Downstream processing of 1,3‐propanediol fermentation broth

The downstream processing of 1,3‐propanediol fermentation broth using flocculation, reactive extraction, and reactive distillation was studied. Cellular debris and soluble protein in the broth were flocculated by combined use of chitosan and polyacrylamide at optimal concentrations of 150 ppm and 70 ppm, respectively; the soluble protein in the broth decreased to 0.06 g L^?1, and the recovery ratio of the supernatant liquor to broth was greater than 99%. 1,3‐Propanediol and other alcohols were extracted from the supernatant liquor by reacting with butyraldehyde. In a four‐stage countercurrent extraction with the volume ratio of the extraction solvent to the aqueous phase being 20:100, more than 99% 1,3‐propanediol acetal (2‐propyl‐1,3‐dioxane) and 2,3‐butanediol acetal (2‐propyl‐4,5‐dimethyl‐1,3‐dioxolane) were recovered from the aqueous phase; 35% of the glycerol acetals were recovered. The acetals produced were hydrolyzed in a reactive distillation column using the strongly acidic cation‐exchange resin as catalyst, the bottom product obtained was a mixture of 1,3‐propanediol (407 g L^?1), 2,3‐butanediol (252 g L^?1), glycerol (277 g L^?1), and glycerol acetals (146 g L^?1). Copyright © 2005 Society of Chemical Industry     

A Kinase‐Independent One‐Pot Multienzyme Cascade for an Expedient Synthesis of Guanosine 5′‐Diphospho‐d‐mannose

Biomimetic synthesis routes towards the important natural d ‐mannosyl donor guanosine 5′‐diphospho‐d ‐mannose (GDP‐Man) rely on kinase‐catalyzed nucleotide triphosphate (NTP)‐dependent phosphorylations of d ‐mannose (Man), to give d ‐mannose 6‐phosphate or α‐d ‐mannose 1‐phosphate (αMan 1‐P) as an intermediate product. A GDP‐Man synthesis not requiring the kinase/NTP system would be practical and cost‐effective. Here, we have developed a multienzyme cascade towards GDP‐Man, characterized in that αMan 1‐P was obtained by a diastereoselective phosphatase‐catalyzed phosphorylation of Man. α‐d ‐Glucose 1‐phosphate (αGlc 1‐P), prepared in situ through phosphorylase‐catalyzed conversion of sucrose in the presence of inorganic phosphate, was used as an expedient phosphoryl donor. The incipient αMan 1‐P and guanosine triphosphate (GTP) were converted into GDP‐Man by a highly manno compared to gluco selective nucleotidyltransferase. Pyrophosphatase was additionally required to hydrolyze the pyrophosphate released from the GTP, thus driving the reaction towards GDP‐Man. The enzymatic cascade was operated with the αMan 1‐P and the GDP‐Man formation decoupled from one another (sequential mode) or having all steps run concurrently (simultaneous mode). Detailed time course analysis revealed that kinetic pull due to the constant removal of the intermediate αMan 1‐P in simultaneous‐mode reactions was important to promote phosphorylation of Man from αGlc 1‐P in high efficiency, avoiding loss of sugar 1‐phosphates by hydrolysis. Under optimized conditions for the one‐pot transformation involving four enzymes, 100 mM (67 g L⁻¹) GDP‐Man was prepared from 140 mM sucrose and phosphate, using 400 mM Man as the phosphoryl acceptor. The product was recovered by anion‐exchange and size‐exclusion chromatography in ≥95% purity in about 50% yield (100 mg). These results demonstrate for the first time the practical use of a phosphorylase‐phosphatase combi‐catalyst as an alternative to the canonical kinase for the anomeric phosphorylation of the sugar substrate in nucleoside diphospho‐sugar synthesis. Phosphorylation from inorganic phosphate via the intermediate αGlc 1‐P rather than from NTP, particularly GTP, appears advantageous specifically in cases where the sugar acceptor is a bulk commodity that can be applied in suitable excess to the phosphatase reaction.

     

Walking a Fine Line with Sucrose Phosphorylase: Efficient Single‐Step Biocatalytic Production of l‐Ascorbic Acid 2‐Glucoside from Sucrose

The 2‐O‐α‐d ‐glucoside of l ‐ascorbic acid (AA‐2G) is a highly stabilized form of vitamin C, with important industrial applications in cosmetics, food, and pharmaceuticals. AA‐2G is currently produced through biocatalytic glucosylation of l ‐ascorbic acid from starch‐derived oligosaccharides. Sucrose would be an ideal substrate for AA‐2G synthesis, but it lacks a suitable transglycosidase. We show here that in a narrow pH window (pH 4.8–6.0, with sharp optimum at pH 5.2), sucrose phosphorylases catalyzed the 2‐O‐α‐glucosylation of l ‐ascorbic acid from sucrose with high efficiency and perfect site‐selectivity. Optimized synthesis with the enzyme from Bifidobacterium longum at 40 °C gave a concentrated product (155 g L^?1; 460 mm ), from which pure AA‐2G was readily recovered in ～50 % overall yield, thus providing the basis for advanced production. The peculiar pH dependence is suggested to arise from a “reverse‐protonation” mechanism in which the catalytic base Glu232 on the glucosyl–enzyme intermediate must be protonated for attack on the anomeric carbon from the 2‐hydroxyl of the ionized l ‐ascorbate substrate.     

Unlocking the Potential of Leloir Glycosyltransferases for Applied Biocatalysis: Efficient Synthesis of Uridine 5′‐Diphosphate‐Glucose by Sucrose Synthase

Despite the unsurpassed selectivity that enzymes usually offer, biocatalytic transformations repeatedly fall short of the robustness and process efficiency demanded for production‐scale chemical synthesis. Nucleotide sugar‐dependent “Leloir” glycosyltransferases (GTs) are fine catalysts of glycosylation but there is concern as to whether reactions from this enzyme class are fit for industrial process development. We demonstrate in this study of sucrose synthase (SuSy; EC 2.4.1.13) that, in order to unlock the synthetic potential of the GT reaction, it was vital to combine a focused, kinetic characteristics‐based enzyme selection with a reaction design properly aligned to thermodynamic constraints. The equilibrium constant (K_eq) for the conversion of sucrose and uridine 5′‐diphosphate (UDP) into the target product UDP‐α‐d ‐glucose and d ‐fructose decreased with increasing pH due to deprotonation of the β‐phosphate group of UDP above the pK_a of ∼6.0. Proton uptake coupled to the glucosyl transfer made it essential that the pH was carefully controlled throughout the reaction. Comparing two SuSys from Acidithiobacillus caldus and Glycine max (soybean), substrate inhibition by UDP superseded catalytic efficiency as the prior selection criterion, demanding choice of the bacterial GT for use at high UDP concentrations. Reaction at the operational pH optimum, determined as 5.0, gave 255 mM (144 g L⁻¹) of UDP‐glucose in 85% yield from UDP. Using an enzyme concentration of only 0.1 g L⁻¹, a space‐time yield of 25 g L⁻¹ h⁻¹ was obtained. The mass‐based turnover number (g product formed per g enzyme added) reached a value of 1440 from a single batch conversion. Therefore, these parameters of the UDP‐glucose synthesis show that the reaction of a GT can be pushed to a process efficiency typically required for implementation in fine chemicals production.

     

Integration of Aqueous Two‐Phase Extraction into Downstream Processing

Aqueous two‐phase extraction (ATPE) is increasingly considered to be a feasible unit operation, e.g., for the capture of monoclonal antibodies or recombinant proteins. So far, knowledge on the applicability of ATPE in antibody processes has been collected mostly in lab‐scale. In contrast, approaches for the integration of ATPE into a downstream process are investigated. A complete process sequence including extraction, washing, ultrafiltration, and ion‐exchange chromatography is discussed and suggested for antibody purification. Excellent antibody purities can be achieved. Additionally, a model is applied that allows early‐on prediction of a multistage ATPE with high prediction accuracy. Finally, an economic evaluation between ATPE and Protein A chromatography is performed, reaching up to five‐fold cost‐saving factors.     

Recovery of lipase by adsorption at the n‐hexadecane–water interface

A novel separation process based on the hydrophobic adsorption at the n‐hexadecane–water interface was developed for the recovery of Acinetobacter radioresistens lipase from a pre‐treated fermentation broth. In a mixture containing water, lipase and n‐hexadecane, a water‐in‐oil emulsion was formed when the n‐hexadecane‐to‐water ratio (o/w ratio) was larger than 3, and a large amount of lipase was found to be adsorbed at the interface. Compared with the oil‐in‐water emulsion (occurring when o/w ratio < 3), the water‐in‐oil emulsion generated smaller droplets and larger interfacial area, and was more stable. The harvested emulsion phase could be centrifuged to give an aqueous, concentrated lipase solution. Adsorption of lipase at the interface could be described by the Langmuir isotherm. For lipase concentrations ranging from 8.4 to 87.2 U cm^?3, a single‐stage adsorption resulted in a six‐ to four‐fold concentration and 16–45% activity recovery, where lipase concentration was the dominant factor. A method using data from a single‐stage adsorption to predict multiple‐stage operation was described, and the agreement between the experimental and the predicted results was good. To improve the enzyme recovery, a multiple‐run adsorption process was proposed. The use of salts enhanced the hydrophobic interaction between lipase and n‐hexadecane. Advantages of the proposed process include simple operation, low operational cost, environmentally friendly, no requirement for pre‐concentration of the enzyme solution, and negligible enzyme denaturation. Copyright © 2003 Society of Chemical Industry     

Asymmetric Aldol Reaction of Thiazole‐Carbaldehydes: Regio‐ and Stereoselective Synthesis of Tubuvalin Analogues

The first organocatalytic enantioselective approach to precursors of tubuvaline (pre‐Tuv) is presented employing a prolinamide‐catalyzed aldol reaction of easily accessible thiazole‐carbaldehyde with methyl isopropyl ketone “on water” in excellent yield as well as regio‐ and enantioselectivities. The analogues of pre‐Tuv were achieved using an L ‐proline‐catalyzed direct asymmetric aldol reaction of substituted thiazole‐carbaldehydes with acetone. A direct and flexible approach to the tubavaline (Tuv) core of tubusylins has been established employing the reductive amination of the pre‐Tuv species. The key aldol reaction should greatly expand the potential of this strategy to the synthesis of natural product tubulysins and a range of analogues.

     

A Recyclable Organocascade Reaction System: Stereoselective Precipitation of Optically Active cis‐δ‐Lactols with Quaternary Stereocenters during the Michael–Hemiacetalization Reaction

A cascade Michael–hemiacetalization reaction between β‐substituted β‐nitroethanols and α,β‐unsaturated aldehydes is described, which provides a convenient and efficient synthesis for cis‐δ‐lactols with quaternary stereocenters in moderate yields with excellent enantioselectivity. Based on the selective precipitation of cis‐δ‐lactols, which were isolated by filtration, the catalytic system in the filtrate can be reused directly and recycled for eight times without any obvious deterioration in enantioselectivity.     

Shear‐Stress‐Mediated Refolding of Proteins from Aggregates and Inclusion Bodies

Recombinant protein overexpression of large proteins in bacteria often results in insoluble and misfolded proteins directed to inclusion bodies. We report the application of shear stress in micrometer‐wide, thin fluid films to refold boiled hen egg white lysozyme, recombinant hen egg white lysozyme, and recombinant caveolin‐1. Furthermore, the approach allowed refolding of a much larger protein, cAMP‐dependent protein kinase A (PKA). The reported methods require only minutes, which is more than 100 times faster than conventional overnight dialysis. This rapid refolding technique could significantly shorten times, lower costs, and reduce waste streams associated with protein expression for a wide range of industrial and research applications.     

The First Catalytic Mannich‐Type Reaction of N‐Alkoxycarbonylamino Sulfones with Silyl Enolates

Bismuth triflate was found to be an efficient catalyst in the Mannich‐type reaction of silyl enolates with N‐alkoxycarbonylamino sulfones. The reaction proceeded smoothly with a low catalyst loading of bismuth triflate (0.5–1.0 mol %) to afford the corresponding protected β‐aminocarbonyl compound in very good yields (up to 96 %).     

Cobalt(II) Phthalocyanine‐Catalyzed Highly Chemoselective Reductive Amination of Carbonyl Compounds in a Green Solvent

Cobalt phthalocyanine has been employed for the highly chemoselective reductive amination of aldehydes and ketones in ethanol as a green solvent. A large range of functional groups such as nitro, acid, amide, ester, nitrile, halogen, lactone, methoxy, hydroxy, alkene, N‐benzyl, O‐benzyl and heterocyclic rings were well tolerated under the present reaction conditions.     

Low‐Temperature Synthesis of Aluminum Nitride from Transition Alumina by Microwave Processing

Aluminum nitride (AlN) was synthesized by carbothermal reduction and nitridation method from a mixture of various transition alumina powders and carbon black using 2.45 GHz microwave irradiation in N₂ atmosphere. We achieved the synthesis of AlN at 1300–1400°C using 2.45 GHz microwave irradiation for 60 min. Our results suggest that θ‐Al₂O₃ is more easily nitrided than γ‐, δ‐, and α‐Al₂O₃. On the other hand, nitridation ratio of samples synthesized in a conventional furnace under nitrogen atmosphere were zero or very low. These results show that 2.45 GHz microwave irradiation enhanced the reduction and nitridation reaction of alumina.     

Visible Light‐Induced Selective Generation of Radicals from Organoborates by Photoredox Catalysis

A new strategy for the generation of carbon‐centered radicals via oxidation of alkyl‐, allyl‐, benzyl‐ and arylborates by visible‐light‐driven single electron transfer (SET) photoredox catalysis has been established. The generated radicals smoothly react with TEMPO and electron‐deficient alkenes to afford C O and C C coupling products, respectively. In this radical initiating system, cyclic organo(triol)borates turn out to be useful radical precursors.     

Asymmetric Rhodium‐Catalyzed 1,4‐ and 1,2‐Additions of Arylboronic Acids to Activated Ketones in Water at Room Temperature Using a Mixed Sulfur‐Olefin Ligand

Performing catalytic enantioselective reactions, especially enantioselective carbon‐carbon bond forming reactions, in water without using any organic solvents is one of the important goals in modern asymmetric synthesis. Herein, we report an efficient enantioselective micellar catalytic approach for the 1,4‐addition of arylboronic acids to cyclic ketones. Noteworthy, applying the same catalytic system we have also developed the first addition of boronic acids to the more challenging α‐keto carbonyl compounds in water, affording tertiary carbinols with high yields and high enantioselectivities. Beside the mild conditions used, the reported processes use as catalyst precursor the robust sulfinamido‐olefin mixed ligand 1 obtained on a multigram scale and in one step from a sugar‐derived sulfinate ester.     

An Odorless,One‐Pot Synthesis of Thioesters from Organic Halides,Thiourea and Benzoyl Chlorides in Water

Thioesterification can be realized via an odorless, one‐pot reaction through the in situ generation of S‐alkylisothiouronium salts from organic halides and thiourea in aqueous Triton X‐100 (TX100) micelles. The protocol is free of foul‐smell thiols and organic solvents, and operates under mild conditions, thereby offering considerable potential for applications in organic synthesis.     

Enzymatic Redox Cascade for One‐Pot Synthesis of Uridine 5′‐Diphosphate Xylose from Uridine 5′‐Diphosphate Glucose

Synthetic ways towards uridine 5′‐diphosphate (UDP)‐xylose are scarce and not well established, although this compound plays an important role in the glycobiology of various organisms and cell types. We show here how UDP‐glucose 6‐dehydrogenase (hUGDH) and UDP‐xylose synthase 1 (hUXS) from Homo sapiens can be used for the efficient production of pure UDP‐α‐xylose from UDP‐glucose. In a mimic of the natural biosynthetic route, UDP‐glucose is converted to UDP‐glucuronic acid by hUGDH, followed by subsequent formation of UDP‐xylose by hUXS. The nicotinamide adenine dinucleotide (NAD⁺) required in the hUGDH reaction is continuously regenerated in a three‐step chemo‐enzymatic cascade. In the first step, reduced NAD⁺ (NADH) is recycled by xylose reductase from Candida tenuis via reduction of 9,10‐phenanthrenequinone (PQ). Radical chemical re‐oxidation of this mediator in the second step reduces molecular oxygen to hydrogen peroxide (H₂O₂) that is cleaved by bovine liver catalase in the last step. A comprehensive analysis of the coupled chemo‐enzymatic reactions revealed pronounced inhibition of hUGDH by NADH and UDP‐xylose as well as an adequate oxygen supply for PQ re‐oxidation as major bottlenecks of effective performance of the overall multi‐step reaction system. Net oxidation of UDP‐glucose to UDP‐xylose by hydrogen peroxide (H₂O₂) could thus be achieved when using an in situ oxygen supply through periodic external feed of H₂O₂ during the reaction. Engineering of the interrelated reaction parameters finally enabled production of 19.5 mM (10.5 g L ⁻¹) UDP‐α‐xylose. After two‐step chromatographic purification the compound was obtained in high purity (>98%) and good overall yield (46%). The results provide a strong case for application of multi‐step redox cascades in the synthesis of nucleotide sugar products.

     

Biomimetic Cyclization of Small Terpenoids Promoted by Zeolite NaY: Tandem Formation of α‐Ambrinol from Geranyl Acetone

Zeolite NaY promotes efficiently the biomimetic cyclization of small acyclic terpenes. Geranyl and neryl acetone undergo a novel tandem 1,5‐diene cyclization/carbonyl‐ene reaction to form the natural product α‐ambrinol isolated in >65% yield.