Repeated Batch Reactions Research Articles

Hydrogen-powered enzymatic valorization using multi-enzyme co-immobilization reactor with polypeptide-based cofactor swing-arm

The chemical valorization of organic matter into high-energy products through reduction using oxidoreductase is a pivotal aspect from an energy perspective. Enzymatic valorization, utilizing the reduction potential of hydrogen contained in inexpensive resources such as coke oven gas (COG) and certain gasified plastics, enables environmentally friendly processes. Here, a strategy was proposed utilizing hydrogenase (H2ase), particularly soluble hydrogenase (SH), for selective utilization of H2 from mixtures without the separation processes. The co-immobilization of SH, elastin-like polypeptide (ELP)-based cofactor swing arm, and mannitol dehydrogenase (MDH) in a microreactor system were demonstrated for enzymatic valorization. The swing arm length and in-resin concentration were optimized to enhance electron mediation efficiency, leading to an improvement in productivity of about 3 mM/hr. Comparable productivity was observed in simulated COG and plastic waste-derived gas, which are promising results for the utilization of such low-purity H2 sources. Furthermore, the system demonstrates robustness and recyclability across maintaining productivity over 6 repeated batch reactions, highlighting its potential for industrial applications. Overall, our study provides valuable insights into the development of sustainable enzymatic microreactors for chemical valorization processes, contributing to greener and more efficient industrial practices.

Reducing residual chlortetracycline in wastewater using a whole-cell biocatalyst

Antibiotic contamination has become an increasingly important environmental problem as a potentially hazardous emergent and recalcitrant pollutant that poses threats to human health. In this study, manganese peroxidase displayed on the outer membrane of Escherichia coli as a whole-cell biocatalyst (E. coli MnP) was expected to degrade antibiotics. The manganese peroxidase activity of the whole-cell biocatalyst was 13.88 ± 0.25 U/L. The typical tetracycline antibiotic chlortetracycline was used to analyze the degradation process. Chlortetracycline at 50 mg/L was effectively transformed via the whole-cell biocatalyst within 18 h. After six repeated batch reactions, the whole-cell biocatalyst retained 87.2 % of the initial activity and retained over 87.46 % of the initial enzyme activity after storage at 25°C for 40 days. Chlortetracycline could be effectively removed from pharmaceutical and livestock wastewater by the whole-cell biocatalyst. Thus, efficient whole-cell biocatalysts are effective alternatives for degrading recalcitrant antibiotics and have potential applications in treating environmental antibiotic contamination.

Self-Sufficient Reusable Biocatalytic System Outfitted with Multiple Oxidoreductases and Flexible Polypeptide-Based Cofactor Swing Arms

Oxidoreductases are widely used in value-added chemical synthesis. Since many oxidoreductases require the consumption of expensive cofactors, multi-enzyme cascade reactions, including cofactor regeneration, have been developed. Although enzymes are frequently immobilized for industrial processes, the immobilization of the small molecule cofactor is challenging. Here, we demonstrate that the co-immobilization of a swing arm cofactor and multiple oxidoreductases can overcome these cofactor limitations. We chose a very flexible protein, elastin-like polypeptide (ELP), fused to a strep-tag II as a genetically encoded swing arm. The Escherichia coli expressed and purified swing arm was conjugated with NAD+ to generate a cofactor swing arm (ELP-NAD+). The cofactor swing arm was co-immobilized on a streptavidin resin with two oxidoreductases fused to a strep-tag II: glucose dehydrogenase (GDH) and mannitol dehydrogenase (MDH). We observed a substantial d-mannitol production (about 1.6 mM) in the three-component (GDH, MDH, and ELP-NAD+) co-immobilized microreactor. The three-component co-immobilized system showed 2 and 3 times higher d-mannitol production than the one with free three components and another with the immobilized enzymes and free cofactor, respectively. Moreover, the product could be easily separated from the co-immobilized reactor and the activity was retained when applied for repeated batch reactions (> seven cycles). These results demonstrate that the genetically encoded cofactor swing arm can be stably co-immobilized with multiple oxidoreductases on a solid support and it can maintain reactivity as a cofactor because of the flexible ELP swing arm.

Direct Isomaltulose Synthesis From Beet Molasses by Immobilized Sucrose Isomerase

Isomaltulose is becoming a focus as a functional sweetener for sucrose substitutes; however, isomaltulose production using sucrose as the substrate is not economical. Low-cost feedstocks are needed for their production. In this study, beet molasses (BM) was introduced as the substrate to produce isomaltulose for the first time. Immobilized sucrose isomerase (SIase) was proved as the most efficient biocatalyst for isomaltulose synthesis from sulfuric acid (H2SO4) pretreated BM followed by centrifugation for the removal of insoluble matters and reducing viscosity. The effect of different factors on isomaltulose production is investigated. The isomaltulose still achieved a high concentration of 446.4 ± 5.5 g/L (purity of 85.8%) with a yield of 0.94 ± 0.02 g/g under the best conditions (800 g/L pretreated BM, 15 U immobilized SIase/g dosage, 40°C, pH of 5.5, and 10 h) in the eighth batch. Immobilized SIase used in repeated batch reaction showed good reusability to convert pretreated BM into isomaltulose since the sucrose conversion rate remained 97.5% in the same batch and even above 94% after 11 batches. Significant cost reduction of feedstock costs was also confirmed by economic analysis. The findings indicated that this two-step process to produce isomaltulose using low-cost BM and immobilized SIase is feasible. This process has the potential to be effective and promising for industrial production and application of isomaltulose as a functional sweetener for sucrose substitute.

Industrial kinetic resolution of d,l-pantolactone by an immobilized whole-cell biocatalyst.

Immobilized whole-cells of Pichia pastoris harboring recombinant d-lactonase were entrapped in calcium alginate gels and used as an efficient biocatalyst for catalytic kinetic resolution of d,l-pantolactone. The immobilized whole-cell biocatalyst exhibited good catalytic stability, which was applied for stereospecific hydrolysis of d-pantolactone for up to 56 repeated batch reactions without obvious loss in the catalytic activity and enantioselectivity.

New Insights into the Deactivation and Stabilization Causes of Plugged AlSBA‐15 in Liquid‐phase Friedel‐Crafts Organic Reactions

AbstractDeactivation is the major challenge of heteroatom‐substituted mesoporous silica materials for large scale applications. Here we report the impact of plugging of mesochannels of AlSBA‐15 on activity and stability of liquid‐phase acylation of anisole with (extremely) low catalyst loadings of 0.15–0.6 wt.% and 0.6–3 wt.% in batch and in repeated batch reactions, respectively. The analysis of property‐catalytic performances showed that deactivation of the plugged AlSBA‐15 was partially from a physical cause by deposition of a uniform nanometer layer of coke on the surface blocking the active sites which could be restored by calcination and partially from an irreversible chemical cause by Al leaching. The catalytic performance of plugged AlSBA‐15 was compared with that of open AlSBA‐[15]. The plugged AlSBA‐15 showed lower but more sustained activity than that by open AlSBA‐15 coming from less and different type of coke deposition. These properties of plugged AlSBA‐15 resulted in two times higher productivity (44.3 vs 23.3 mg‐product/mg‐catalyst) than that by open AlSBA‐[15] . The plugging of mesochannels thus reduced the amount of coke deposition and stabilized the catalyst; however, it exerted no effect on Al leaching. In conclusion, understanding the role of plugging of mesochannels in stabilization and Al leaching and coke deposition in deactivation are useful to design and synthesize efficient solid acid catalysts for liquid‐phase organic reactions.

Reducing residual antibiotic levels in animal feces using intestinal Escherichia coli with surface-displayed erythromycin esterase

Antibiotics are widely used in livestock and poultry industries, which results in large quantities of antibiotic residues in manure that influences subsequent treatments. In this study, an Escherichia coli strain was engineered to display erythromycin esterase on its cell surface. The engineered strain (E. coli ereA) efficiently degraded erythromycin by opening the macrocyclic 14-membered lactone ring in solution. Erythromycin (50 mg/L) was completely degraded in a solution by E. coli ereA (1 × 109 CFU/mL) within 24 h. E. coli ereA retained over 86.7 % of the initial enzyme activity after 40 days of storage at 25 °C, and 78.5 % of the initial activity after seven repeated batch reactions in solution at 25 °C. Mice were fed with E. coli ereA and real-time quantitative PCR data showed that E. coli ereA colonized in the mice large intestine. The mice group fed E. coli ereA exhibited 83.13 % decrease in erythromycin levels in their feces compared with the mice group not fed E. coli ereA. E. coli ereA eliminated antibiotics from the source preventing its release into the environment. The surface-engineered strain therefore is an effective alternative agent for treating recalcitrant antibiotics, and has the potential to be applied in livestock and poultry industries.

Enzymatic synthesis of bio-based wax esters from palm and soybean fatty acids using crude lipases produced on agricultural residues

The increased demand for the consumption of vegetable oils has led to the generation of high quantities of by-products, such as palm and soybean fatty acid distillates (PFAD, SFAD), as a result of the refining process. In this study PFAD and SFAD were used as raw materials for the enzymatic production of wax esters in a solvent-free system. A fungal crude lipase was produced by solid state fermentation using babassu cake as substrate and then fermented solids were used as biocatalyst for wax esters synthesis using cetyl and oleyl alcohols. The highest wax esters production of 80% was achieved for the SFAD-cetyl, SFAD-oleyl and PFAD-oleyl esters using 8.8 U/g of biocatalyst at 50 °C. The proposed biocatalysis could be economically feasible since the crude lipase was efficiently reused for up to five repeated batch reactions. The physicochemical properties of the wax esters were also studied. Differential scanning calorimetry and the determination of acid, iodine and saponification values showed that the wax esters have similar properties to natural waxes. This study demonstrated the valorization of by-products from vegetable oil production processes as an alternative feedstock for the synthesis of bio-based wax esters with potential use in food, cosmetics and chemical applications.

Synthesis, characterization and optimization of a two-step immobilized lipase

Lipase (Candida sp. 99–125) was immobilized by a two-step process, adsorption and subsequent entrapment. The formation mechanism of immobilized lipase was investigated by SEM and EDS. With the support of diatomite, the internal network of immobilized lipase showed more porosity and was fully developed. The dimension of the network was more uniform in the present of gelatine. The maximum biodiesel yield was 92% at 40 °C in a 24 h reaction. The biodiesel yield was maintained at about 70% after 11 repeated batch reactions. This enzyme immobilization method has potential applications in biodiesel and other lipase catalyzed synthesis process.

An intensified reaction technology for high levulinic acid concentration from lignocellulosic biomass

An intensified reaction technology was investigated to achieve high concentrations of levulinic acid (LA) from eucalyptus wood. In the new process, the woody biomass was first pretreated in mild conditions to hydrolyze hemicellulose and recover xylose. The pretreated wood with a high content of cellulose was further treated under relatively severe conditions to produce LA and formic acid. The reaction solution was reused in treatment of new batches of biomass, resulting in high concentrations of LA (105 g/L) and formic acid (39 g/L), respectively. The lignin and humins were recovered as solid byproducts, which had an estimated high heating value of 25–27 MJ/kg in comparison with 18 MJ/kg of original wood. In the sequential repeated batch reactions, however, the LA yield deteriorated batch by batch. It was found out that the old LA in the reaction solution inhibited formation of new LA from glucose. In addition, some LA at high concentrations was lost due to its interactions with other intermediates and byproducts.

Increased Production of Food-Grade d-Tagatose from d-Galactose by Permeabilized and Immobilized Cells of Corynebacterium glutamicum, a GRAS Host, Expressing d-Galactose Isomerase from Geobacillus thermodenitrificans.

The generally recognized as safe microorganism Corynebacterium glutamicum expressing Geobacillus thermodenitrificans d-galactose isomerase (d-GaI) was an efficient host for the production of d-tagatose, a functional sweetener. The d-tagatose production at 500 g/L d-galactose by the host was 1.4-fold higher than that by Escherichia coli expressing d-GaI. The d-tagatose-producing activity of permeabilized C. glutamicum (PCG) cells treated with 1% (w/v) Triton X-100 was 2.1-fold higher than that of untreated cells. Permeabilized and immobilized C. glutamicum (PICG) cells in 3% (w/v) alginate showed a 3.1-fold longer half-life at 50 °C and 3.1-fold higher total d-tagatose concentration in repeated batch reactions than PCG cells. PICG cells, which produced 165 g/L d-tagatose after 3 h, with a conversion of 55% (w/w) and a productivity of 55 g/L/h, showed significantly higher d-tagatose productivity than that reported for other cells. Thus, d-tagatose production by PICG cells may be an economical process to produce food-grade d-tagatose.

Cell Surface Display Fungal Laccase as a Renewable Biocatalyst for Degradation of Persistent Micropollutants Bisphenol A and Sulfamethoxazole.

Fungal laccases have high activity in degrading various persistent organic pollutants. However, using enzymes in solution for water treatment has limitations of nonreusability, short enzyme lifetimes, and high cost of single use. In this study, we developed a new type of biocatalyst by immobilizing fungal laccase on the surface of yeast cells using synthetic biology techniques. The biocatalyst, referred to as surface display laccase (SDL), had an enzyme activity of 104 ± 3 mU/g dry cell (with 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS)). The SDL retained over 90% of the initial enzyme activity after 25 days storage at room temperature, while, in contrast, activity of free laccase declined to 60% of its initial activity. The SDL could be reused with high stability as it retained 74% of initial activity after eight repeated batch reactions. Proof-of-concept evaluations of the effectiveness of SDL in treating contaminants of emerging concern were performed with bisphenol A and sulfamethoxazole. Results from contaminant degradation kinetics and the effects of redox mediator amendment provided insights into the factors affecting the efficacy of the SDL system. This study reports, for the first time, the development of a surface display enzyme biocatalyst as an effective and renewable alternative for treating recalcitrant organic micropollutants.

Purification and Immobilization of a Novel Enantioselective Lipase from Tsukamurella tyrosinosolvents for Efficient Resolution of Ethyl 2-(2-oxopyrrolidin-1-yl) Butyrate.

A highly enantioselective lipase from Tsukamurella tyrosinosolvents E105 was purified via ultrasonic extraction, precipitation, and chromatographic steps. The enzyme was purified about 38-fold with the recovery yield of 9% and was confirmed as a dimer protein consisting of two identical subunits with a molecular mass of 24kDa. The purified lipase was used to catalyze resolution of racemic ethyl 2-(2-oxopyrrolidin-1-yl) butyrate to (S)-2-(2-oxopyrrolidin-1-yl) butyric acid. The maximum activity of such lipase was obtained at pH7.5, 35°C, and the highest relative activity (156.80%) was observed in the presence of 0.5mM Co2+. Subsequently, the lipase was encapsulated within a mixture of 3% sodium alginate and 0.8% carrageenan, and then cross-linked with 0.6% glutaraldehyde to enhance its biocatalytic capability and stability. Comparing with 36.9% product yield and 97.5% product ee of free lipase, the highest product yield of 46.3% and ee of 98.5% for immobilized lipase were achieved with the presence of 20mM substrate. In addition, the reusability of immobilized lipase was also investigated, which could maintain 63.7% of its initial conversion yield after seven repeated batch reactions. Thus, the evaluated enantioselective lipase in this work has a good potential for further industrial application.

Synthesis of galacto-oligosaccharides from lactose using immobilized cells of Kluyveromyces marxianus NCIM 3551

Sodium alginate immobilized cells of Kluyveromyces marxianus NCIM 3551 cells were used for synthesis of galacto-oligosaccharides (GOS) using lactose as a substrate through transgalactosylation approach. The immobilized cells retained 84.4% of the original β-galactosidase activity displayed by free cells. An increase in thermo-stability and a slight shift in pH optimum was observed for β-galactosidase in immobilized cells. Maximum GOS concentration of 72.0g/L and specific GOS productivity 60.0g/L/U was obtained using free cells as opposed to 42.6g/L and productivity of 35.6g/L/U for immobilized cells. A repeated batch reaction was carried out using free and immobilized cells for 10 cycles, of 3h each, using 20% lactose (w/v) with a nearly similar productivity of 24.0g/L/h and 21.3g/L/h respectively. Based on the stability of the immobilized cells, continuous synthesis of GOS was carried out for 10 days in packed bed bioreactor using immobilized cells at a loading of 28.8 units/reactor volume. A yield of 64.0g/L of GOS and a productivity of 21.3g/L/h was achieved at an optimized flow rate of 1.0mL/min. The system here demonstrated the feasibility of using whole cells for production of GOS in a packed bed reactor.

Metal Catalysts Recycling and Heterogeneous/Homogeneous Catalysis

Heterogeneous metal catalysts rather than homogeneous ones are recommended for industrial applications after considering their performance in activity, separation, and recycling [1]. The recycling of metal catalysts is important from economic and environmental points of view. When supported and bulk metal catalysts are used in liquid-phase organic reactions, there is a possibility that active metal species are leaching away into the liquid phases [2,3]. The metal leaching would make it difficult for the catalysts to maintain their desired initial performance for repeated batch reactions and during continuous ones. The metal leaching would also cause some undesired contamination of products by the metal species dissolved in the reaction mixture, and the separation of the metal contaminants would be required to purify the products. Therefore, various novel methods have been proposed so far to immobilize/stabilize the active metal species and to separate/collect/reuse the dissolved metal species [4]. In addition, knowledge on the heterogeneous and homogeneous natures of organic reactions using heterogeneous catalysts is important to discuss their reaction mechanisms and catalytically working active species. [...]

Improved production of isomaltulose by a newly isolated mutant of Serratia sp. cells immobilized in calcium alginate.

Isomaltulose, also known as palatinose, is produced by sucrose isomerase and has been highlighted as a sugar substitute due to a number of advantageous properties. For the massive production of isomaltulose, high resistance to sucrose and stability of sucrose isomerase as well as sucrose conversion yields would be critical factors. We describe a series of screening procedures to isolate the mutant strain of Serratia sp. possessing enhanced isomaltulose production with improved stability. The new Serratia sp. isolated from a series of screening procedures allowed us to produce isomaltulose from 60% sucrose solution, with over 90% conversion yield. Moreover, when this strain was immobilized in calcium alginate beads and placed in a medium containing 60% sucrose, it showed over 70% sucrose conversion yields for 30 cycles of repeated-batch reactions. Thus, improved conversion activity and stability of the newly isolated Serratia sp. strain in the present study would be highly valuable for industries related to isomaltulose production.

Biodiesel production from jatropha oil catalyzed by immobilized Burkholderia cepacia lipase on modified attapulgite

Lipase from Burkholderia cepacia was immobilized on modified attapulgite by cross-linking reaction for biodiesel production with jatropha oil as feedstock. Effects of various factors on biodiesel production were studied by single-factor experiment. Results indicated that the best conditions for biodiesel preparation were: 10 g jatropha oil, 2.4 g methanol (molar ratio of oil to methanol is 1:6.6) being added at 3h intervals, 7 wt% water, 10 wt% immobilized lipase, temperature 35°C, and time 24h. Under these conditions, the maximum biodiesel yield reached 94%. The immobilized lipase retained 95% of its relative activity during the ten repeated batch reactions. The half-life time of the immobilized lipase is 731 h. Kinetics was studied and the Vmax of the immobilized lipases were 6.823 mmol L(-1). This immobilized lipase catalyzed process has potential industrial use for biodiesel production to replace chemical-catalyzed method.

Electrospun polyacrylonitrile nanofibrous membranes for chitosanase immobilization and its application in selective production of chitooligosaccharides

Polyacrylonitrile nanofibrous membranes (PANNFM) were prepared by electrospinning from 10wt.% of PAN solution and its surface was modified by amidination reaction. A new chitosan degrading enzyme from Aspergillus sp. was covalently immobilized on PANNFM. Immobilization efficiency of 80% was achieved by activating PANNFM surface for 30min followed by 2h treatment with enzyme solution. The optimum temperature and pH for immobilized enzyme were 50°C and 5.8, respectively. The immobilized chitosanase retained >70% activity after ten repeated batch reaction and could be stored up to 60days at 4°C with minor loss in activity. Chitosan hydrolysis using different substrates were studied using immobilized chitosanase in batch conditions. Continuous selective production of chitooligosaccharides (dimer to hexamer) by changing the temperature was achieved by PANNFM-chitosanase.

Gradually accumulating beneficial mutations to improve the thermostability of N-carbamoyl-d-amino acid amidohydrolase by step-wise evolution

To further enhance repeated batch reactions with immobilized N-carbamoyl-D-amino acid amidohydrolase (DCase), which can be used for the industrial production of D-amino acids, the stability of high soluble mutant DCase-M3 from Ralstonia pickettii CGMCC1596 was improved by step-wise evolution. In our previous report, six thermostability-related sites were identified by error-prone PCR. Based on the above result, an improved mutant B5 (Q12L/Q23L/H248Q/T262A/T263S) was obtained through two rounds of DNA shuffling, showing a 10°C increase in the T (50) (defined as the temperature at which heat treatment for 15 min reduced the initial activity by 50%) compared with the parental enzyme DCase-M3. Furthermore, several thermostability-related sites (Met(31), Asn(93), Gln(207), Asn(242), Glu(266), Thr(271), Ala(273)) on B5 were identified using amino acid consensus approach based on sequence alignment of homologous DCases. These sites were further investigated by iterative saturation mutagenesis (ISM), and a combinational mutant D1 (Q12L/Q23L/Q207E/N242G/H248Q/T262A/T263S/E266D/T271I/A273P) that enhanced the T(50) by about 16°C over DCase-M3 was obtained. Oxidative stability assay showed that the most heat-resisting mutant displayed only a slight increase in resistance to hydrogen peroxide. Comparative characterization showed that D1 not only maintained its characteristic high solubility but also shared similar k(cat) and K(m) values and optimum reaction pHs with the parental enzyme. The significantly improved mutants in the immobilized form are expected to be applied in the industrial production of D-p-hydroxyphenylglycine.

Biodiesel production from pomace oil by using lipase immobilized onto olive pomace

In the present work, microbial lipase from Thermomyces lanuginosus was immobilized by covalent binding onto olive pomace. Immobilized support material used to produce biodiesel with pomace oil and methanol. The properties of the support and immobilized derivative were evaluated by scanning electron microscopy (SEM). The maximum immobilization of T. lanuginosus was obtained as 18.67mg/g support and the highest specific activity was 10.31U/mg protein. The properties of immobilized lipase were studied. The effects of protein concentration, pH and buffer concentration on the immobilization and lipase activity were investigated. Biodiesel production using the immobilized lipase was realized by a three-step addition of methanol to avoid strong substrate inhibition. Under the optimized conditions, the maximum biodiesel yield was 93% at 25°C in 24h reaction. The immobilized enzyme retained its activity during the 10 repeated batch reactions.