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1.
Front Bioeng Biotechnol ; 11: 1280464, 2023.
Article in English | MEDLINE | ID: mdl-38033815

ABSTRACT

The U.S. Department of Energy has listed levulinic acid (LA) as one of the top 12 compounds derived from biomass. LA has gained much attention owing to its conversion into enantiopure 4-aminopentanoic acid through an amination reaction. Herein, we developed a coupled-enzyme recyclable cascade employing two transaminases (TAs) for the synthesis of (S)-4-aminopentanoic acid. TAs were first utilized to convert LA into (S)-4-aminopentanoic acid using (S)-α-Methylbenzylamine [(S)-α-MBA] as an amino donor. The deaminated (S)-α-MBA i.e., acetophenone was recycled back using a second TAs while using isopropyl amine (IPA) amino donor to generate easily removable acetone. Enzymatic reactions were carried out using different systems, with conversions ranging from 30% to 80%. Furthermore, the hybrid nanoflowers (HNF) of the fusion protein were constructed which afforded complete biocatalytic conversion of LA to the desired (S)-4-aminopentanoic acid. The created HNF demonstrated storage stability for over a month and can be reused for up to 7 sequential cycles. A preparative scale reaction (100 mL) achieved the complete conversion with an isolated yield of 62%. Furthermore, the applicability of this recycling system was tested with different ß-keto ester substrates, wherein 18%-48% of corresponding ß-amino acids were synthesized. Finally, this recycling system was applied for the biosynthesis of pharmaceutical important drug sitagliptin intermediate ((R)-3-amino-4-(2,4,5-triflurophenyl) butanoic acid) with an excellent conversion 82%.

2.
Front Chem ; 10: 839636, 2022.
Article in English | MEDLINE | ID: mdl-35295971

ABSTRACT

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 resolution of the 1-phenylpropan-1-amine was performed using benzaldehyde as an amino acceptor, which is cheaper than pyruvate. Our results highlight the utility of ncAAs for designing enzymes with enhanced functionality beyond the limit of 20 canonical amino acids.

3.
Biotechnol Adv ; 53: 107868, 2021 12.
Article in English | MEDLINE | ID: mdl-34774927

ABSTRACT

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.


Subject(s)
Amines , Amino Acids , Biocatalysis , Enzymes/metabolism
4.
Front Bioeng Biotechnol ; 9: 757062, 2021.
Article in English | MEDLINE | ID: mdl-34692666

ABSTRACT

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 biocatalyst. The whole-cell reactions were performed with varying substrate concentrations (10-100 mM), resulting in excellent conversions (ranging from 72 to 91%) into the desired product. Finally, the applicability of this cascade was highlighted on Gram scale, indicating production of 70% of the sitagliptin intermediate with 61% isolated yield. The protocol reported herein may be considered an alternative to existing methods with respect to the use of cheaper amine donors as well as improved synthesis of (R) and (S) enantiomers with the use of non-chiral amino donors.

5.
Biotechnol Bioeng ; 118(8): 3263-3268, 2021 08.
Article in English | MEDLINE | ID: mdl-33990942

ABSTRACT

Here, we report a bienzymatic cascade to produce ß-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 ß-amino acid (229 mM). Sitagliptin phosphate was chemically synthesized from ß-amino acids with 82% yield and > 99% purity.


Subject(s)
Escherichia coli , Esterases , Genetic Engineering , Microorganisms, Genetically-Modified , Promoter Regions, Genetic , Sitagliptin Phosphate/metabolism , Transaminases , Escherichia coli/genetics , Escherichia coli/metabolism , Esterases/genetics , Esterases/metabolism , Microorganisms, Genetically-Modified/genetics , Microorganisms, Genetically-Modified/metabolism , Transaminases/genetics , Transaminases/metabolism
6.
Chem Rev ; 121(10): 6173-6245, 2021 05 26.
Article in English | MEDLINE | ID: mdl-33886302

ABSTRACT

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.


Subject(s)
Amino Acids/biosynthesis , Glucosidases/metabolism , Metalloproteins/metabolism , Amino Acids/chemistry , Biocatalysis , Molecular Structure , Protein Engineering
7.
Angew Chem Int Ed Engl ; 60(7): 3481-3486, 2021 02 15.
Article in English | MEDLINE | ID: mdl-33140477

ABSTRACT

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 and 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.


Subject(s)
Amines/metabolism , Nylons/metabolism , Amines/chemistry , Metabolic Engineering , Nylons/chemistry
8.
Chem Commun (Camb) ; 55(100): 15133-15136, 2019 Dec 28.
Article in English | MEDLINE | ID: mdl-31789331

ABSTRACT

Herein we report the development of an efficient cellular system for the in vivo biosynthesis of Tyr-analogs and their concurrent incorporation into target proteins by the residue-specific approach. This system makes use of common phenol derivatives and the tyrosine phenol lyase machinery to create various tyrosine analogues that impart desired properties on the target proteins. Biosynthesized 2-fluorotyrosine was incorporated into three industrially important enzymes which resulted in enhanced thermostability.


Subject(s)
Protein Engineering , Tyrosine Phenol-Lyase/metabolism , Tyrosine/biosynthesis , Biocatalysis , Fluorometry , Oxidoreductases/genetics , Oxidoreductases/metabolism , Transaminases/genetics , Transaminases/metabolism , Tyrosine/analogs & derivatives , Tyrosine Phenol-Lyase/genetics
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