Dual-function transaminases with hybrid nanoflower for the production of value-added chemicals from biobased levulinic acid.

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%.

Non-Canonical Amino Acid-Based Engineering of (R)-Amine Transaminase.

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.

Synthesis of Sitagliptin Intermediate by a Multi-Enzymatic Cascade System Using Lipase and Transaminase With Benzylamine as an Amino Donor.

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.

Promoter engineering-mediated Tuning of esterase and transaminase expression for the chemoenzymatic synthesis of sitagliptin phosphate at the kilogram-scale.

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.

Identification and structure-activity relationship studies of small molecule inhibitors of the human cathepsin D.

Cathepsin D, an aspartyl protease, is an attractive therapeutic target for various diseases, primarily cancer and osteoarthritis. However, despite several small molecule cathepsin D inhibitors being developed, that are highly potent, most of them show poor microsomal stability, which in turn limits their clinical translation. Herein, we describe the design, optimization and evaluation of a series of novel non-peptidic acylguanidine based small molecule inhibitors of cathepsin D. Optimization of our hit compound 1a (IC50 = 29 nM) led to the highly potent mono sulphonamide analogue 4b (IC50 = 4 nM), however with poor microsomal stability (HLM: 177 and MLM: 177 µl/min/mg). To further improve the microsomal stability while retaining the potency, we carried out an extensive structure-activity relationship screen which led to the identification of our optimised lead 24e (IC50 = 45 nM), with an improved microsomal stability (HLM: 59.1 and MLM: 86.8 µl/min/mg). Our efforts reveal that 24e could be a good starting point or potential candidate for further preclinical studies against diseases where Cathepsin D plays an important role.

An Integrated Cofactor/Co-Product Recycling Cascade for the Biosynthesis of Nylon Monomers from Cycloalkylamines.

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.