Repetitive Batches Research Articles

Intensification of a biocatalytic oxidation under fine bubble aeration in a rotating bed reactor

The utilization of oxidases suffers from insufficient oxygen availability due to its low solubility in gas-liquid multiphase systems. To overcome this limitation, oxygen is continuously supplied to the reaction medium during the course of the reaction, however, it causes significant gas consumption. In this study, a novel experimental set-up was established by installing a sintered frit porous sparger in the SpinChem® rotating bed reactor. Covalently immobilized glucose oxidase on epoxy-functionalized carriers was used in the rotating bed reactor with an activity yield of the immobilization of over 98%. For fine bubble aeration, the volumetric mass transfer coefficient kLa (67.3 h−1) tripled compared to macrobubble aeration (22.5 h−1), while the volume-specific aeration rate remained constant. A maximum reaction yield of 96.5% was reached. The reaction rate was improved by a factor of 12.9 for the fine bubble aeration compared to macrobubble aeration under identical conditions. With fine bubble aeration, the oxygen consumption was reduced by 87.5% over macrobubble aeration, establishing the same enzyme-specific reaction rates at a comparable kLa. Validation was carried out in repetitive batches, achieving the same glucose oxidase specific activity. This study demonstrates the advantages of fine bubble aeration and presents promising results for addressing the sustainable biotransformation processes.

Economical alternatives for the production of fungal β-1,3-glucanase using easily obtainable industrial substrates

The β-1,3-glucanases synthesized by filamentous fungi have wide applicability in the food, chemical, and pharmaceutical industries. However, its obtainment can be costly, especially due to substrates used to induce its synthesis. Therefore, the objective of this work was to produce β-1,3-glucanase by T. harzianum Rifai using free and immobilized cells in synthetic and plant sponges, using different inducing substrates that could provide better cost-effectiveness for the industrial production of the enzyme. The Petri dish zymogram technique proved to be efficient for screening substrates inducing β -1,3-glucanases against species of filamentous fungi. It was possible to perform the immobilization of T. harzianum in a synthetic sponge allowing the realization of repetitive batches for enzymatic production. All tested substrates resulted in the synthesis of β-1,3-glucanase, including succinoglycan, proposed innovatively in this study. Fungal biomass resulted in the best inducing substrate under conditions of free and immobilized cells, with a production of β-1,3-glucanases of 0.73 U and 0.80 U of β -1,3-glucanases. The substrates corn starch and cassava showed promise in the production of β-1,3-glucanase and maintained production until the fourth batch was evaluated, with values of 0.51 U and 0.46 U of β-1.3-glucanases, respectively. The results obtained in this study showed that the zymogram is a practical method for screening substrates induced by the fungus T. harzianum. Corn starch and cassava are accessible and low-cost sources for β-1,3-glucanase synthesis in repetitive batches, including the use of immobilized and free cells.

Gram‐Scale Production of GDP‐β‐l‐fucose with Multi‐Enzyme Cascades in a Repetitive‐Batch Mode

AbstractGuanosine 5’‐diphospho‐β‐l‐fucose (GDP‐Fuc) is an important and expensive nucleotide sugar only available in low quantities. We established two processes for the gram‐scale production of GDP‐Fuc with multi‐enzyme cascades. We used the bifunctional enzyme fucokinase/GDP‐fucose pyrophosphorylase from Bacteroides fragilis either with an ATP‐regeneration system using polyphosphate or an excess of ATP. The optimized enzyme modules were scaled up to a 20 mL repetitive‐batch production with 15 and 12 repetitive batches for ATP‐regeneration and ATP excess, respectively. With ATP‐regeneration a final product amount of 1.1 g (31 % yield) GDP‐Fuc was obtained. The production process with ATP excess reached 1.6 g (57 % yield). Exceptional high total turnover numbers of 27 gP*gE−1 and 31 gP*gE−1, as well as space‐time‐yields of 0.083 gp*L−1*h−1 and 0.217 gp*L−1*h−1 were reached for each process respectively. Furthermore, we established a product purification protocol that enables a 95 % removal of free phosphates from the product solution, to ease further purification.

Repetitive Synthesis of High-Molecular-Weight Hyaluronic Acid with Immobilized Enzyme Cascades.

Industrial hyaluronic acid (HA) production comprises either fermentation with Streptococcus strains or extraction from rooster combs. The hard‐to‐control product quality is an obstacle to these processes. Enzymatic syntheses of HA were developed to produce high‐molecular‐weight HA with low dispersity. To facilitate enzyme recovery and biocatalyst re‐use, here the immobilization of cascade enzymes onto magnetic beads was used for the synthesis of uridine‐5’‐diphosphate‐α‐d‐N‐acetyl‐glucosamine (UDP‐GlcNAc), UDP‐glucuronic acid (UDP‐GlcA), and HA. The combination of six enzymes in the UDP‐sugar cascades with integrated adenosine‐5'‐triphosphate‐regeneration reached yields between 60 and 100 % for 5 repetitive batches, proving the productivity. Immobilized HA synthase from Pasteurella multocida produced HA in repetitive batches for three days. Combining all seven immobilized enzymes in a one‐pot synthesis, HA production was demonstrated for three days with a HA concentration of up to 0.37 g L−1, an average M W of 2.7–3.6 MDa, and a dispersity of 1.02–1.03.

Economic model for obtaining cyclodextrins from commercial cgtase

A repetitive batch process was employed followed by membrane ultrafiltration system to produce low-cost cyclodextrins (CDs) using commercial enzymes Toruzyme® cyclomaltodextrin glucanotransferase (CGTase) and its kinetic parameters were determined. The ultrafiltration system enabled the removalof inhibitory products from the reaction medium, allowing the enzyme to be recovered for reuse. A 10 kDa membrane was used to separate the different CDs produced by the CGTase. The substrates evaluated were maltodextrin, corn starch and cassava starch at 5, 10 and 15% (w/V), in the presence and absence of 10% (V/V) ethanol. After reaction for 132 h, 10% (w/V) cassava starch in the presence of ethanol provided the best results with 32.1 mg/mL of β-CD. Maximum production occurred after 72 h of reaction, with a yield of 87.4% of β-CD and an α-CD, β-CD and γ-CD production ratio of 1:1:0.08 g, respectively. When eight repetitive batches of 72 h followed by ultrafiltration and crystallization of β-CD were performed, 2.1 g of precipitate was obtained with a purity of 67.6% β-CD. The supernatant from the crystallization process was lyophilized and resulted in 35.3% α-CD. The developed model can be used industrially for the production of low cost CDs from easily obtained raw material.

PEG-mediated recyclable borylative coupling of vinyl boronates with olefins

This paper reports on the first green and sustainable repetitive batch borylative coupling of vinyl boronates with olefins by the effective immobilization of the [Ru(CO)Cl(H)(PCy3)2] catalyst in poly(ethylene glycols) with different molecular weights (Mw = 600–2000) and ending groups (OH, OMe, OSiMe3) or in biphasic poly(ethylene glycols)/supercritical CO2 (PEGs/scCO2) systems. Within this process, (E)-alkenyl boronates were obtained with high yields and excellent stereo-, regioselectivities. The best strategies permitted to carry out cross-coupling of vinyl boronates with styrene for up to 8–16 repetitive batches respectively, by applying 2 or 4 mol% of Ru-catalyst. The described methods enable the reuse of the TM-catalyst and solvents and the reduction or elimination of the use of volatile organic solvents and metal content in the final products, and obtain high cumulative TON values (up to 440). The biphasic systems also allowed the simplification of the separation procedure by effective product extraction in CO2 stream.

Gas Chromatographic Enantioseparation of Fluorinated Anesthetics: Single-Column Performance and Scale-up Estimation

Results of a study devoted to provide the pure enantiomers of isoflurane and desflurane from racemic mixtures using gas chromatography are presented. For that purpose, a cyclodextrin-based selector described in earlier work was immobilized on porous glass beads. The adsorption isotherms were determined and applied to predict operating parameters which provide the highest possible productivity of the separation. The analysis included evaluation of the performance of larger columns applying simplifying scale-up considerations. Using repetitive batches, the method can provide per day with a laboratory scale column approximately 1 g pure enantiomer. Selected model predictions were validated experimentally.

Extractive Bioconversion of Gamma-Cyclodextrin and Recycling of Cyclodextrin Glycosyltransferase in Liquid Biphasic System Using Thermo-Separating Polymer.

An extractive bioconversion conducted on soluble starch with cyclodextrin glycosyltransferase (CGTase) enzyme in ethylene oxide-propylene oxide (EOPO)/potassium phosphates liquid biphasic system (LBS) to extract gamma-cyclodextrin (γ-CD) was examined. A range of EOPO (with potassium phosphates) molecular weights was screen to investigate the effect of the latter on the partioning efficency of CGTase and γ-CD. The results show that the optimal top phase γ-CD yield (74.4%) was reached in 35.0% (w/w) EOPO 970 and 10.0% (w/w) potassium phosphate with 2.0% (w/w) sodium chloride. A theoretical explanation for the effect of NaCl on γ-CD was also presented. After a 2 h bioconversion process, a total of 0.87 mg/mL concentration of γ-CD was produced in the EOPO/ phosphates LBS top phase. After the extraction of top phase from LBS, four continuous repetitive batches were successfully conducted with relative CGTase activity of 1.00, 0.86, 0.45, and 0.40 respectively.

Different strategies for cyclodextrin production: Ultrafiltration systems, CGTase immobilization and use of a complexing agent

The study comparatively evaluated diverse strategic models of cyclodextrin (CD) production by the CGTase of Bacillus firmus strain 37: continuous production and repetitive batches in ultrafiltration systems; immobilization of CGTase on curdlan and vegetable sponge natural supports; the use of the glycyrrhizin complexing agent to modulate CGTase selectivity in favor of γ-CD production. All strategies had in common the possibility of separation of CGTase from its inhibitory products and its reuse. In the continuous production model, at 48 h of assay, the highest productivity and selectivity for β-CD were obtained, 1.47 mmol/L/h and 92.8%, respectively. Glycyrrhizin was able to modulate the production of γ-CD with selectivity of 61.2% for 30-h batches. The comparative evaluation of the different strategic models for obtaining CDs showed particularities that should be considered, and most of the models studied returned satisfactory yields as well as excellent selectivity.

Reaction engineering of biocatalytic (S)-naproxen synthesis integrating in-line process monitoring by Raman spectroscopy

Biocatalytic (S)-naproxen synthesis using an (S)-selective arylmalonate decarboxylase mutant (AMDase G74C/M159L/C188G/V43I/A125P/V156L, AMDase-CLGIPL) exposes a promising environmentally friendly alternative to conventional chemical synthesis strategies.

HaloTag™: Evaluation of a covalent one-step immobilization for biocatalysis

Easy, fast and gentle immobilization for the efficient reuse of important biocatalysts is highly demanded. We used the commercially available HaloTag™ technology (Promega), so far relatively unknown in the context of biocatalysis, to immobilize the benzaldehyde lyase from P. fluorescence (PfBAL). Immobilization mediated by this fusion tag proceeds rapidly within minutes from crude extracts yielding covalently attached enzymes in high purity, making expensive and laborious previous chromatographic purification steps obsolete, which strongly reduces the costs for biocatalyst immobilization. Further, we introduce a novel design of HaloTag fusions and demonstrate the positive effect of the tag on soluble expression and activity of PfBAL. The immobilized biocatalyst was stable at 4°C for months and was successfully reused in several repetitive batches for the carboligation of aggressive aldehydes.

Diastereoselective Synthesis of an Industrially Relevant 4-Aminopentanoic Acid by Asymmetric Catalytic Hydrogenation in a Biphasic­ System Using Aqueous Sodium Hydroxide as Substrate Phase

A ’basic solution' for multiphase catalysis: The diastereoselective synthesis of a pharmaceutically relevant 4-aminopentanoic acid derivative has been studied using a chiral homogeneous catalyst in tailored biphasic reaction media. Different polar solvents were investigated as the stationary phase for the well-established Ru–Mandyphos catalyst in combination with aqueous NaOH as the substrate and product phase. Facile product isolation and effective recycling of the catalyst phase were demonstrated at gram-scale. In particular, up to 3200 turnovers have been achieved in seven repetitive batches with a diastereoselectivity of 87–96% using [EMIM][NTf2]/NaOHaq as the biphasic system.

Improvement of synthetic activity and stability of a commercial lipase in a low-water system via immobilization of hydrated lipase aggregates

Abstract Activation and immobilization of a commercial lipase from Candida cylindracea were investigated to improve its catalytic performance for esterification reaction between 1-octanol and three butyric acid derivatives in low-water solvent-free systems. Lipase powder was suspended in 1-octanol, which is the alcohol substrate of the esterification reaction, and then an appropriate amount of water (10% to 1-octanol in volume) was added to form hydrated lipase aggregates. Lipase exhibited high activity in this aggregated state, in contrast with the very low activity of nonhydrated lipase powder. We believe that activation using this hydration-aggregation pretreatment was based on interfacial activation at the 1-octanol-water interface. The hydrated lipase aggregates had paste-like morphology and could be immobilized on nonwoven fabrics by physical compression. Polypropylene exhibited the best specific activity among the tested nonwoven fabric materials. A stirred tank batch bioreactor was developed using lipase-immobilizing polypropylene nonwoven fabric sheets. This bioreactor stably maintained its reaction performance in repetitive-batch (maximum 30 times, for a total of 2000 h) esterification with three different acid substrates. The ester yields were over 90% throughout the all repetitive batches. During repeated reaction, the immobilized lipase maintained its water content at a constant level, thus exhibiting catalytic activity in nonaqueous reaction systems.

Ultrafiltration system for cyclodextrin production in repetitive batches by CGTase from Bacillus firmus strain 37.

This study aimed to improve the yield of cyclodextrins (CDs) production in repetitive batches. An innovative ultrafiltration system was used to remove the inhibitory products that accumulated in the medium and to recover the enzyme. The assays were performed with the CGTase from Bacillus firmus strain 37 in purified, semi-purified, and crude extract forms. Maltodextrin (10% w/v) and corn starch (5% w/v) were used as substrates. After eight repetitive 24-h batches, the yield of β-CD obtained with the purified enzyme and the corn starch substrate was 0.54 mmol/L/h, which was 36% greater than that observed with the 10% maltodextrin substrate. The crude CGTase extract with the corn starch substrate showed a productivity of 0.38 mmol/L/h, which was 29% lower than using the purified enzyme and the corn starch substrate but 7% higher than using the purified enzyme and the maltodextrin substrate. The crude extract, assayed with the corn starch substrate in the presence of 10% ethanol reached 0.43 mmol/L/h productivity, which was 12% higher compared to the assay without ethanol. The semi-purified enzyme was assayed with the corn starch substrate in the presence of 10% ethanol for eight batches lasting 12 h and an excellent selectivity for the β-CD was obtained, reaching a mean percentage of 96.0%. Therefore, this ultrafiltration system enabled several batches of CD production, with efficient removal of products inhibitory to the CGTase and recovery of the enzyme. The possibility of industrial application of this system is promising.

Production of recombinant Aedes albopictus akirin in Pichia pastoris using an aqueous two-phase semicontinuous fermentation process

The yeast, Pichia pastoris, has been successfully used as an efficient system to produce heterologous proteins. The secretion of the recombinant Aedes albopictus mosquito akirin (AKR) from P. pastoris using an extractive bioconversion in an aqueous two-phase system (ATPS) fed-batch fermentation process allowed the conduction of vaccination trials to assay its protective efficacy against different arthropod vector species. Herein, we report the development of a semicontinuous process for the extractive bioconversion in an ATPS coupled with a simple and easy to scale up process for polymer recycling as an effective alternative to improve production of recombinant A. albopictus AKR in P. pastoris. Six repetitive batches of the extractive bioconversion were conducted in a semicontinuous process by replacing 2/3 of the culture and recycling the top polymeric phase every 72h. Overall process in a 5-L fermentor yielded a protein concentration in the bottom phase of 0.46±0.13mgmL−1 and 6g of total purified protein in 490h with a reduction of 25% of the operation time and 27% of the operation cost when compared to the fed-batch ATPS process. These results improve the production process for mosquito AKR and suggest the possibilities to develop similar processes for the expression of other recombinant proteins in P. pastoris.

Evolutionary engineering strategies to enhance tolerance of xylose utilizing recombinant yeast to inhibitors derived from spruce biomass

BackgroundOne of the crucial factors for a sustainable and economical production of lignocellulosic based bioethanol is the availability of a robust fermenting microorganism with high tolerance to inhibitors generated during the pretreatment of lignocellulosic raw materials, since these inhibitors are known to severely hinder growth and fermentation.ResultsA long-term adaptation in repetitive batch cultures in shake flasks using a cocktail of 12 different inhibitors and a long-term chemostat adaptation using spruce hydrolysate were used as evolutionary engineering strategies to improve the inhibitor tolerance in the metabolically engineered xylose utilizing Saccharomyces cerevisiae strain, TMB3400. The yeast was evolved for a period of 429 and 97 generations in repetitive batch cultures and chemostat cultivation, respectively. During the evolutionary engineering in repetitive batch cultures the maximum specific growth rate increased from 0.18 h-1 to 0.33 h-1 and the time of lag phase was decreased from 48 h to 24 h. In the chemostat adaptation, after 97 generations, the specific conversion rates of HMF and furfural were found to be 3.5 and 4 folds higher respectively, compared to rates after three generations. Two evolved strains (RK60-5, RKU90-3) and one evolved strain (KE1-17) were isolated from evolutionary engineering in repetitive batches and chemostat cultivation, respectively. The strains displayed significantly improved growth performance over TMB3400 when cultivated in spruce hydrolysate under anaerobic conditions, the evolved strains exhibited 25 to 38% increase in specific consumption rate of sugars and 32 to 50% increased specific ethanol productivity compared to TMB3400. The evolved strains RK60-5 and RKU90-3 were unable to consume xylose under anaerobic conditions, whereas, KE1-17 was found to consume xylose at similar rates as TMB3400.ConclusionUsing evolutionary engineering strategies in batch and chemostat cultivations we have generated three evolved strains that show significantly better tolerance to inhibitors in spruce hydrolysate and displayed a shorter time for overall fermentation of sugars compared to the parental strain.

Identification of suitable ionic liquids for application in the enzymatic hydrolysis of rutin by an automated screening

An automated method in milliliter scale was developed for the screening of process parameters concerning the hydrolysis of the flavonoid rutin catalyzed by the rhamnosidase activity of naringinase from Penicillium decumbens. Besides the effect of additives such as ionic liquids and low molecular salts, the productivity in a multiple phase system as well as the recyclability of the enzyme in repetitive batches were studied. The hydrophobic ionic liquid (IL) trihexyl(tetradecyl)phosphonium bis(trifluormethylsulfonyl)imide [P(h(3))t][Tf(2)N] was identified to combine the most favorable characteristics out of 23 investigated ILs with regard to enzyme compatibility, substrate solubility and enzyme partition coefficient. Also, for the corresponding cations 1-ethyl-3-methylimidazolium [EMIM], 1-butyl-3-methylimidazolium [BMIM], 1-butyl-1-methylpyrrolidinium [BMPL] and 1-octyl-3-methylimidazolium [OMIM], the entity with the [Tf(2)N] anion was best tolerated by the naringinase. With increasing IL content, higher space time yields with up to 1.5 g/(L h) for 80% (v/v) [P(h(3))t][Tf(2)N] were achieved. Enhanced specific enzyme activity was observed in the presence of Ca(2+) ions. By addition of [P(h(3))t][Tf(2)N] and calcium chloride, the reactive aqueous phase was successfully used in three repetitive batches with full conversion.

Kinetic resolution of 2-chloro-3,3,3-trifluoropropanoic acid esters catalyzed by lipase from Candida rugosa

By screening around 30 commercially available lipases and esterases, two enzymes, C. rugosa lipase and P. fluorescens esterase, were found to posess catalytic activity and enantioselectivity (E˜10) for the hydrolysis of 2-chloro-3,3,3-trifluoropropanoic acid (CTFPA) methyl and ethyl ester. Both enzymes were tentatively assigned to be (S)-selective based on the assumption that they have the same stereopreference as in the hydrolysis of methyl 2-chloropropanoate, which is a non-fluorinated analogue of CTFPA. The enzymes were applied in the kinetic resolution of CTFPA ethyl ester and 95% ee of the remaining ester could be achieved at 60% conversion. The crosslinked enzyme aggregate (CLEA) of C. rugosa lipase was found to catalyze enantioselective transesterification (E˜40) of CTFPA methyl ester with ethanol. By conducting the transesterification in a 10-mL packed-bed reactor containing CLEA, it was possible to convert racemic CTFPA methyl ester into the mixture of (S)-methyl and (R)-ethyl esters with 82% and 90% ee, respectively, at 4.0 g/L-1/h-1 space-time yield, which decreased to 1.0 g/L-1/h-1 after four repetitive batches.

Catalytic Reactions in Surfactant Systems:Product Isolation and Catalyst Recycling

The potential of surfactant based reaction media was studied with different homogeneous catalytic reactions. Micellar systems with the surfactants (p-tert-octylphenoxy) polyethoxyethanol (Triton X-100) and dodecyl sulfate sodium salt were used as reaction media for the enantioselective catalytic hydrogenation of dimethyl itaconate (DMI) with the Rh catalyst complexed with the chiral ligand (2S,4S)-1-tert-butoxycarbonyl-4-diphenylphosphino-2-(diphenylphosphinometyl)-pyrrolidine (BPPM) at 30 °C and 1.1 bar, obtaining an enantiomeric excess (ee) of up to 69%. After complete hydrogenation was achieved, micellar enhanced ultrafiltration (MEUF) was used to recycle the catalyst achieving up to 95% retention. A microemulsion system stabilized with the surfactant Triton X-100 was used as alternative reaction media for the hydrogenation of DMI with a Rh catalyst complexed with the water-soluble tris(3-sulfophenyl)phosphine trisodium salt (TPPTS) at 50 °C and 1.1 bar. With the Triton X-100 system, phase separation by temperature induced separation allowed for up to four repetitive batches of DMI hydrogenations, resulting in a TON of 1530. Suzuki coupling for the synthesis of 4′-methyl-2-biphenylcarbonitrile proceeded faster in a narrow range alkylpolyglycol ether (Novel 8 = Novel 1216CO-8 Ethoxylate) three-phase system than in an dioctyl sulfosuccinate sodium salt (AOT) two-phase system, demonstrating the retarding effect of the salinization on the reaction rate.

Novel immobilization routes for the covalent binding of an alcohol dehydrogenase from Rhodococcus ruber DSM 44541

Immobilization of the alcohol dehydrogenase ADH-‘A’ from Rhodococcus ruber DSM 44541 has been performed with different amino-functionalized carrier materials. The procedure included the activation of the carrier with glutaraldehyde and subsequent covalent binding to the enzyme. The porous glass beads TRISOPERL ® and TRISOPOR ®, magnetic particles, and detonation nanodiamonds were used as carriers in these experiments. In all cases, the immobilization was successful with almost quantitative immobilization yields; subsequently the activity for the reduction of acetophenone was lower compared to the activity of the free biocatalyst. Activity yields of 40% and 60% were obtained. The immobilized biocatalysts showed high stabilities in repetitive batches.