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Pagar Amol D.,조성찬,정서희,윤현석,윤형돈 한국생물공학회 2023 KSBB Journal Vol.38 No.1
Non-canonical amino acids (ncAAs) have shown considerable potential in enzyme engineering to improve catalytic activity, modulate enzyme active sites, probe complex enzyme mechanisms, create new to nature enzymes, and enhance thermostability. In this study, we report site-specific incorporation of the photocrosslinking ncAA: p-benzoyl-phenylalanine (pBpA) at the dimeric interface of the transaminase from Vibrio fluvialis (TAVF) and the effect of bulky ncAA incorporation on its functionality has been evaluated. Incorporating the pBpA at selected sites of TAVF led to the destabilization of the functional dimer and improper folding of variants. Two mutants, V31pBpA and K73pBpA, were isolated in the soluble form. The incorporation of bulky pBpA showed detrimental effects on the enzyme activity. Also, we determined the photocrosslinking efficiency of the incorporated pBpA, which showed a cross-linked variant with only 31pBpA highlighting the importance of the precise selection of the incorporation site for ncAA incorporation. Here we demonstrated the ncAA incorporation at a single site of the enzyme and its effects on the enzyme activity.
Recent Advances in Enzyme Engineering through Incorporation of Unnatural Amino Acids
원유미,Amol D. Pagar,Mahesh D. Patil,Philip E. Dawson,윤형돈 한국생물공학회 2019 Biotechnology and Bioprocess Engineering Vol.24 No.4
The development of new enzyme engineering technologies has been actively pursued as the industrial use of biocatalysts is rapidly increasing. Traditional enzyme engineering has been limited to changing the functional properties of enzymes by replacing one amino acid with the other 19 natural amino acids. However, the incorporation of unnatural amino acids (UAAs) has been exploited to manipulate efficient enzymes for biocatalysis. This has been an effective enzyme engineering technique by complementing and extending the limits of traditional enzymatic functional changes. This review paper describes the basic functions of the new functional groups of UAAs used in enzyme engineering and the utilization of UAAs in the formation of chemical bonds in the proteins. The recent developments of UAA-mediated enzymology and its applicability in industry, pharmaceutical and other research areas to overcome the limitations of existing enzymes is also emphasized.
Biocatalytic Cascade for Synthesis of Sitagliptin Intermediate Employing Coupled Transaminase
Taresh P. Khobragade,Pagar Amol D.,GIRI PRITAM DEVIDAS,SARAKSHARADCHANGDEO,전현우,주상우,고영환,박부수,윤형돈 한국생물공학회 2023 Biotechnology and Bioprocess Engineering Vol.28 No.2
Transaminases (TAs) are employed in synthesizing various enantiopure β‐amino acids, key precursors for various pharmaceuticals. Sitagliptin, an oral hyperglycemic drug, is a well-known example. Herein, we developed the coupled enzyme cascade to synthesize the sitagliptin intermediate by fusing two different TAs to regenerate the amino donor. In a cascade system, ethyl 3-oxo-4-(2,4,5- trifluorophenyl) butanoate (1) was converted by esterase from Pseudomonas stutzeri (EstPS) to respective β-keto acid (2) which was subsequently converted by first TA to sitagliptin intermediate (3) using (S)-α-MBA as an amino donor and the acetophenone formed in the reaction was recycled by the second TA to (S)-α-MBA. A single wholecell system was established by the co-expression of esterase and TA fusion protein. The whole-cell biotransformation reaction was performed with varying substrate concentrations from 50-200 mM. The excellent conversion of the product was achieved, ranging from 62-to 100% at the expense of only 25 mM (S)-α-MBA. Notably, our designed system with fusion protein can produce ~5-fold higher product at the expense of 0.5 equivalent (S)-α-MBA. Finally, a preparative scale reaction was performed with 98% conversion.