Amol Pagar - Academia.edu (original) (raw)

Papers by Amol Pagar

Biotechnology and Bioprocess Engineering

Green Chemistry

We report a multienzymatic cascade for the synthesis of 1,6-hexamethylenediamine (HMD) and relate... more We report a multienzymatic cascade for the synthesis of 1,6-hexamethylenediamine (HMD) and related α,ω-diamines from corresponding cycloalkanols.

Frontiers in Chemistry, 2022

Non-canonical amino acids (ncAAs) have been utilized as an invaluable tool for modulating the act... more Non-canonical amino acids (ncAAs) have been utilized as an invaluable tool for modulating the active site of the enzymes, probing the complex enzyme mechanisms, improving catalytic activity, and designing new to nature enzymes. Here, we report site-specific incorporation of p-benzoyl phenylalanine (pBpA) to engineer (R)-amine transaminase previously created from d-amino acid aminotransferase scaffold. Replacement of the single Phe88 residue at the active site with pBpA exhibits a significant 15-fold and 8-fold enhancement in activity for 1-phenylpropan-1-amine and benzaldehyde, respectively. Reshaping of the enzyme’s active site afforded an another variant F86A/F88pBpA, with 30% higher thermostability at 55°C without affecting parent enzyme activity. Moreover, various racemic amines were successfully resolved by transaminase variants into (S)-amines with excellent conversions (∼50%) and enantiomeric excess (>99%) using pyruvate as an amino acceptor. Additionally, kinetic resoluti...

GFP was performed by adding IPTG. After 12 h incubation using this cell system, orange-red color ... more GFP was performed by adding IPTG. After 12 h incubation using this cell system, orange-red color of the harvested cells was obtained, indicating the incorporation of biosynthesized Dopa into GFP. The expression of TPL and GFP was also confirmed by SDS-PAGE (Figure S1). These results encouraged us to further optimize the experimental protocol using fluorescence intensity analysis. It was estimated that higher the biosynthesis of Dopa, higher would be the incorporation efficiency and thus fluorescence intensity. Figure S1. Coomassie-stained SDS-PAGE showing expression of TPL in all three constructs but absence of GFP expression in constructs 1 and 2 due to failure in biosynthesized Dopa incorporation. Third construct demonstrates TPL as well as GFP expression indicating Dopa biosynthesis and its residue specific incorporation into GFP in absence of Tyr. Construct 1: E. coli. Tyr auxotroph harboring pBAD33-TPL and pQE80L-GFP. Construct 2: E. coli. Tyr auxotroph harboring pQE80L-TPL and pBAD33-GFP Construct 3: E. coli. Tyr auxotroph pHCEIIB-TPL and pD422-NH-GFP

Biotechnology Advances, 2021

Improvement in intrinsic enzymatic features is in many instances a prerequisite for the scalable ... more Improvement in intrinsic enzymatic features is in many instances a prerequisite for the scalable applicability of many industrially important biocatalysts. To this end, various strategies of chemical modification of enzymes are maturing and now considered as a distinct way to improve biocatalytic properties. Traditional chemical modification methods utilize reactivities of amine, carboxylic, thiol and other side chains originating from canonical amino acids. On the other hand, noncanonical amino acid- mediated 'click' (bioorthogoal) chemistry and dehydroalanine (Dha)-mediated modifications have emerged as an alternate and promising ways to modify enzymes for functional enhancement. This review discusses the applications of various chemical modification tools that have been directed towards the improvement of functional properties and/or stability of diverse array of biocatalysts.

Biotechnology and Bioengineering, 2021

Here, we report a bienzymatic cascade to produce β‐amino acids as an intermediate for the synthes... more Here, we report a bienzymatic cascade to produce β‐amino acids as an intermediate for the synthesis of the leading oral antidiabetic drug, sitagliptin. A whole‐cell biotransformation using recombinant Escherichia coli coexpressing a esterase and transaminase were developed, wherein the desired expression level of each enzyme was achieved by promotor engineering. The small‐scale reactions (30 ml) performed under optimized conditions at varying amounts of substrate (100–300 mM) resulted in excellent conversions of 82%–95% for the desired product. Finally, a kilogram‐scale enzymatic reaction (250 mM substrate, 220 L) was carried out to produce β‐amino acid (229 mM). Sitagliptin phosphate was chemically synthesized from β‐amino acids with 82% yield and > 99% purity.

Green Chemistry, 2021

We developed a multienzyme biocatalytic cascade with high atom efficiency and a self-sufficient r... more We developed a multienzyme biocatalytic cascade with high atom efficiency and a self-sufficient redox network for the synthesis of nylon monomers without adding auxiliary enzymes to recycle cofactors.

Frontiers in Bioengineering and Biotechnology, 2021

Herein, we report the development of a multi-enzyme cascade using transaminase (TA), esterase, al... more Herein, we report the development of a multi-enzyme cascade using transaminase (TA), esterase, aldehyde reductase (AHR), and formate dehydrogenase (FDH), using benzylamine as an amino donor to synthesize the industrially important compound sitagliptin intermediate. A panel of 16 TAs was screened using ethyl 3-oxo-4-(2,4,5-trifluorophenyl) butanoate as a substrate (1). Amongst these enzymes, TA from Roseomonas deserti (TARO) was found to be the most suitable, showing the highest activity towards benzylamine (∼70%). The inhibitory effect of benzaldehyde was resolved by using AHR from Synechocystis sp. and FDH from Pseudomonas sp., which catalyzed the conversion of benzaldehyde to benzyl alcohol at the expense of NAD(P)H. Reaction parameters, such as pH, buffer system, and concentration of amino donor, were optimized. A single whole-cell system was developed for co-expressing TARO and esterase, and the promoter engineering strategy was adopted to control the expression level of each bi...

Chemical Reviews, 2021

The two main strategies for enzyme engineering, directed evolution and rational design, have foun... more The two main strategies for enzyme engineering, directed evolution and rational design, have found widespread applications in improving the intrinsic activities of proteins. Although numerous advances have been achieved using these ground-breaking methods, the limited chemical diversity of the biopolymers, restricted to the 20 canonical amino acids, hampers creation of novel enzymes that Nature has never made thus far. To address this, much research has been devoted to expanding the protein sequence space via chemical modifications and/or incorporation of noncanonical amino acids (ncAAs). This review provides a balanced discussion and critical evaluation of the applications, recent advances, and technical breakthroughs in biocatalysis for three approaches: (i) chemical modification of cAAs, (ii) incorporation of ncAAs, and (iii) chemical modification of incorporated ncAAs. Furthermore, the applications of these approaches and the result on the functional properties and mechanistic study of the enzymes are extensively reviewed. We also discuss the design of artificial enzymes and directed evolution strategies for enzymes with ncAAs incorporated. Finally, we discuss the current challenges and future perspectives for biocatalysis using the expanded amino acid alphabet.

Angewandte Chemie, 2020

We report a highly atom-efficient integrated cofactor/co-product recycling cascade employing cycl... more We report a highly atom-efficient integrated cofactor/co-product recycling cascade employing cycloalkylamines as multifaceted starting materials for the synthesis of nylon building blocks. Reactions using E. coli whole cells as well as purified enzymes produced excellent conversions ranging from >80 to 95% into desired ω-amino acids, respectively with varying substrate concentrations. The applicability of this tandem biocatalytic cascade was demonstrated to produce the corresponding lactams by employing engineered biocatalysts. For instance, ε-caprolactam, a valuable polymer building block was synthesized with 75% conversion from 10 mM cyclohexylamine by employing whole-cell biocatalysts. This cascade could be an alternative for bio-based production of ω-amino acids and corresponding lactam compounds.

Catalysis Letters, 2020

Medium to long chain aliphatic amino alcohols have numerous applications in polymer-based industr... more Medium to long chain aliphatic amino alcohols have numerous applications in polymer-based industries. We herein report a biocatalytic reduction of amino fatty acids into corresponding amino alcohols using carboxylic acid reductase and E coli endogenous aldehyde reductases. Interestingly, employing this reaction in tandem with minimizing the hurdle of substrate insolubility, the biocatalysts successfully produced 5.76 mM 12-amino-1-dodecanol from 10 mM of corresponding 12-amino-dodecanoic acid. Finally, up to 9.17 mM of 8-amino-1-octanol was obtained in 24 h on a preparative scale reaction of 50 mM substrate. Graphic Abstract

Chemical Communications, 2019

A cellular system for the in vivo biosynthesis of Tyr-analogs and their concurrent incorporation ... more A cellular system for the in vivo biosynthesis of Tyr-analogs and their concurrent incorporation into target proteins is reported.

Biotechnology and Bioprocess Engineering, 2019

The development of new enzyme engineering technologies has been actively pursued as the industria... more 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.

Catalysts, 2019

Amine dehydrogenases (AmDHs) efficiently catalyze the NAD(P)H-dependent asymmetric reductive amin... more Amine dehydrogenases (AmDHs) efficiently catalyze the NAD(P)H-dependent asymmetric reductive amination of prochiral carbonyl substrates with high enantioselectivity. AmDH-catalyzed oxidative deamination can also be used for the kinetic resolution of racemic amines to obtain enantiopure amines. In the present study, kinetic resolution was carried out using a coupled-enzyme cascade consisting of AmDH and alanine dehydrogenase (AlaDH). AlaDH efficiently catalyzed the conversion of pyruvate to alanine, thus recycling the nicotinamide cofactors and driving the reaction forward. The ee values obtained for the kinetic resolution of 25 and 50 mM rac-α-methylbenzylamine using the purified enzymatic systems were only 54 and 43%, respectively. The use of whole-cells apparently reduced the substrate/product inhibition, and the use of only 30 and 40 mgDCW/mL of whole-cells co-expressing AmDH and AlaDH efficiently resolved 100 mM of rac-2-aminoheptane and rac-α-methylbenzylamine into the correspo...

Bioorganic Chemistry, 2018

Two new compounds, lasdiplactone (1) and lasdiploic acid (2) and one known compound 3 were isolat... more Two new compounds, lasdiplactone (1) and lasdiploic acid (2) and one known compound 3 were isolated from the chloroform extract of cell free filtrate of the endophytic fungus Lasiosdiplodia pseudotheobromae. The structures of new compounds were determined by interplay of spectral techniques (IR, mass, 1 H NMR, 13 C NMR, DEPT, and 2D NMR). The absolute configuration at C-4 position of 1 was established as S using a process similar to modified Mosher's method. The absolute configuration of 2 was established by comparing its ECD spectrum with the calculated ECD spectra of all possible isomers. In the in vitro XO inhibition assay, the highest inhibition was exhibited by 3 with an IC 50 of 0.38 ± 0.13 μg/ml, followed by 2 with an IC 50 of 0.41 ± 0.1 μg/ml and the least in 1. The oxidized form of 1 also showed high XO inhibition with IC 50 of 0.35 ± 0.13 μg/ml.

Chemistry & Biology, 2004

natural base. In comparison to the conventional methods using modified ribonucleotides in place o... more natural base. In comparison to the conventional methods using modified ribonucleotides in place of one of the natural ribonucleotides [2-10], such as 5-substituted uracil nucleotides, or by chemical RNA synthesis, the specific transcription would be a more powerful tool for