acids to form Aspartame, an artificial sweetner. The enzyme is a thermostable metalloprotease (Majumder and Kanekar, 2012; Majumder et al., 2013). Thermoactinomyces thalpophilus was reported to produce TLP for the first time which had commercial importance with respect to production of aspartame. Thermolysin is a thermostable metalloprotease, which has a role in biotransformation of amino acids into an important condensation product like Aspartame (Rao et al., 1998). The enzyme actinokinase is produced by thermophilic actinomycete, Streptomyces megasporus isolated from thermal spring in Maharashtra, India. It dissolves blood clot but does not affect other plasma proteins. Thus the enzyme has potential application in treatment of myocardial infarction (Chitte and Dey, 2000; Chitte and Dey, 2002; Chitte et al., 2011).
Biologically Triggered Injectable Hydrogels as Intelligent Scaffolds
Published in Gilson Khang, Handbook of Intelligent Scaffolds for Tissue Engineering and Regenerative Medicine, 2017
Yoon Ki Joung, Kyung Min Park, Ki Dong Parkc
In addition to HRP and TGase, other enzymes have also been exploited for in situ hydrogel formation. B. Xu et al. reported the first example of supramolecular hydrogels that respond to ligand–receptor interactions.60 He designed a compound, N-(flurenyl-9-methoxycarbonyl) (Fmoc)-D-Ala-D-Ala, as a receptor, targeting ligand–receptor binding with vancomycin (Van) as its ligand. The Fmoc-dipeptides are stereospecific compounds, where Van shows no response to its enantiomer, and cause hydrogelation by π-π overlap of these amino acids. The microstructure was shown to be a nanofibrous network of stacked amino acids. They also reported another example of the enzymatic formation of supramolecular hydrogels.61,62 In addition, they used an enzymatic reaction to convert the ionic group on an amino acid derivative into a neutral group, resulting in the formation of a small-molecular hydrogelator and supramolecular hydrogels. Gelation was processed by the dephosphorylation of the PO43– of Fmoc tyrosine phosphate under basic conditions, which are mediated by alkaline phosphatase, one of the components of the kinase/phosphatase switches that regulate the protein activity. They prepared amphiphilic Fmoc–amino acid derivatives as hydrogelators to adjust the hydrophilic/hydrophobic balance of amino acids for hydrogelation. In a recent paper, Ulijn et al. reported a novel approach of enzyme-triggered hydrogelation using proteases, which are enzymes that normally hydrolyze peptide bonds in aqueous media, to perform the reverse reaction to produce an amphiphilic peptide hydrogelator that self assembles to form a nanofibrous structure.63 He employed thermolysin from Thermoprotelolyticus rokko as a suitable enzyme to test this approach. Thermolysin has been used in reverse hydrolysis reactions and has a well-known preference for the hydrophobic/aromatic residues on the amine side of the peptide bond. He prepared Fmocconjugated peptide amphiphiles that self-assemble into nanofibrous structures derived from π-stacking of the highly conjugated fluorenyl group further stabilized by the formation of helical structures. He applied this system to a novel PEG-based hydrogel particle that was responsive to enzymes for controlled release.64 A copolymer of PEG and acrylamide form a hydrogel particle incorporated with Fmocpeptides with an enzyme-cleavable linker.
Gelation-based visual detection of analytes
Published in Soft Materials, 2019
Wangkhem Paikhomba Singh, Rajkumar Sunil Singh
A reversed hydrolysis method for hydrogelation of peptides was reported by Ulijn’s group (43). Normally, proteases hydrolyze peptide substrates into smaller fragments. However, it is also known that certain proteases (e.g. thermolysin) are capable of performing the reverse reaction of linking of smaller peptides to generate a larger peptide molecule. The authors hypothesized that linking of non-gelling Fmoc-amino acids with dipeptides may afford some gelling Fmoc-tripeptides. Different Fmoc-protected amino acids and the dipeptide FF were incubated in presence of thermolysin at pH 7.2. Some of the tripeptides formed were found to induce hydrogelation. These were Fmoc-AFF, Fmoc-VFF, Fmoc-LFF, and Fmoc-FFF. The use of an esterase, subtilisin, in an enzyme-mediated hydrogelation was also reported by the same group (44). Four Fmoc-dipeptide methyl esters (Fmoc-YT-OMe, Fmoc-YS-OMe, Fmoc-YN-OMe, and Fmoc-YQ-OMe) were treated with subtilisin and monitored for their hydrogelation ability. Two of the resultant Fmoc-dipeptides (Fmoc-YS and Fmoc-YN) were able to form hydrogels at pH 8.