Retro-aldol Cleavage Research Articles

Recent advances in pyridoxal phosphate-dependent enzymes and their applications

Pyridoxal phosphate (PLP), the active form of vitamin B6, is an important coenzyme in various enzyme-catalyzed reactions. PLP-dependent enzymes can catalyze a variety of chemical reactions, such as racemization, decarboxylation, β-addition, β-elimination, retro-aldol cleavage, transamination, and α-elimination. They are biologically synthesized a powerful tool for a variety of natural amino acids, non-natural amino acids and their related compounds. This article details the structural features and catalytic mechanisms of typical PLP-dependent enzymes such as ω-transaminase, lysine decarboxylase, threonine aldolase, and L-tyrosine phenol-lyase, and reviews the research progress in molecular modification and industrial applications of these enzymes. Finally, this article provides an outlook on the future development of PLP-dependent enzymes, including in vivo regeneration system and industrial applications of PLP cofactors, and discusses the tremendous potential of these enzymes in biocatalytic applications.

Shy is a proteobacterial steroid hydratase which catalyzes steroid side chain degradation without requiring a catalytically inert partner domain

Shy (side chain hydratase) and Sal (side chain aldolase), are involved in successive reactions in the pathway of bile acid side chain catabolism in Proteobacteria. Untagged Shy co-purified with His-tagged Sal indicating that the two enzymes form a complex. Shy contains a MaoC and a DUF35 domain. When co-expressed with Sal, the DUF35 domain but not the MaoC domain of Shy was observed to co-purify with Sal, indicating Sal interacts with Shy through its DUF35 domain. The MaoC domain of Shy (ShyMaoC) remained catalytically viable and could hydrate cholyl-enoyl-CoA with similar catalytic efficiency as in the Shy-Sal complex. Sal expressed with the DUF35 domain of Shy (Sal-ShyDUF35) was similarly competent for the retroaldol cleavage of cholyl-3-OH-CoA. ShyMaoC showed a preference for C5 side chain bile acid substrates, exhibiting low activity towards C3 side chain substrates. The ShyMaoC structure was determined by X-ray crystallography, showing a hot dog fold with a short central helix surrounded by a twisted anti-parallel β-sheet. Modeling and mutagenesis studies suggest that the bile acid substrate occupies the large open cleft formed by the truncated central helix and repositioning of the active site housing. ShyMaoC therefore contains two substrate binding sites per homodimer, making it distinct from previously characterized MaoC steroid hydratases that are (pseudo)-heterodimers with one substrate binding site per dimer. The characterization of Shy provides insight into how MaoC family hydratases have adapted to accommodate large polycyclic substrates that can facilitate future engineering of these enzymes to produce novel steroid pharmaceuticals.

Carbon-Supported Ru-Ni and Ru-W Catalysts for the Transformation of Hydroxyacetone and Saccharides into Glycol-Derived Primary Amines.

Nitrogen-containing molecules are used for the synthesis of polymers, surfactants, agrochemicals, and dyes. In the context of green chemistry, it is important to form such compounds from bioresource. Short-chain primary amines are of interest for the polymer industry, like 2-aminopropanol, 1-aminopropan-2-ol, and 1,2-diaminopropane. These amines can be formed through the amination of oxygenated substrates, preferably in aqueous phase. This is possible with heterogeneous catalysts, however, effective systems that allow reactions under mild conditions are lacking. We report an efficient catalyst Ru-Ni/AC for the reductive amination of hydroxyacetone into 2-aminopropanol. The catalyst has been reused during 3 cycles demonstrating a good stability. As a prospective study, extension to the reactivity of (poly)carbohydrates has been realized. Despite a lesser efficiency, 2-aminopropanol (9 % yield of amines) has been formed from fructose, the first example from a carbohydrate. This was possible using a 7.5 %Ru-36 %WxC/AC catalyst, composition allowing a one-pot retro-aldol cleavage into hydroxyacetone and reductive amination. The transformation of cellulose through sequential reactions with a combination of 30 %W2C/AC and 7.5 %Ru-36 %WxC/AC system gave 2 % of 2-aminopropanol, corresponding to the first example of the formation of this amine from cellulose with heterogeneous catalysts.

Kinetics on the valorization of hexoses with Sn-USY catalysts in methanolic media: glycosidation vs retroaldol cleavage

The catalytic performance of potassium-exchanged Sn-containing USY zeolite ([K]Sn-USY) for the transformation of hexoses into methyl lactate in methanol has been tackled. This work explores the effects of temperature and...

Aqueous microdroplets promote C-C bond formation and sequences in the reverse tricarboxylic acid cycle.

The reverse tricarboxylic acid cycle (rTCA) is a central anabolic network that uses carbon dioxide (CO2) and may have provided complex carbon substrates for life before the advent of RNA or enzymes. However, non-enzymatic promotion of the rTCA cycle, in particular carbon fixation, remains challenging, even with primordial metal catalysis. Here, we report that the fixation of CO2 by reductive carboxylation of succinate and α-ketoglutarate was achieved in aqueous microdroplets under ambient conditions without the use of catalysts. Under identical conditions, the aqueous microdroplets also facilitated the sequences in the rTCA cycle, including reduction, hydration, dehydration and retro-aldol cleavage and linked with the glyoxylate cycle. These reactions of the rTCA cycle were compatible with the aqueous microdroplets, as demonstrated with two-reaction and four-reaction sequences. A higher selectivity giving higher product yields was also observed. Our results suggest that the microdroplets provide an energetically favourable microenvironment and facilitate a non-enzymatic version of the rTCA cycle in prebiotic carbon anabolism.

Characterization of thermostable serine hydroxymethyltransferase for β-hydroxy amino acids synthesis

β-hydroxy amino acids, such as serine, threonine, and phenylserine, are important compounds for medical purposes. To date, there has been only limited exploration of thermostable serine hydroxylmethyltransferase (SHMT) for the synthesis of these amino acids, despite the great potential that thermostable enzymes may offer for commercial use due to their high stability and catalytic efficiencies. ITBSHMT_1 (ITB serine hydroxylmethyltransferase clone number 1) from thermophilic and methanol-tolerant bacteria Pseudoxanthomonas taiwanensis AL17 was successfully cloned. Biocomputational analysis revealed that ITBSHMT_1 contains Pyridoxal-3′-phosphate and tetrahydrofolatebinding residues. Structural comparisons show that ITBSHMT_1 has 5 additional residues VSRQG on loop near PLP-binding site as novel structural feature which distinguish this enzyme with other characterized SHMTs. In silico mutation revealed that the fragment might have very essential role in maintaining of PLP binding on structure of ITBSHMT_1. Recombinant protein was produced in Escherichia coli Rosetta 2(DE3) in soluble form and purified using NiNTA affinity chromatography. The purified protein demonstrated the best activity at 80 °C and pH 7.5 based on the retro aldol cleavage of phenylserine. Activity decreased significantly in the presence of 3 mM transition metal ions but increased in the presence of 30 mM β-mercaptoethanol. ITBSHMT_1 demonstrated Vmax, Km, Kcat, and Kcat/Km at 242 U/mg, 23.26 mM, 186/s, and 8/(mM.s), respectively. The aldol condensation reaction showed the enzyme’s best activity at 80 °C for serine, threonine, or phenylserine, with serine synthesis showing the highest specific activity. Biocomputational analysis revealed that high intramolecular interaction within the 3D structure of ITBSHMT_1 might be correlated with the enzyme’s high thermal stability. The above data suggest that ITBSHMT_1 is a potential and novel enzyme for the production of various β-hydroxy amino acids.

L-Serine Biosensor-Controlled Fermentative Production of l-Tryptophan Derivatives by Corynebacterium glutamicum.

Simple Summaryl-tryptophan is an amino acid found in proteins. Its derivatives, such as hydroxylated or halogenated l-tryptophans, find applications in the chemical and pharmaceutical industries, for example, in therapeutic peptides. Biotechnology provides a sustainable way for the production of l-tryptophan and its derivatives. In the final reaction of l-tryptophan biosynthesis in bacteria, such as Corynebacterium glutamicum, another amino acid, l-serine, is incorporated. Here, we show that C. glutamicum TrpB is able to convert indole derivatives, which were added to cells synthesizing l-serine, to the corresponding l-tryptophan derivatives. The gene trpB was expressed under the control of the l-serine-responsive transcriptional activator SerR in the C. glutamicum cells engineered for this fermentation process.l-Tryptophan derivatives, such as hydroxylated or halogenated l-tryptophans, are used in therapeutic peptides and agrochemicals and as precursors of bioactive compounds, such as serotonin. l-Tryptophan biosynthesis depends on another proteinogenic amino acid, l-serine, which is condensed with indole-3-glycerophosphate by tryptophan synthase. This enzyme is composed of the α-subunit TrpA, which catalyzes the retro-aldol cleavage of indole-3-glycerol phosphate, yielding glyceraldehyde-3-phosphate and indole, and the β-subunit TrpB that catalyzes the β-substitution reaction between indole and l-serine to water and l-tryptophan. TrpA is reported as an allosteric actuator, and its absence severely attenuates TrpB activity. In this study, however, we showed that Corynebacterium glutamicum TrpB is catalytically active in the absence of TrpA. Overexpression of C. glutamicum trpB in a trpBA double deletion mutant supported growth in minimal medium only when exogenously added indole was taken up into the cell and condensed with intracellularly synthesized l-serine. The fluorescence reporter gene of an l-serine biosensor, which was based on the endogenous transcriptional activator SerR and its target promoter PserE, was replaced by trpB. This allowed for l-serine-dependent expression of trpB in an l-serine-producing strain lacking TrpA. Upon feeding of the respective indole derivatives, this strain produced the l-tryptophan derivatives 5-hydroxytryptophan, 7-bromotryptophan, and 5-fluorotryptophan.

Mechanism-Based Inactivation of Mycobacterium tuberculosis Isocitrate Lyase 1 by (2R,3S)-2-Hydroxy-3-(nitromethyl)succinic acid.

The isocitrate lyase paralogs of Mycobacterium tuberculosis (ICL1 and 2) are essential for mycobacterial persistence and constitute targets for the development of antituberculosis agents. We report that (2R,3S)-2-hydroxy-3-(nitromethyl)succinic acid (5-NIC) undergoes apparent retro-aldol cleavage as catalyzed by ICL1 to produce glyoxylate and 3-nitropropionic acid (3-NP), the latter of which is a covalent-inactivating agent of ICL1. Kinetic analysis of this reaction identified that 5-NIC serves as a robust and efficient mechanism-based inactivator of ICL1 (kinact/KI = (1.3 ± 0.1) × 103 M-1 s-1) with a partition ratio <1. Using enzyme kinetics, mass spectrometry, and X-ray crystallography, we identified that the reaction of the 5-NIC-derived 3-NP with the Cys191 thiolate of ICL1 results in formation of an ICL1-thiohydroxamate adduct as predicted. One aspect of the design of 5-NIC was to lower its overall charge compared to isocitrate to assist with cell permeability. Accordingly, the absence of the third carboxylate group will simplify the synthesis of pro-drug forms of 5-NIC for characterization in cell-infection models of M. tuberculosis.

Characteristic flavor formation of thermally processed N-(1-deoxy-α-d-ribulos-1-yl)-glycine: Decisive role of additional amino acids and promotional effect of glyoxal

The role of amino acids and α-dicarbonyls in the flavor formation of Amadori rearrangement product (ARP) during thermal processing was investigated. Comparisons of the volatile compounds and their concentrations when N-(1-deoxy-α-d-ribulos-1-yl)-glycine reacted with different amino acids or glyoxal (GO) at 100 °C were executed. Additional amino acids, such as glycine (Gly), in ARP models contributed to the diversity of furanoids by the chain elongation of the derived formaldehyde. Whereas the monoanion of additional glutamic acid acted as nucleophile, favored 2-ethyl-3,5-dimethylpyrazine and 2,5-dimethylpyrazine formation; the nonionized amino group of additional lysine were involved in α-dicarbonyls formation, causing pyrazine and methylpyrazine accumulation in the ARP model. Moreover, the high dosage and pH stabilization of additional GO probably promoted the ARP degradation and deoxyosones retro-aldol cleavage, resulting in methylpyrazine rather than furanoids formation. The present work provided the guidance for the controlled flavor formation of ARP in industrial application.

Heterologous gene expression and characterization of two serine hydroxymethyltransferases from Thermoplasma acidophilum.

Serine hydroxymethyltransferase (SHMT) and threonine aldolase are classified as fold type I pyridoxal-5'-phosphate-dependent enzymes and engaged in glycine biosynthesis from serine and threonine, respectively. The acidothermophilic archaeon Thermoplasma acidophilum possesses two distinct SHMT genes, while there is no gene encoding threonine aldolase in its genome. In the present study, the two SHMT genes (Ta0811 and Ta1509) were heterologously expressed in Escherichia coli and Thermococcus kodakarensis, respectively, and biochemical properties of their products were investigated. Ta1509 protein exhibited dual activities to catalyze tetrahydrofolate (THF)-dependent serine cleavage and THF-independent threonine cleavage, similar to other SHMTs reported to date. In contrast, the Ta0811 protein lacks amino acid residues involved in the THF-binding motif and catalyzes only the THF-independent cleavage of threonine. Kinetic analysis revealed that the threonine-cleavage activity of the Ta0811 protein was 3.5times higher than the serine-cleavage activity of Ta1509 protein. In addition, mRNA expression of Ta0811 gene in T. acidophilum was approximately 20times more abundant than that of Ta1509. These observations suggest that retroaldol cleavage of threonine, mediated by the Ta0811 protein, has a major role in glycine biosynthesis in T. acidophilum.

Effluent decontamination by the ibuprofen-mineralizing strain, Sphingopyxis granuli RW412: Metabolic processes.

The high global consumption of ibuprofen and its limited elimination by wastewater treatment plants (WWTPs), has led to the contamination of aquatic systems by this common analgesic and its metabolites. The potentially negative environmental and public health effects of this emerging contaminant have raised concerns, driving the demand for treatment technologies. The implementation of bacteria which mineralize organic contaminants in biopurification systems used to decontaminate water or directly in processes in WWTPs, is a cheap and sustainable means for complete elimination before release into the environment. In this work, an ibuprofen-mineralizing bacterial strain isolated from sediments of the River Elbe was characterized and assayed to remediate different ibuprofen-polluted media. Strain RW412, which was identified as Sphingopyxis granuli, has a 4.48Mb genome which includes plasmid sequences which harbor the ipf genes that encode the first steps of ibuprofen mineralization. Here, we confirm that these genes encode enzymes which initiate CoA ligation to ibuprofen, followed by aromatic ring activation by a dioxygenase and retroaldol cleavage to unequivocally produce 4-isobutylcatechol and propionyl-CoA which then undergo further degradation. In liquid mineral salts medium, the strain eliminated more than 2mM ibuprofen within 74h with a generation time of 16h. Upon inoculation into biopurification systems, it eliminated repeated doses of ibuprofen within a few days. Furthermore, in these systems the presence of RW412 avoided the accumulation of ibuprofen metabolites. In ibuprofen-spiked effluent from a municipal WWTP, ibuprofen removal by this strain was 7 times faster than by the indigenous microbiota. These results suggest that this strain can persist and remain active under environmentally relevant conditions, and may be a useful innovation to eliminate this emerging contaminant from urban wastewater treatment systems.

L-Threonine Transaldolase Activity Is Enabled by a Persistent Catalytic Intermediate.

l-Threonine transaldolases (lTTAs) are a poorly characterized class of pyridoxal-5'-phosphate (PLP) dependent enzymes responsible for the biosynthesis of diverse β-hydroxy amino acids. Here, we study the catalytic mechanism of ObiH, an lTTA essential for biosynthesis of the β-lactone natural product obafluorin. Heterologously expressed ObiH purifies as a mixture of chemical states including a catalytically inactive form of the PLP cofactor. Photoexcitation of ObiH promotes the conversion of the inactive state of the enzyme to the active form. UV-vis spectroscopic analysis reveals that ObiH catalyzes the retro-aldol cleavage of l-threonine to form a remarkably persistent glycyl quinonoid intermediate, with a half-life of ∼3 h. Protonation of this intermediate is kinetically disfavored, enabling on-cycle reactivity with aldehydes to form β-hydroxy amino acids. We demonstrate the synthetic potential of ObiH via the single step synthesis of (2S,3R)-β-hydroxyleucine. To further understand the structural features underpinning this desirable reactivity, we determined the crystal structure of ObiH bound to PLP as the Schiff's base at 1.66 Å resolution. This high-resolution model revealed a unique active site configuration wherein the evolutionarily conserved Asp that traditionally H-bonds to the cofactor is swapped for a neighboring Glu. Molecular dynamics simulations combined with mutagenesis studies indicate that a structural rearrangement is associated with l-threonine entry into the catalytic cycle. Together, these data explain the basis for the unique reactivity of lTTA enzymes and provide a foundation for future engineering and mechanistic analysis.

Engineering aldolases for asymmetric synthesis.

Formation of carbon-carbon bonds is central to synthetic chemistry. The aldol reaction provides the chemistry to fuse a nucleophilic enolate with an electrophilic aldehyde to form a new CC bond between two newly formed asymmetric centers. A major challenge in the reaction is steering the stereochemistry of the product. Aldolases are lyases that catalyze aldol reactions as well as the retro-aldol cleavage, and are abundant in cellular metabolism. Due to the often exquisite stereoselectivity in aldolase catalyzed carboligation reactions, these enzymes are gaining increased interest as potentially important tools in asymmetric synthesis of new useful compounds. Fructose 6-phosphate aldolase from Escherichia coli (FSA) is of special interest because of its very unusual independence of phosphorylated reactant substrates. The current text describes the protein engineering of FSA, applying principles of directed evolution, for the generation, production and characterization of new aldolase variants. A range of new enantiopure polyhydroxylated compounds were produced applying isolated FSA variants.

Thermal and IR-Fourier transform spectroscopic study of water-soluble carbohydrate-arylamine condensation products in neutral media

Production of synthetic analogues of natural compounds, including high-molecular ones, as well as study of processes of their structure formation, is a pressing task of organic substances technology. To a large extent, this applies to synthetic humic substances, important products characterized by plant growth effect, chelating and other important properties. Simple carbohydrates and aromatic amines as oxygen and nitrogen-containing constituents during condensation in ethanol media produce water-fractionated products, the soluble fractions of which are studied herein by thermal degradation in combination with IR-Fourier transform spectroscopy. Spectral band profile is confirmed mainly by aliphatic structure with high degree of functionalization by carboxyl, hydroxyl and amine groups. As the isolated water-soluble solid products are thermally decomposed in the range of 100-180 °С temperatures in an inert atmosphere, the intensity of bands at 1030 and 1090 cm-1 decreases, and the intensity of the first band drops to almost zero at 180 °С. This experimental fact indicates the progress of thermal dehydration processes, indicating the presence of hydroxyl functions in the structure of the products. The latter are both OH-groups of carbohydrate residue and groups, formed during condensation processes. Additionally, the intensity of the absorption band in the region of 1600 cm-1 corresponding to the fluctuations of double bonds resulting from dehydration is increased. It can be assumed that unlike products of insoluble fractions, as well as products of acid-catalyzed condensations, water-soluble products are formed in neutral ethanol media, the main processes of formation of which are processes of direct retro-aldol cleavage of N-glycosylamines with subsequent condensation of decomposition products. Such a set of processes is an alternative to the experimental difficulty of Amadori rearrangement and lead to formation of products structure differ from one for acid catalyzed condensation products.

Bioconjugation with Aminoalkylhydrazine for Efficient Mass Spectrometry-Based Detection of Small Carbonyl Compounds.

Bioconjugation through oxime or hydrazone formation is a versatile strategy for covalent labeling of biomolecules in vitro and in vivo. In this work, a mass spectrometry-based method was developed for the bioconjugation of small carbonyl compounds (CCs) with an aminoalkylhydrazine to form stable hydrazone conjugates that are readily detectable with electrospray ionization mass spectrometry (ESI-MS). Out of all hydrazine reagents tested, 2-(dimethylamino)ethylhydrazine (DMAEH) was selected for further analysis due to the fastest reaction rates observed. A thorough study of the reaction kinetics between structurally varied short-chain CCs and DMAEH was performed with the second-order reaction rate constants spanning in the range of 0.23–208 M–1 s–1. In general, small aldehydes reacted faster than the corresponding ketones. Moreover, a successful reaction monitoring of a deoxyribose-5-phosphate aldolase-catalyzed reversible retro–aldol cleavage of deoxyribose was demonstrated. Thus, the developed method shows potential also for ESI-MS-based enzyme kinetics studies.

Functional Characterization of YdjH, a Sugar Kinase of Unknown Specificity in Escherichia coli K12.

The ydj gene cluster is annotated to catalyze the catabolism of an unknown carbohydrate. Previously, YdjI, a class II aldolase, was shown to catalyze the retro-aldol cleavage of l-glycero-l-galacto-octuluronate-1-phosphate into DHAP and l-arabinuronate. In this report, the functional characterization of YdjH is presented. YdjH catalyzes the phosphorylation of 2-keto-monosaccharides at the C1 hydroxyl group with a substrate profile significantly more stringent than that of YdjI. Similar to YdjI, YdjH shows a strong preference for higher-order monosaccharides (seven to nine carbons) with a carboxylate terminus. The best substrate was determined to be l-glycero-l-galacto-octuluronate, yielding l-glycero-l-galacto-octuluronate-1-phosphate with a kcat of 16 s-1 and a kcat/Km of 2.1 × 104 M-1 s-1. This is apparently the first reported example of kinase activity with eight-carbon monosaccharides. Two crystal structures of YdjH were previously determined to 2.15 and 1.8 Å resolution (Protein Data Bank entries 3H49 and 3IN1 ). We present an analysis of the active site layout and use computational docking to identify potential key residues in the binding of l-glycero-l-galacto-octuluronate.

New Stereoselective Biocatalysts for Carboligation and Retro-Aldol Cleavage Reactions Derived from d-Fructose 6-Phosphate Aldolase.

d-Fructose 6-phosphate aldolase (FSA) catalyzes the asymmetric cross-aldol addition of phenylacetaldehyde and hydroxyacetone. We conducted structure-guided saturation mutagenesis of noncatalytic active-site residues to produce new FSA variants, with the goal of widening the substrate scope of the wild-type enzyme toward a range of para- and meta-substituted arylated aldehydes. After a single generation of mutagenesis and selection, enzymes with diverse substrate selectivity scopes were identified. The kinetic parameters and stereoselectivities for a subset of enzyme/substrate combinations were determined for the reactions in both the aldol addition and cleavage reaction directions. The achieved collection of new aldolase enzymes provides new tools for controlled asymmetric synthesis of substituted aldols.

Milk protein cleaves C–C bonds

Enzymes that can form or break carbon-carbon bonds are sought-after biocatalysts. Tuning them to work on all manner of substrates can be challenging. Now, however, chemists at Texas A&M University have shown that the most abundant protein in whey, β-lactoglobulin, can catalyze retroaldol cleavage of α,β-unsaturated aldehydes (Org. Biomol. Chem. 2018, DOI: 10.1039/c8ob00139a). This milk protein’s natural function isn’t known, but Coran M. H. Watanabe’s team previously showed that it can promote condensation of α,β-unsaturated aldehydes. Watanabe’s group surmised that β-lactoglobulin would have retroaldolase activity as well, because its active-site lysine resembles that of another group’s de novo-designed retroaldolase. The team’s experiments showed this to be the case. The enzyme works best on substrates with hydrophobic phenyl and naphthyl side chains, thus complementing the selectivity of similar enzymes that work best on hydrophilic substrates. The team increased the enzyme’s yield by blocking a compet...

Kinetics of homogeneous and heterogeneous reactions in the reductive aminolysis of glucose with dimethylamine

The reductive aminolysis of glucose with dimethylamine (DMA) as aminating agent has been investigated experimentally as well as via kinetic model construction. A fed-batch reactor configuration was used at following conditions: temperatures ranging between 383 K–398 K, total pressures from 6.0 MPa to 7.5 MPa, an overall catalyst to glucose ratio of 9–31 gcat mol−1, an overall DMA to glucose ratio of 12–24 mol mol−1 and an overall hydrogen to glucose ratio of 5–10 mol mol−1. Such a reactor configuration, combined with a controlled feeding rate of the sugar, allowed to significantly avoid degradation reactions. The main desired products, i.e., dimethylaminoethanol (DMAE) and tetramethylethylenediamine (TMEDA), were obtained after amination followed by retro-aldol cleavage with maximum selectivity, amounting up to 15% and 60% respectively. Retro-aldol cleavage after amination proceeds at lower temperatures, evidenced by an activation energy of 60 kJ mol−1, than without amination, where activation energies amounting to 140 kJ mol−1 have been reported. A higher catalyst to glucose ratio leads to more parallel side products such as N,N-dimethylglucamine and 4‐dimethylamino-1,2,3-butanetriol. The effects of temperature and catalyst to glucose ratio were much more pronounced than that of the total pressure or the ratio of the aminating agent to glucose. The developed kinetic model is based on the most prominent homogeneous bulk phase and heterogeneously catalyzed reactions and accounts quantitatively for degradation reactions. A statistically and physically significant model which could satisfactorily reproduce the experimental observations, was thus obtained.

Biocatalysis with the milk protein β-lactoglobulin: promoting retroaldol cleavage of α,β-unsaturated aldehydes

Enzymes with a hydrophobic binding site and an active site lysine have been suggested to be promiscuous in their catalytic activity. β-Lactoglobulin (BLG), the principle whey protein found in milk, possesses a central calyx that binds non-polar molecules. Here, we report that BLG can catalyze the retro-aldol cleavage of α,β-unsaturated aldehydes making it a naturally occurring protein capable of catalyzing retro-aldol reactions on hydrophobic substrates. Retroaldolase activity was seen to be most effective on substrates with phenyl or naphthyl side-chains. Use of a brominated substrate analogue inhibitor increases the product yield by a factor of three. BLG's catalytic activity and its ready availability make it a prime candidate for the development of commercial biocatalysts.