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%.

Chemical modification of enzymes to improve biocatalytic performance.

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.

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.

Recent Advances in Biocatalysis with Chemical Modification and Expanded Amino Acid Alphabet.

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.

Machine Learning Modeling for Ultrasonication-Mediated Fermentation of Penicillium brevicompactum to Enhance the Release of Mycophenolic Acid.

Described here is the modeling used to improve the mycophenolic acid (MPA) titer from Penicillium brevicompactum using central composite design and a comparatively newer, data-centric approach method k-nearest-neighbor algorithm. The two models for enhancing MPA production using P. brevicompactum were compared with respect to ultrasonic stimulation. During the ultrasonic treatment, we studied different independent factors such as ultrasound power, irradiation duration, treatment frequency and duty cycle to determine their ability to enhance the MPA titer value. The optimized factors such as a treatment time of 10 min (50% duty cycles) with a 12-h interlude at fixed ultrasonic power and frequency (200 W, 40 kHz) were used for ultrasonic treatment of a mycelial culture from the 2nd to 10th day of fermentation. Thus the production of MPA was improved 1.64-fold under the optimized sonication conditions compared with the non-sonicated batch fermentation (non-optimized conditions).

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.

Liposomal Delivery of Mycophenolic Acid With Quercetin for Improved Breast Cancer Therapy in SD Rats.

The present study explores the influence of mycophenolic acid (MPA) in combination therapy with quercetin (QC) (impeding MPA metabolic rate) delivered using the liposomal nanoparticles (LNPs). Mycophenolic acid liposome nanoparticles (MPA-LNPs) and quercetin liposome nanoparticles (QC-LNPs) were individually prepared and comprehensively characterized. The size of prepared MPA-LNPs and QC-LNPs were found to be 183 ± 13 and 157 ± 09.8, respectively. The in vitro studies revealed the higher cellular uptake and cytotoxicity of combined therapy (MPA-LNPs + QC-LNPs) compared to individual ones. Moreover pharmacokinetics studies in female SD-rat shown higher T 1 / 2 value (1.94 fold) of combined therapy compared to MPA. Furthermore, in vivo anticancer activity in combination of MPA-LNPs and QC-LNPs was also significantly higher related to other treatments groups. The combination therapy of liposomes revealed the new therapeutic approach for the treatment of breast cancer.

Mycophenolate co-administration with quercetin via lipid-polymer hybrid nanoparticles for enhanced breast cancer management.

Mycophenolic acid (MPA) has promising anticancer properties; however, it has limited clinical applications in vivo due to hydrophobic nature, high first-pass metabolism, lack of targeting, etc. These associated problems could be addressed by developing a suitable delivery vehicle, inhibiting the first-pass metabolism and additive/synergistic pharmacodynamic effect. Thus, MPA loaded highly stable lipid polymer hybrid nanoparticles (LPNs) were developed and investigated with the combination of quercetin (QC), a CYP 450 inhibitor cum anticancer. LPNs of MPA and QC (size; 136 ±â€¯12 and 176 ±â€¯35 nm, respectively) demonstrated higher cellular uptake and cytotoxicity of combination therapy (MPA-LPN + QC-LPN) compared to individual congeners in MCF-7 cells. In vivo pharmacokinetics demonstrated 2.17 fold higher T1/2 value and significantly higher pharmacodynamic activity in case of combination therapy compared to free MPA. In nutshell, the combinatory therapeutic regimen of MPA and QC could be a promising approach in improved breast cancer management.

In vivo biosynthesis of tyrosine analogs and their concurrent incorporation into a residue-specific manner for enzyme engineering.

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.

Production of 12-hydroxy dodecanoic acid methyl ester using a signal peptide sequence-optimized transporter AlkL and a novel monooxygenase.

In this study, a signal peptide of AlkL was replaced with other signal peptides to improve the soluble expression and thereby facilitate the transport of dodecanoic acid methyl ester (DAME) substrate into the E. coli. Consequently, AlkL with signal peptide FadL (AlkLf) showed higher transport activity toward DAME. Furthermore, the promoter optimization for the efficient heterologous expression of the transporter AlkLf and alkane monooxygenase (AlkBGT) system was conducted and resulted in increased ω-oxygenation activity of AlkBGT system. Moreover, bioinformatic studies led to the identification of novel monooxygenase from Pseudomonas pelagia (Pel), which exhibited 20% higher activity towards DAME as substrate compared to AlkB. Finally, the construction of a chimeric transporter and the expression of newly identified monooxygenase enabled the production of 44.8 ±â€¯7.5 mM of 12-hydroxy dodecanoic acid methyl ester (HADME) and 31.8 ±â€¯1.7 mM of dodecanedioic acid monomethyl ester (DDAME) in a two-phase reaction system.

Machine learning modelling for the ultrasonication-mediated disruption of recombinant E. coli for the efficient release of nitrilase.

The ultrasonication-mediated cell disruption of recombinant E. coli was modeled using three machine learning techniques namely Multiple linear regression (MLR), Multi-layer perceptron (MLP) and Sequential minimal optimization (SMO). The four attributes were cellmass concentration (g/L), acoustic power (A), duty cycle (%) and treatment time of sonication (min). For the three responses (nitrilase, total protein release and cell disruption) MLP model was found to be at par with RSM model in terms of generalization as well as prediction capability. Nitrilase release was significantly influenced by the cellmass concentration so was in case of total protein release. Fraction of cells disrupted was heavily influenced by acoustic power and sonication time. Almost 32 U/mL nitrilase could be released for 300 g/L cellmass concentration when sonicated at 225 W for 1 min with 20% duty cycle.

Purification and characterization of arginine deiminase from Pseudomonas putida: Structural insights of the differential affinities of l-arginine analogues.

Arginine deiminase (ADI) from Pseudomonas putida was purified using ammonium sulphate precipitation, anion exchange and hydrophobic interaction chromatography. Influence of various chemical compounds (metal ions, reducing agents, sulphydryl agents, and surfactants) on the catalytic activity of ADI was determined was evaluated on the purified ADI. The enzyme displayed high sensitivity towards thiol binding metal ions, chemicals acting on sulfhydryl group, and most of the surfactants. Substrate specificity studies exhibited that among the eight substrate analogues tested, canavanine had the highest affinity for ADI, followed by d-arginine and guanidine. Canavanine decreased the ADI activity up to 50% at its lowest concentration tested (10 mM), while d-arginine decreased the ADI activity up to ∼4% at its highest tested concentration (200 mM). Differential affinities of the structural analogues of arginine towards ADI were further studied by molecular modeling methods, which included homology modeling, molecular docking and molecular dynamic simulations. The molecular docking studies revealed the critical importance of residues Arg 243, Asp 166, Asp 280, Gly 299 and His 278. RMSDs for protein-ligand complexes were within a range of 1-3 Å, suggesting that the complexes were stable throughout the molecular dynamic simulation. The formation of strong hydrogen bonds by residues Asn 160, Asp166, Arg 185, Arg243, Asp280 and Gly 399 in l-arginine were preserved in the case of d-arginine and canavanine and was responsible for higher affinity towards ADI. Calculations of the substrate binding energies revealed that binding energies ΔGbind and ΔGvdw play a critical role for the differential affinities of various substrate analogues towards P. putida ADI.

Enzymatic synthesis of sitagliptin intermediate using a novel ω-transaminase.

Enantiopure ß-amino acids are essential precursors of various pharmaceuticals, agrochemicals and other industrially important chemicals. In this study, we selected sixteen potential ω-Transaminases (ω-TAs) by BLAST and phylogenetic tree analysis. These ω-TAs were cloned, purified and tested for their reactivity for the synthesis of model ß-amino acid (R)-3-amino-4-(2,4,5-triflurophenyl) butanoic acid [3-ATfBA], a key precursor for sitagliptin. In an enzymatic cascade, lipase converted ß-ketoester substrate to ß-keto acid, which was subsequently aminated by the selected ω-TA to its corresponding ß-amino acid. A potent enzyme from Ilumatobacter coccineus (ω-TAIC) was identified for the production of 3-ATfBA. The pH dependency of the product inhibition suggested that lowering the reaction pH to 7.0 can circumvent the inhibition of ω-TAIC by 3-ATfBA and about 92.3% conversion of 100 mM ß-keto ester substrate could be achieved. The applicability of this enzymatic system was further evaluated at the scale of 140 mM, wherein 3-ATfBA was generated with excellent conversion (81.9%) and enantioselectivity (99% ee). Furthermore, ω-TAIC was successfully used for the synthesis of various ß-amino acids from their corresponding ß-keto ester substrates.

Combined effect of attrition and ultrasound on the disruption of Pseudomonas putida for the efficient release of arginine deiminase.

Disruption of Pseudomonas putida KT2440 by ultrasound treatment in a bath sonicator, in presence of the glass beads, was carried out for the release of arginine deiminase (ADI) and the results were compared with that of by Dyno-mill. The release of ADI depended mainly on the bead size and cellmass concentration being disrupted in bead mill. Nearly 23 U mL-1 ADI was released when slurry with a cell-mass concentration of 250 g L-1 was disintegrated for 9 min with 80% bead loading (0.25 mm) in Dyno-mill. Marginally higher amount of ADI (24.1 U mL-1 ) was released by the bath sonication of 250 g L-1 cellmass slurry for 30 min with the beads (0.1 mm) and a sonication power of 170 W. The glass beads, suspended along with the cellmass slurry in bath sonicator, efficiently disrupted the microbial cells to release ADI. Variation in the kinetic constants for the performance parameters implied that ADI release and cell disruption kinetics is a function of disruption technique used and the process variables thereof. Estimation of location factor suggested that selective release of ADI can be achieved. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1185-1194, 2018.

Biosynthesis of the Nylon 12 Monomer, ω-Aminododecanoic Acid with Novel CYP153A, AlkJ, and ω-TA Enzymes.

Bioplastics are derived from renewable biomass sources, such as vegetable oils, cellulose, and starches. An important and high-performance member of the bioplastic family is Nylon 12. The biosynthesis of ω-amino dodecanoic acid (ω-AmDDA), the monomer of Nylon 12 from vegetable oil derivatives is considered as an alternative to petroleum-based monomer synthesis. In this study, for the production of ω-AmDDA from dodecanoic acid (DDA), the cascade of novel P450 (CYP153A), alcohol dehydrogenase (AlkJ), and ω-transaminase (ω-TA) is developed. The regioselective ω-hydroxylation of 1 mM DDA with near complete conversion (>99%) is achieved using a whole-cell biocatalyst co-expressing CYP153A, ferredoxin reductase and ferredoxin. When the consecutive biotransformation of ω-hydroxy dodecanoic acid (ω-OHDDA) is carried out using a whole-cell biocatalyst co-expressing AlkJ and ω-TA, 1.8 mM ω-OHDDA is converted into ω-AmDDA with 87% conversion in 3 h. Finally, when a one-pot reaction is carried out with 2 mM DDA using both whole-cell systems, 0.6 mM ω-AmDDA is produced after a 5 h reaction. The results demonstrated the scope of the potential cascade reaction of novel CYP153A, AlkJ, and ω-TA for the production of industrially important bioplastic monomers, amino fatty acids, from FFAs.

Ultrasonic disruption of Pseudomonas putida for the release of arginine deiminase: Kinetics and predictive models.

The responses of the ultrasound-mediated disruption of Pseudomonas putida KT2440 were modelled as the function of biomass concentration in the cell suspension; the treatment time of sonication; the duty cycle and the acoustic power of the sonicator. For the experimental data, the response surface (RSM), the artificial neural network (ANN) and the support vector machine (SVM) models were compared for their ability to predict the performance parameters. The satisfactory prediction of the unseen data of the responses implied the proficient generalization capabilities of ANN. The extent of the cell disruption was mainly dependent on the acoustic power and the biomass concentration. The cellmass concentration in the slurry most strongly influenced the ADI and total protein release. Nearly 28U/mL ADI was released when a biomass concentration of 300g/L was sonicated for 6min with an acoustic power of 187.5W at 40% duty cycle. Cell disruption obeyed first-order kinetics.

Production of Mycophenolic Acid by Penicillium brevicompactum Using Solid State Fermentation.

Solid-state fermentation using the microfungus Penicillium brevicompactum for the production of mycophenolic acid is reported in this paper. Of the initial substrates tested (whole wheat, cracked wheat, long grain Basmati rice, and short grain Parmal rice), Parmal rice proved to be the best. Under initial conditions, using steamed Parmal rice with 80% (w/w) initial moisture content, a maximum mycophenolic acid concentration of 3.4 g/kg substrate was achieved in 12 days of fermentation at 25 °C. The above substrate was supplemented with the following additional nutrients (g/L packed substrate): glucose 40.0, peptone 54.0, KH2PO4 8.0, MgSO4⋅7H2O 2.0, glycine 7.0, and methionine 1.65 (initial pH 5.0). A small amount of a specified trace element solution was also added. The final mycophenolic acid concentration was increased to nearly 4 g/kg substrate by replacing glucose with molasses. Replacing Parmal rice with rice bran as substrate further improved the mycophenolic acid production to nearly 4.5 g/kg substrate.

Disruption of Pseudomonas putida by high pressure homogenization: a comparison of the predictive capacity of three process models for the efficient release of arginine deiminase.

Disruption of Pseudomonas putida KT2440 by high-pressure homogenization in a French press is discussed for the release of arginine deiminase (ADI). The enzyme release response of the disruption process was modelled for the experimental factors of biomass concentration in the broth being disrupted, the homogenization pressure and the number of passes of the cell slurry through the homogenizer. For the same data, the response surface method (RSM), the artificial neural network (ANN) and the support vector machine (SVM) models were compared for their ability to predict the performance parameters of the cell disruption. The ANN model proved to be best for predicting the ADI release. The fractional disruption of the cells was best modelled by the RSM. The fraction of the cells disrupted depended mainly on the operating pressure of the homogenizer. The concentration of the biomass in the slurry was the most influential factor in determining the total protein release. Nearly 27 U/mL of ADI was released within a single pass from slurry with a biomass concentration of 260 g/L at an operating pressure of 510 bar. Using a biomass concentration of 100 g/L, the ADI release by French press was 2.7-fold greater than in a conventional high-speed bead mill. In the French press, the total protein release was 5.8-fold more than in the bead mill. The statistical analysis of the completely unseen data exhibited ANN and SVM modelling as proficient alternatives to RSM for the prediction and generalization of the cell disruption process in French press.

Use of response surface method for maximizing the production of arginine deiminase by Pseudomonas putida.

Statistically designed experiments were used to optimize the production of arginine deiminase (ADI) by Pseudomonas putida KT2440 in batch culture. A Plackett-Burman design involving eleven factors showed that ADI production was most influenced by the initial pH and the initial concentrations of glucose and yeast extract. A central composite experimental design showed that the optimal values of these factors were 8.0, 10 g/L and 12.5 g/L, respectively. The other components of the optimal culture medium were bacto peptone 7.5 g/L, Triton X-100 0.30% (v/v), and arginine 3 g/L, for a culture temperature of 25 °C. Compared with the basal medium, the ADI activity in the optimized medium had nearly 4.5-fold increase (4.31 U/mL). The optimized medium was then used for a further study of ADI production in a 14 L stirred tank bioreactor. The agitation speed and the aeration rates were varied to determine suitable values of these variables.

Production of mycophenolic acid by Penicillium brevicompactum-A comparison of two methods of optimization.

Production of mycophenolic acid (MPA) by submerged fermentation using the microfungus Penicillium brevicompactum MTCC 8010 is reported here. Screening experiments were used to identify: the suitable media composition; the optimal initial pH; and the optimal incubation temperature to maximize the production of MPA in batch cultures. The initial concentrations of the selected sources of carbon (glucose), nitrogen (peptone) and the precursors (methionine, glycine) were then optimized by: (1) one-at-a-time variation of factors; and (2) a central composite design (CCD) of experiments, in a 12-day batch culture at an initial pH of 5.0, an incubation temperature of 25 °C, and an agitation speed of 200 rpm. The medium optimized using the one-at-a-time variation yielded a peak MPA titer of 1232 ± 90 mg/L. The medium optimized by the CCD method yielded a 40% higher MPA titer of 1737 ± 55 mg/L. The latter value was nearly 9-fold greater than the titer achieved prior to optimization.