Immobilization of an amino acid racemase for application in crystallization‐based chiral resolutions of asparagine monohydrate

Integration of racemization and a resolution process is an attractive way to overcome yield limitations in the production of pure chiral molecules. Preferential crystallization and other crystallization‐based techniques usually produce low enantiomeric excess in solution, which is a constraint for coupling with racemization. We developed an enzymatic fixed bed reactor that can potentially overcome these unfavorable conditions and improve the overall yield of preferential crystallization. Enzyme immobilization strategies were investigated on covalent‐binding supports. The amino acid racemase immobilized in Purolite ECR 8309F with a load of 35 mg‐enzyme/g‐support showed highest specific activity (approx. 500 U/g‐support) and no loss in activity in reusability tests. Effects of substrate inhibition observed for the free enzyme were overcome after immobilization. A packed bed reactor with the immobilized racemase showed good performance in steady state operation processing low enantiomeric excess inlet. Kinetic parameters from batch reactor experiments can be successfully used for prediction of packed bed reactor performance. Full conversions could be achieved for residence times above 1.1 min. The results suggest the potential of the prepared racemase reactor to be combined with preferential crystallization to improve resolution of asparagine enantiomers.     

Substrate spectrum of mandelate racemase: Part 1: Variation of the α-hydroxy acid moiety

Enzymatic racemization of mandelic acid derivatives modified at the α-hydroxy acid moiety was achieved using mandelate racemase [EC 5.1.2.2]. Whereas α-amino acid derivatives, such as phenyl glycine and mandelic acid hydrazide were not accepted, the mandelic acid amide was racemized at an acceptable rate. The latter was significantly enhanced by an electron-withdrawing substituent in the phenyl moiety. Based on the catalytic mechanism of the enzyme, the relative activities of non-natural substrates could be explained by steric and electronic reasons.     

A control strategy of partial nitritation in a fixed bed biofilm reactor

To achieve an appropriate mixture of ammonium and nitrite for anaerobic ammonium oxidation (ANAMMOX), 50% partial nitritation was optimized in a fixed bed biofilm reactor treating synthetic wastewater. Results suggested that 50% partial nitritation could be achieved by stepwise increases of influent NH₄⁺-N at pH of 7.8 ± 0.2, temperature of 30 ± 1 °C and dissolved oxygen (DO) of 0.5-0.8 mg l⁻¹. Hydraulic retention time (HRT) and influent alkalinity did significantly affect partial nitritation. At HRT 12 h, 50% partial nitritation could be kept stable, regardless of influent NH₄⁺-N variation, by controlling the influent HCO₃⁻/NH₄⁺ molar ratio at 1:1. The fluorescent in situ hybridization (FISH) results indicated the abundance of evolution of ammonia-oxidizing bacteria (AOB) and the nitrite-oxidizing bacteria (NOB) coincided well with the performance of partial nitritation. Furthermore, the AOB were highly affiliated with Nitrosomonas spp. and Nitrosospira spp. dominated (64.1%) in the biofilm with a compact structure during the stable 50% partial nitritation period.     

Substrate spectrum of mandelate racemase: Part 2. (Hetero)-aryl-substituted mandelate derivatives and modulation of activity

Efficient enzymatic racemization of 2-hydroxy-2-heteroaryl-acetic acid derivatives by mandelate racemase under mild conditions is reported for the first time. (i) Steric limitations for aryl-substituted mandelate derivatives were elucidated to be particularly striking for o-substituents, whereas m- and p-analogues were freely accepted, as well as heteroaryl- and naphthyl-analogs. (ii) The electronic character of substituents was found to play an important role: whereas electron-withdrawing substituents dramatically enhanced the racemization rates, electron-donating analogs caused a depletion. This effect could be ascribed to an α-carbanion-stabilization in accordance with the known enzyme mechanism. The latter was modeled by comparison of gas phase deprotonation energies as a useful parameter to describe resonance stabilization. The calculated data nicely correlate with the experimentally observed activities for a specific substrate as long as other parameters, such as steric restrictions, are absent or play a minor role.     

Perchlorate reduction by a mixed culture in an up-flow anaerobic fixed bed reactor

Wolinella succinogenes HAP-1 is a Gram-negative microaerophile which reduces perchlorate to chloride by the proposed pathway ClO₄ to ClO₃ to ClO₂ to Cl + O₂. A cost-effective perchlorate treatment process has been established using a consortium of facultative anaerobic organisms and W. succinogenes HAP-1. The mixed anaerobic bacterial culture containing W. succinogenes HAP-1 was examined for the ability to form a biofilm capable of perchlorate reduction. An up-flow anaerobic fixed bed reactor (UAFBR) was packed with diatomaceous earth pellets and operated at residence times of 1.17 and 0.46 h to insure a viable biofilm had attached to the diatomaceous earth pellets. Reduction rates of perchlorate to chloride in the UAFBR could be maintained at 1 g of perchlorate reduced h⁻¹ L⁻¹. Studies with the same bacterial consortium in continuously stirred tank reactors (CSTR) generally reduced 0.5–0.7 g of perchlorate h⁻¹. Viable cell counts were performed periodically on the diatomaceous earth pellets and demonstrated that the W. succinogenes HAP-1 population was maintained at 28–47% of the total microbial population. Scanning electron micrographs demonstrated that the external and internal surfaces of the diatomaceous pellets were densely colonized with microbial cells of multiple cell types. This is the first report of an anaerobic mixed culture forming a biofilm capable of perchlorate reduction. Received 22 May 1997/ Accepted in revised form 07 January 1998     

Immobilization of galactosyltransferase and continuous galactosylation of glycoproteins in a reactor

We immobilized human milk galactosyltransferase covalently to CNBr- and tresylchloride-activated Sepharose. The enzyme was also immobilized non-covalently to Concanavalin A-Sepharose and to monoclonal anti-galactosyltransferase antibodies which were boundvia their Fc-fragment to Protein G-Sepharose. With the covalent methods, up to 72% of the enzyme could be bound to the carrier, but more than 90% of the specific activity was lost. In contrast, non-covalent immobilization yielded only about 50% immobilization efficiency, but 21% and 25% of specific activity, respectively, could be recovered. The stability of immobilized galactosyltransferase as compared to native enzyme was considerably increased: at room temperature, 55% of initial immobilized activity was lost after 65 hours compared to 95% of loss of soluble enzyme activity. Immobilized galactosyltransferase was then used for continuous galactosylation of the glycoproteins ovalbumin, endo H-treated yeast invertase and bovine serum albumin-N-acetylglucosamine in a slurry reactor. 55%, 35% and 25%, respectively, of all acceptor sites on these glycoproteins could be galactosylated by this method.     

Immobilization of laccase from the white rot fungus Coriolopsis polyzona and use of the immobilized biocatalyst for the continuous elimination of endocrine disrupting chemicals

Laccase from the white rot fungus strain Coriolopsis polyzona was immobilized covalently on the diatomaceous earth support Celite^® R-633 using different strategies. A first methodology involved the sequential activation of the support surface with γ-aminopropyltriethoxysilane followed by the reaction of the functionalized surface with glutaraldehyde (GLU) or glyoxal (GLY) and the immobilization of laccase on the activated surface. Another strategy tested the simultaneous internal cross-linking of the protein with GLU or GLY and the immobilization of the laccase on the silanized surface. Finally, these two strategies were modified to test the impact of the concomitant addition of bovine serum albumin (BSA) as a stabilizing agent during the immobilization steps. The highest laccase activity and the greatest degree of activity recovery (tested using 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as the substrate) were achieved by the sequential immobilization procedure using GLU as the cross-linking agent. The solid catalysts featuring internal cross-linking of the protein showed significantly higher stability against several denaturants. The Michaelis–Menten kinetic parameters with respect to ABTS revealed a higher affinity for this substrate in the case of the sequential procedure compared to the simultaneous approach. The biocatalyst formed using GLU in the sequential procedure was applied in a packed bed reactor for the continuous treatment of 5 mg l⁻¹ solutions of the endocrine disrupting chemicals (EDCs) nonylphenol (NP), bisphenol A (BPA) and triclosan (TCS) through repeated batch treatments. All of these EDCs could be eliminated at a contact time of less than 200 min by using, respectively, 3.75 units (U) of laccase activity for BPA and TCS and 1.88 U for NP. These performances of elimination were maintained over five consecutive treatment cycles using the same biocatalyst. This system could also remove these EDCs from 100 mg l⁻¹ solutions. The Michaelis–Menten kinetic parameters with respect to these chemicals showed a decreasing affinity of the solid biocatalyst for NP, TCS and BPA in that order.     

Pilot-scale culture ofCoffea arabica in a novel loop fluidised bed reactor

A novel bubble free loop fluidized bed reactor for plant cell cultures was developed and tested usingCoffea arabica as a model cell line. The effects of main operational parameters like morphology and size of inoculum, oxygen supply as well as recirculation of sparingly soluble gases on cell growth and alkaloid production rates in this reactor were studied and the results were compared with standard shake flask experiments. By on-line monitoring of biomass and oxygen uptake rates the main kinetic parameters for cell growth and alkaloid production were evaluated. It was demonstrated that the novel reactor is easy to run and is particularly adequate for measuring kinetic parameters necessary for scale up.     

Anaerobic biodegradation of diesel fuel-contaminated wastewater in a fluidized bed reactor

Diesel fuel spills have a major impact on the quality of groundwater. In this work, the performance of an Anaerobic Fluidized Bed Reactor (AFBR) treating synthetic wastewater is experimentally evaluated. The wastewater comprises tap water containing 100, 200 and 300 mg/L of diesel fuel and nutrients. Granular, inert, activated carbon particles are employed to provide support for biomass inside the reactor where diesel fuel is the sole source of carbon for anaerobic microorganisms. For different rates of organic loading, the AFBR performance is evaluated in terms of the removal of diesel fuel as well as chemical oxygen demand (COD) from wastewater. For the aforementioned diesel fuel concentrations and a wastewater flow rate of 1,200 L/day, the COD removal ranges between 61.9 and 84.1%. The concentration of diesel fuel in the effluent is less than 50 mg/L, and meets the Level II groundwater standards of the MUST guidelines of Alberta.     

Model-based characterization of an amino acid racemase from Pseudomonas putida DSM 3263 for application in medium-constrained continuous processes

The amino acid racemase with broad substrate specificity from Pseudomonas putida DSM 3263 was overproduced and characterized with respect to application in an integrated multi-step process (e.g., dynamic kinetic resolution) that--theoretically--would allow for 100% chemical yield and 100% enantiomeric excess. Overexpression of the racemase gene in Escherichia coli delivered cell free extract with easily sufficient activity (20-50 U mg(-1) total protein) for application in an enzyme membrane reactor (EMR) setting. Model-based experimental analysis of a set of enzyme assays clearly indicated that racemization of the model substrates D- or L-methionine could be accurately described by reversible Michaelis-Menten kinetics. The corresponding kinetic parameters were determined from progress curves for the entire suitable set of aqueous-organic mixtures (up to 60% methanol and 40% acetonitrile) that are eligible for an integrated process scheme. The resulting kinetic expression could be successfully applied to describe enzyme membrane reactor performance under a large variety of settings. Model-based calculations suggested that a methanol content of 10% and an acetonitrile content of 20% provide maximum productivity in EMR operations. However product concentrations were decreased in comparison to purely aqueous operation due to decreasing solubility of methionine with increasing organic solvent content. Finally, biocatalyst stability was investigated in different solvent compositions following a model-based approach. Buffer without organic content provided excellent stability at moderate temperatures (20-35 degrees C) while addition of 20% acetonitrile or methanol drastically reduced the half-life of the racemase.     

Biodiesel production using Aspergillus niger as a whole‐cell biocatalyst in a packed‐bed reactor

Enzymatic lipase transesterification of palm oil to biodiesel in a packed‐bed reactor (PBR) using a novel strain of the fungus Aspergillus niger, immobilized within polyurethane biomass support particles (BSPs), was investigated. A three‐step addition of methanol was used to reduce lipase inhibition by immiscible methanol. The influence of water content and PBR flow rate was investigated. FAME yield was enhanced with an increase of PBR flow rate in the range of 0.15–30 L h^?1, where inefficient mixing of the reaction mixture at lower flow rates resulted in low conversion rates i.e. 69% after 72‐h reaction. Adding the third mole equivalent of methanol resulted in lipase inhibition due to methanol migration into the accumulated glycerol layer. Glutaraldehyde (GA) solution (0.5 vol.%) was used to stabilize lipase activity, which led to a high FAME yield (>90%) in the PBR after 72‐h of reaction time at a flow rate of 15 L h^?1, and a water content of 15%. Moreover, a high conversion rate (>85%) was maintained after four palm oil batch conversion cycles in the PBR. In contrast, lipase activity of non‐GA‐treated cells decreased with each PBR batch cycle, where only 70% FAME was produced after the forth PBR cycle. Transesterification of palm oil in a PBR system using BSPs‐immobilized A. niger as a whole‐cell biocatalyst is a viable process for enzymatic biodiesel production.     

Precipitation and recovery of metal sulfides from metal containing acidic wastewater in a sulfidogenic down-flow fluidized bed reactor

This study reports the feasibility of recovering metal precipitates from a synthetic acidic wastewater containing ethanol, Fe, Zn, and Cd at an organic loading rate of 2.5 g COD/L-day and a COD to sulfate ratio of 0.8 in a sulfate reducing down-flow fluidized bed reactor. The metals were added at increasing loading rates: Fe from 104 to 320 mg/L-day, Zn from 20 to 220 mg/L-day, and Cd from 5 to 20 mg/L-day. The maximum COD and sulfate removals attained were 54% and 41%, respectively. The biofilm reactor was operated at pH as low as 5.0 with stable performance, and no adverse effect over COD consumption or sulfide production was observed. The metals precipitation efficiencies obtained for Fe, Zn, and Cd exceeded 99.7%, 99.3%, and 99.4%, respectively. The total recovered precipitate was estimated to be 90% of the theoretical mass expected as metal sulfides. The precipitate was mainly recovered from the bottom of the reactor and the equalizer. The analysis of the precipitates showed the presence of pyrite (FeS2), sphalerite (ZnS) and greenockite (CdS); no metal hydroxides or carbonates in crystalline phases were identified. This study is the first in reporting the feasibility to recover metal sulfides separated from the biomass in a sulfate reducing process in one stage.     

Enzyme-assisted physicochemical enantioseparation processes: Part I. Production and characterization of a recombinant amino acid racemase

The demand for enantiopure substances, e.g. for pharmaceutical applications or fine chemical production, continues to increase. This has led to the development of numerous stereoselective synthesis methods. Nevertheless a large number of chemical syntheses still result in racemic mixtures making a subsequent enantioseparation step necessary and thus are restricted to a maximum yield of 50%. Our work focuses on strategies to overcome this limitation by combining physicochemical separation processes with enzymatic racemization of the unwanted enantiomer in order to produce enantiopure amino acids. This paper deals with the production and characterization of a suitable amino acid racemase with broad substrate specificity (EC 5.1.1.10) from Pseudomonas putida which we cloned into Escherichia coli. Two enzyme lyophilizates of different purity were obtained from which the crude (CL) was sufficient for the racemization of methionine (Met) and the pure (PL) was used for asparagine (Asn). Racemization reactions of D-/L-Asn in H₂O and D-/L-Met in 95 vol.% 100 mM KP_i-buffer, 5 vol.% methanol (MeOH) at different pH values and temperatures were characterized. The studied range of reaction parameters was chosen in dependency on planned enantioseparation processes. We found increasing V_max values when temperature was risen stepwise from 20 to 40 °C for both systems and when pH was shifted from 6 to 8 for the Met system. The presented results provide the basis for engineering enzyme-assisted physicochemical enantioseparation processes.     

Performance and population analysis of a non‐sterile trickle bed reactor inoculated with Caldicellulosiruptor saccharolyticus,a thermophilic hydrogen producer

Non‐axenic operation of a 400 L trickle bed reactor inoculated with the thermophile Caldicellulosiruptor saccharolyticus, yielded 2.8 mol H₂/mol hexose converted. The reactor was fed with a complex medium with sucrose as the main substrate, continuously flushed with nitrogen gas, and operated at 73°C. The volumetric productivity was 22 mmol H₂/(L filterbed h). Acetic acid and lactic acid were the main by‐products in the liquid phase. Production of lactic acid occurred when hydrogen partial pressure was elevated above 2% and during suboptimal fermentation conditions that also resulted in the presence of mono‐ and disaccharides in the effluent. Methane production was negligible. The microbial community was analyzed at two different time points during operation. Initially, other species related to members of the genera Thermoanaerobacterium and Caldicellulosiruptor were present in the reactor. However, these were out‐competed by C. saccharolyticus during a period when sucrose was completely used and no saccharides were discharged with the effluent. In general, the use of pure cultures in non‐sterile industrial applications is known to be less useful because of contamination. However, our results show that the applied fermentation conditions resulted in a culture of a single dominant organism with excellent hydrogen production characteristics. Biotechnol. Bioeng. 2009;102: 1361–1367. © 2008 Wiley Periodicals, Inc.     

Selection of Type I and Type II methanotrophic proteobacteria in a fluidized bed reactor under non-sterile conditions

Type II methanotrophs produce polyhydroxybutyrate (PHB), while Type I methanotrophs do not. A laboratory-scale fluidized bed reactor was initially inoculated with a Type II Methylocystis-like dominated culture. At elevated levels of dissolved oxygen (DO, 9 mg/L), pH of 6.2–6.5 with nitrate as the N-source, a Methylobacter-like Type I methanotroph became dominant within the biofilms which did not produce PHB. A shift to biofilms capable of PHB production was achieved by re-inoculating with Type II Methylosinus culture, providing dissolved N₂ as the N-source, and maintaining a low influent DO (2.0 mg/L). The resulting biofilms contained both Types I and II methanotrophs. Batch tests indicated that biofilm samples grown with N₂ became dominated by Type II methanotrophs and produced PHB. Enrichments with nitrate or ammonium were dominated by Type I methanotrophs without PHB production capability. The key selection factors favoring Type II were N₂ as N-source and low DO.     

Lipase kinetics: hydrolysis of triacetin by lipase from Candida cylindracea in a hollow-fiber membrane reactor

The aptitude of a hollow-fiber membrane reactor to determine lipase kinetics was investigated using the hydrolysis of triacetin catalyzed by lipase from Canadida cylindracea as a model system. The binding of the lipase to the membrane appears not to be very specific (surface adsorption), and probably its conformation is hardly altered by immobilization, resulting in an activity comparable to that of the enzyme in its native form. The reaction kinetics defined on the membrane surface area were found to obey Michaelis-Menten kinetics. The specific activity of the lipase in the membrane reactor was found to be significantly higher than in an emulsion reactor. The activity and stability of the enzyme immobilized on a hydrophilic membrane surface seem not to be influenced significantly by the choice of the membrane material. The hollow-fiber membrane reactor is a suitable tool to assess lipase kinetics in a fast and convenient way.     

Enzymatic synthesis of ethyl esters from waste oil using mixtures of lipases in a plug‐flow packed‐bed continuous reactor

This work describes the continuous synthesis of ethyl esters via enzymatic catalysis on a packed‐bed continuous reactor, using mixtures of immobilized lipases (combi‐lipases) of Candida antarctica (CALB), Thermomyces lanuginosus (TLL), and Rhizomucor miehei (RML). The influence of the addition of glass beads to the reactor bed, evaluation of the use of different solvents, and flow rate on reaction conditions was studied. All experiments were conducted using the best combination of lipases according to the fatty acid composition of the waste oil (combi‐lipase composition: 40% of TLL, 35% of CALB, and 25% of RML) and soybean oil (combi‐lipase composition: 22.5% of TLL, 50% of CALB, and 27.5% of RML). The best general reaction conditions were found to be using tert‐butanol as solvent, and the flow rate of 0.08 mL min^?1. The combi‐lipase reactors operating at steady state for over 30 days (720 h), kept conversion yields of ～50%, with average productivity of 1.94 g_{ethyl esters} h^?1, regardless of the type of oil in use. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:952–959, 2018     

Proposed pathways for the reduction of a reactive azo dye in an anaerobic fixed bed reactor

Some process has been proposed for azo dye degradation and anaerobic bioreactors are one of them, since for their reduction, the dye has to be the electron acceptor. An anaerobic fixed bed bioreactor packed with activated carbon (AC) is proposed to degradate the Reactive Red 272 azo dye. In the present paper a dye degradation mechanism in an anaerobic environment is explained. It is very important to consider the interaction dye-microorganism-AC, because the groups in the AC surface take part in the reaction besides being an excellent carrier for microorganism and an adsorbent for the dye. The aromatic compounds produced in the dye reduction are partially degraded as a function of inlet dye concentration and reactor residence time. In anaerobic environment the aromatic compounds are decomposed through hydroxylation, carboxylation and redox reactions, due to enzymatic reactions.     

Investigating the performance of an up-flow anoxic fixed-bed bioreactor and a sequencing anoxic batch reactor for the biodegradation of hydrocarbons in petroleum-contaminated saline water

This work presents the biodegradation of petroleum hydrocarbons in an upflow anoxic fixed-bed bioreactor (UAnFB) and a sequencing anoxic batch reactor (SAnBR). The performances of the UAnFB and the SAnBR in the removal of petroleum hydrocarbons (TPH) were investigated as a function of inlet concentration at a hydraulic retention time of 24 h. The UAnFB had higher robustness and adapted better towards the transition in TPH concentration. The average TPH removal rates for concentrations of 950, 1450, and 2500 mg L⁻¹ were 99.9%, 99.6%, and 93.7%, respectively, for the UAnFB and 99.7%, 98.5%, and 87.7%, respectively, for the SAnBR. The highest rates of TPH biodegradation at a loading rate of 104 g m⁻³ h⁻¹ in the UAnFB and the SAnBR were 97.5 and 91.3 g m⁻³ h⁻¹, respectively. The UAnFB was more efficient than the SAnBR in biodegrading aromatic hydrocarbons. Accordingly, the UAnFB is an efficient and viable technology for the treatment of hydrocarbon-laden streams.