Self-sufficient Biocatalysts Research Articles

Facile Asymmetric Syntheses of Non-Natural Amino Acid (S)-Cyclopropylglycine by the Developed NADH-Driven Biocatalytic System

A self-sufficient bifunctional enzyme integrating reductive amination and coenzyme regeneration activities was developed and successfully employed to synthesize (S)-cyclopropylglycine with an improved reaction rate 2.1-fold over the native enzymes and a short bioconversion period of 6 h at a high substrate concentration of 120 g·L−1 and space–time yield of (S)-cyclopropylglycine up to 377.3 g·L−1·d−1, higher than that of any previously reported data. Additionally, (S)-cyclopropylglycine could be continuously synthesized for 90 h with the enzymes packed in a dialysis tube, providing 634.6 g of (S)-cyclopropylglycine with >99.5% ee and over 95% conversion yield up to 12 changes. These results confirmed that the newly developed NADH-driven biocatalytic system could be utilized as a self-sufficient biocatalyst for industrial application in the synthesis of (S)-cyclopropylglycine, which provides a chiral center and cyclopropyl fragment for the frequent synthesis of preclinical/clinical drug molecules.

Overcoming Biochemical Limitations of Galactose Oxidase through the Design of a Solid-Supported Self-Sufficient Biocatalyst.

Galactose Oxidase (GalOx) has gained significant interest in biocatalysis due to its ability for selective oxidation beyond the natural oxidation of galactose, enabling the production of valuable derivatives. However, the practical application of GalOx has been hindered by the limited availability of active and stable biocatalysts, as well as the inherent biochemical limitations such as oxygen (O2 ) dependency and the need for activation. In this study, we addressed these challenges by immobilizing GalOx into agarose-based and Purolite supports to enhance its activity and stability. Additionally, we identified and quantified the oxygen supply limitation into solid catalysts by intraparticle oxygen sensing showing a trade-off between the amount of protein loaded onto the solid support and the catalytic effectiveness of the immobilized enzyme. Furthermore, we coimmobilized a heme-containing protein along with the enzyme to function as an activator. To evaluate the practical application of the immobilized GalOx, we conducted the oxidation of galactose in an instrumented aerated reactor. The results showcased the efficient performance of the immobilized enzyme in the 8 h reaction cycle. Notably, the GalOx immobilized into dextran sulfate-activated agarose exhibited improved stability, overcoming the need for a soluble activator supply, and demonstrated exceptional performance in galactose oxidation. These findings offer promising prospects for the utilization of GalOx in technical biocatalytic applications.

Glycosylation of polyphenolic compounds: Design of a self-sufficient biocatalyst by co-immobilization of a glycosyltransferase, a sucrose synthase and the cofactor UDP

Glycosyltransferases catalyze the regioselective glycosylation of polyphenolic compounds, increasing their solubility without altering their antioxidant properties. Leloir-type glycosyltransferases require UDP-glucose as a cofactor to glycosylate a hydroxyl of the polyphenol, which is expensive and unstable. To simplify these processes for industrial implementation, the preparation of self-sufficient heterogeneous biocatalysts is needed. In this study, a glycosyltransferase and a sucrose synthase (as an UDP-regenerating enzyme) were co-immobilized onto porous agarose-based supports coated with polycationic polymers: polyethylenimine and polyallylamine. In addition, the UDP cofactor was strongly ionically adsorbed and co-immobilized with the enzymes, eliminating the need to add it separately. Thus, the optimal self-sufficient heterogeneous biocatalyst was able to catalyze the glycosylation of three polyphenolic compounds (piceid, phloretin and quercetin) with in situ regeneration of the UDP-glucose, allowing multiple consecutive reaction cycles without the addition of exogenous cofactor. A TTN value of 50 (theoretical maximum) was obtained in the reaction of piceid glycosylation, after 5 reaction cycles, using the self-sufficient biocatalyst based on an improved sucrose synthase variant. This result was 5-fold higher than the obtained using soluble cofactor and the co-immobilized enzymes, and much higher than those reported in the literature for similar processes.

Self-sufficient biocatalysts constructed using chitin-based microspheres

The concomitant recycling of enzymes and co-factors is a big challenge in biocatalysis. In this study, co-immobilization of the enzyme and the co-factor pyridoxal 5′-phosphate was achieved using chitin-based microspheres as the support. Chitin/chitosan composite microspheres were prepared and then modified with polyethyleneimine. Three pyridoxal 5′-phosphate-independent enzymes were used to demonstrate that the self-sufficient biocatalysts based on chitin/chitosan composite microspheres and polyethyleneimine modified microspheres displayed good generalizability for high recycling efficiency of both enzyme and pyridoxal 5′-phosphate. Furthermore, the self-sufficient biocatalysts were applied in the continuous flow production of cadaverine and maintained good continuous catalysis without the exogenous addition of pyridoxal 5′-phosphate, suggesting that this co-immobilization platform shows promise for application in industrial biocatalysis.

Expression and Characterization of Monomeric Recombinant Isocitrate Dehydrogenases from Corynebacterium glutamicum and Azotobacter vinelandii for NADPH Regeneration.

The enzymatic transformation of various chemicals, especially using NADPH-dependent hydroxylase, into more soluble and/or high value-added products has steadily garnered increasing attention. However, the industrial application of these NADPH-dependent hydroxylases has been limited due to the high cost of the cofactor NADPH. As an alternative, enzymatic NADPH-regeneration systems have been developed and are frequently used in various fields. Here, we expressed and compared two recombinant isocitrate dehydrogenases (IDHs) from Corynebacterium glutamicum and Azotobacter vinelandii in Escherichia coli. Both enzymes were hyper-expressed in the soluble fraction of E. coli and were single-step purified to apparent homogeneity with yields of more than 850 mg/L. These enzymes also functioned well when paired with NADPH consumption systems. Specifically, NADPH was regenerated from NADP+ when an NADPH-consuming cytochrome P450 BM3 from Bacillus megaterium was incorporated. Therefore, both enzymes could be used as alternatives to the commonly used regeneration system for NADPH. These enzymes also have promising potential as genetic fusion partners with NADPH-dependent enzymes due to the monomeric nature of their quaternary structure, thereby resulting in self-sufficient biocatalysts via NADPH regeneration in a single polypeptide with NADPH-dependent activity.

Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity

Fusion of multiple enzymes to multifunctional constructs has been recognized as a viable strategy to improve enzymatic properties at various levels such as stability, activity and handling. In this study, the genes coding for cytochrome P450 BM3 from B. megaterium and formate dehydrogenase from Pseudomonas sp. were fused to enable both substrate oxidation catalyzed by P450 BM3 and continuous cofactor regeneration by formate dehydrogenase within one construct. The order of the genes in the fusion as well as the linkers that bridge the enzymes were varied. The resulting constructs were compared to individual enzymes regarding substrate conversion, stability and kinetic parameters to examine whether fusion led to any substantial improvements of enzymatic properties. Most noticeably, an activity increase of up to threefold was observed for the fusion constructs with various substrates which were partly attributed to the increased diflavin reductase activity of the P450 BM3. We suggest that P450 BM3 undergoes conformational changes upon fusion which resulted in altered properties, however, no NADPH channeling was detected for the fusion constructs.

Chitin biopolymer mediates self-sufficient biocatalyst of pyridoxal 5′-phosphate and L-lysine decarboxylase

Enzyme cofactors are essential in biocatalysis for many enzymes. However, their high commercial cost limits the industrial application of cofactor-dependent enzymes. We overcame this challenge by constructing a self-sufficient biocatalyst that co-immobilised pyridoxal 5′-phosphate (PLP) and L-lysine decarboxylase (LDC) using natural polymer chitin as the functional material. First, degree of deacetylation (DDA) of chitin was regulated to enhance PLP adsorption and immobilisation. Then, the enzyme was immobilised via fusion of a chitin-binding domain. Chitin with 36% DDA efficiently co-immobilised LDC and PLP. Laser scanning confocal microscope and scanning electron microscope proved that the LDC and PLP were immobilised on the chitin. The self-sufficient biocatalyst retained over 55% of its original activity after five cycles. The proposed strategy provides a new way to achieve co-immobilisation of PLP-dependent enzymes.

Asymmetric synthesis of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate using a self-sufficient biocatalyst based on carbonyl reductase and cofactor co-immobilization.

tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate [(3R,5S)-CDHH] is the key chiral intermediate to synthesize the side chain of the lipid-lowering drug rosuvastatin. Carbonyl reductases showed excellent activity for the biosynthesis of (3R,5S)-CDHH. The requirement of cofactor NADH/NADPH leads to high cost for the industrial application of carbonyl reductases. In this study, a self-sufficient biocatalyst based on carbonyl reductase and NADP+ co-immobilization strategy was developed on an amino resin carrier LX-1000HAA (SCR-NADP+@LX-1000HAA). The self-sufficient biocatalyst achieved in situ cofactor regeneration and showed the activity recovery of 77.93% and the specific activity of 70.45U/g. Asymmetric synthesis of (3R,5S)-CDHH using SCR-NADP+@LX-1000HAA showed high enantioselectivity (>99% e.e.) and yield (98.54%). Batch reactions were performed for ten cycles without extra addition of NADP+, and the total yield of (3R,5S)-CDHH achieved at 10.56g/g biocatalyst. The present work demonstrated the potential of the self-sufficient biocatalyst for the asymmetric biosynthesis of rosuvastatin intermediate.

Redox self-sufficient biocatalyst system for conversion of 3,4-Dihydroxyphenyl-L-alanine into (R)- or (S)-3,4-Dihydroxyphenyllactic acid.

We developed an efficient multi-enzyme cascade reaction to produce (R)- or (S)-3,4-Dihydroxyphenyllactic acid [(R)- or (S)-Danshensu, (R)- or (S)-DSS] from 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) in Escherichia coli by introducing tyrosine aminotransferase (tyrB), glutamate dehydrogenase (cdgdh) and D-aromatic lactate dehydrogenase (csldhD) or L-aromatic lactate dehydrogenase (tcldhL). First, the genes in the pathway were overexpressed and fine-tuned for (R)- or (S)-DSS production. The resulting strain, E. coli TGL 2.1 and E. coli TGL 2.2, which overexpressed tyrB with the stronger T7 promoter and cdgdh, csldhD or tcldhL with the weaker Trc promoter, E. coli TGL 2.1 yielded 57% increase in (R)-DSS production: 59.8 ± 2.9mM. Meanwhile, E. coli TGL 2.2 yielded 54% increase in (S)-DSS production: 52.2 ± 2.4mM. The optimal concentration of L-glutamate was found to be 20mM for production of (R)- or (S)-DSS. Finally, L-DOPA were transformed into (R)- or (S)-DSS with an excellent enantiopure form (enantiomeric excess > 99.99%) and productivity of 6.61mM/h and 4.48mM/h, respectively.

A novel self-sufficient biocatalyst based on transaminase and pyridoxal 5′-phosphate covalent co-immobilization and its application in continuous biosynthesis of sitagliptin

Transaminases (TAs, EC 2.6.1.X) are a class of promising catalysts for the synthesis of chiral amines. The addition of exogenous expensive cofactor, pyridoxal 5′-phosphate (PLP) leads to the high cost and complicated reaction composition and thus limits the industrial implementation of TAs. In this study, a novel self-sufficient biocatalyst was developed based on transaminase and PLP covalent co-immobilization with the activity recovery of 83.6% and the specific activity of 343.0 U/g catalyst. The self-sufficient BgTA biocatalyst was employed in the continuous biosynthesis of (R)-sitagliptin in a recirculating packed bed reactor (RPBR) for 700 batches reaction without extra addition of PLP. The yield and e.e. of (R)-sitagliptin for each batch were above 90% and 99% respectively. The space-time yield was 40.0 g/(L·h) which was higher than ever reported. This is the first report about the covalent co-immobilization of TA and PLP with high activity and stability, and our work also contributed to the economic flexibility of chiral amine pharmaceutical intermediates biosynthesis.

Enrichment and Coimmobilization of Cofactors and His-Tagged ω-Transaminase into Nanoflowers: A Facile Approach to Constructing Self-Sufficient Biocatalysts

ω-Transaminase (ω-TA), a cofactor-dependent enzyme, has been shown to be an outstanding biocatalyst for the production of chiral amines. The need for improved enzyme stability and cofactor (PLP) recyclability is an important goal in the biocatalysis process. In this study, a novel self-sufficient biocatalyst, ω-TA-PLP@Co3(PO4)2, was constructed by coimmobilizing ω-TA and PLP into Co3(PO4)2 nanoflowers for the first time. The preparation process of ω-TA-PLP@Co3(PO4)2 was investigated in detail, and the formation mechanism was clarified. The resulting ω-TA-PLP@Co3(PO4)2 was characterized by various analytical techniques, such as scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and X-ray photoelectron spectroscopy (XPS). Compared with free ω-TA, the ω-TA-PLP@Co3(PO4)2 showed improved catalytic efficiency, thermal stability, pH stability, and storage stability. Furthermore, ω-TA-PLP@Co3(PO4)2 could maintain about 72% of initial overall activity after 12 catalytic cycles. All thes...

An Engineered Self-Sufficient Biocatalyst Enables Scalable Production of Linear α-Olefins from Carboxylic Acids

Fusing the decarboxylase OleTJE and the reductase domain of P450BM3 creates a self-sufficient protein, OleT-BM3R, which is able to efficiently catalyze oxidative decarboxylation of carboxylic acids into linear α-olefins (LAOs) under mild aqueous conditions using O2 as the oxidant and NADPH as the electron donor. The compatible electron transfer system installed in the fusion protein not only eliminates the need for auxiliary redox partners, but also results in boosted decarboxylation reactivity and broad substrate scope. Coupled with the phosphite dehydrogenase-based NADPH regeneration system, this enzymatic reaction proceeds with improved product titers of up to 2.51 g L–1 and volumetric productivities of up to 209.2 mg L–1 h–1 at low catalyst loadings (∼0.02 mol %). With its stability and scalability, this self-sufficient biocatalyst offers a nature-friendly approach to deliver LAOs.

Wolfgang Kroutil

Angewandte Chemie International EditionVolume 55, Issue 18 p. 5372-5372 Author ProfileFree Access Wolfgang Kroutil First published: 01 March 2016 https://doi.org/10.1002/anie.201509882AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract “When I was eight, I wanted to be a surgeon. Chemistry is fun because there is always something new and it is full of surprises ...” This and more about Wolfgang Kroutil can be found on page 5372. 1 Table 1. Wolfgang Kroutil Date of birth: August 5, 1972 Position: Professor, Institute of Chemistry, University of Graz E-mail: wolfgang.kroutil@uni-graz.at Homepage: http://biocatalysis.uni-graz.at/ Education: 1991–1995 Diploma and master's studies at the Graz University of Technology 1998 PhD with Kurt Faber and Stanley M. Roberts at the Graz University of Technology and Exeter University, respectively 1998–1999 Postdoc, Novartis Crop Protection, Basel Awards: 2003 Science Award, Austrian Chemical Society; 2012 Biocat Award, 6th International Congress of Biocatalysis; 2015 Biotrans Award, 12th Biotrans—International Symposium on Biocatalysis and Biotransformations Current research interests: Biocatalytic synthesis, asymmetric synthesis, enzymes in organic synthesis, enzyme mechanisms, C−C bond formation, redox enzymes, cascades, systems biocatalysis Hobbies: Gardening, swimming, handcraft, skiing, traveling When I was eight, I wanted to be a surgeon. Chemistry is fun because there is always something new and it is full of surprises. My favorite painter is Salvador Dalí. My favorite season is spring, when nature awakens after a cold winter. My biggest inspiration is watching nature. I get advice from friends, my wife and my daughter. My favorite way to spend a holiday is with my family and without the internet. My favorite composers include Andrew Lloyd Webber, Giuseppe Verdi, and Wolfgang Amadeus Mozart. The talent I would like to be gifted with is playing the violin. My favorite molecule is water, because it enables life and does not behave as expected. If I could be anyone for a day, I would be Yoda (Star Wars). My 5 top papers: References 1“Introducing an In Situ Capping Strategy in Systems Biocatalysis to Access 6-Aminohexanoic acid”: J. H. Sattler, M. Fuchs, F. G. Mutti, B. Grischek, P. Engel, J. Pfeffer, J. M. Woodley, W. Kroutil, Angew. Chem. Int. Ed. 2014, 53, 14153; Angew. Chem. 2014, 126, 14377. (Circumvents product inhibition to form one of the most important building blocks for polymers.) 2“Redox Self-Sufficient Biocatalyst Network for the Amination of Primary Alcohols”: J. H. Sattler, M. Fuchs, K. Tauber, F. G. Mutti, K. Faber, J. Pfeffer, T. Haas, W. Kroutil, Angew. Chem. Int. Ed. 2012, 51, 9156; Angew. Chem. 2012, 124, 9290. (Biocatalytic “borrowing-hydrogen” concept for the amination of primary alcohols.) 3“Biocatalytic Enantioselective Oxidative C−C Coupling by Aerobic C−H Activation”: J. H. Schrittwieser, V. Resch, J. H. Sattler, W.-D. Lienhart, K. Durchschein, A. Winkler, K. Gruber, P. Macheroux, W. Kroutil, Angew. Chem. Int. Ed. 2011, 50, 1068; Angew. Chem. 2011, 123, 1100. (Opened novel synthetic routes to berberine alkaloids.) 4“Orchestration of Concurrent Oxidation and Reduction Cycles for Stereoinversion and Deracemisation of sec-Alcohols”: C. V. Voss, C. C. Gruber, K. Faber, T. Knaus, P. Macheroux, W. Kroutil, J. Am. Chem. Soc. 2008, 130, 13969. (Proceeds with oxidative and reductive cofactor recycling systems running concomitantly without compartmentalization.) 5“Formal Asymmetric Biocatalytic Reductive Amination”: D. Koszelewski, I. Lavandera, D. Clay, G. M. Guebitz, D. Rozzell, W. Kroutil, Angew. Chem. Int. Ed. 2008, 47, 9337; Angew. Chem. 2008, 120, 9477. (Stereoselective biocatalyzed amination of ketones at the expense of ammonia and a reducing agent.) Volume55, Issue18April 25, 2016Pages 5372-5372 ReferencesRelatedInformation

An artificial self-sufficient cytochrome P450 directly nitrates fluorinated tryptophan analogs with a different regio-selectivity.

Aromatic nitration is an immensely important industrial process to produce chemicals for a variety of applications, but it often suffers from multiple unsolved challenges. Enzymes as biocatalysts have been increasingly used for organic chemistry synthesis due to their high selectivity and environmental friendliness, but nitration has benefited minimally from the development of biocatalysis. In this work, we aimed to develop TxtE as practical biocatalysts for aromatic nitration. TxtE is a unique class I cytochrome P450 enzyme that nitrates the indole of l-tryptophan. To develop cost-efficient nitration processes, we fused TxtE with the reductase domains of CYP102A1 (P450BM3) and of P450RhF to create class III self-sufficient biocatalysts. The best engineered fusion protein was comparable with wild type TxtE in terms of nitration performance and other key biochemical properties. To demonstrate the application potential of the fusion enzyme, we nitrated 4-F-dl-tryptophan and 5-F-l-tryptophan in large scale enzymatic reactions. Tandem MS/MS and NMR analyses of isolated products revealed altered nitration sites. To our knowledge, these studies represent the first practice in developing biological nitration approaches and lay a solid basis to the use of TxtE-based biocatalysts for the production of valuable nitroaromatics.

Recent Advances in Cofactor Regeneration Systems Applied to Biocatalyzed Oxidative Processes

Nowadays, the design of sustainable processes applicable at industrial scale is highly desirable due to environmental reasons. The use of biocatalytic reactions to carry out oxidative transformations is one of the possible strategies framed in here due to the mildness and usually high selectivities achieved with these methods. Anyway, while implementing this type of system at industrial scale several drawbacks must be overcome in order to obtain efficient setups. Historically, one of the main issues has been the cofactor-dependency of these biocatalysts to be active. Herein, we will show the state-of-the-art concerning the recent efforts developed in the design of (potentially) efficient methods to regenerate the cofactor in oxidative transformations. Thus, the more studied enzymatic methods will be discussed to highlight some recent examples dealing with the co-expression of both oxidative and recycling enzymes in one host or the development of self-sufficient biocatalysts. Furthermore, novel applications of these systems to couple two productive synthetic reactions will also be reviewed. Subsequently, we will focus on some recent examples related with the employment of electrochemical, photochemical, and chemical strategies to carry out the nicotinamide coenzyme or the flavin/heme prosthetic group regeneration. In the case of biocatalysts that use NAD(P) as electron shuttle, these systems have also been employed to replace it, allowing the design of nicotinamide- free recycling setups. In all cases, the (dis)advantages that these methodologies present will be briefly discussed.