Enzyme Column Research Articles

Evaluation of Proteolytic Digestion Efficiency in Hydrogen Exchange-Mass Spectrometry Experiments Using the Digestible Peptide Score.

In a hydrogen exchange-mass spectrometry (HX-MS) experiment, the enzymatic proteolysis of the deuterated protein is an essential step. Often the differences in the performance between different digestion protocols or between immobilized protease columns can be challenging to evaluate. To compare differences in the performance of immobilized protease columns, a new digestion efficiency metric known as digestible peptide scoring (DPS) was developed and is presented in this work. The measured response fraction of substance P peptide is used to assign a value between 0% and 100% based on the fraction of substance P digested by the enzyme, using angiotensin II as an undigested internal standard. In this work, the DPS approach was tested using multiple immobilized pepsin batches prepared using different protocols. The results demonstrate the repeatability of DPS values for batches prepared using the same conditions and the ability of the DPS evaluations to provide unique values when the immobilization conditions were altered. Protein digestions obtained with a higher scoring column were better than digestions obtained using a lower scoring column. The DPS evaluation is simple and quickly provides an unambiguous assessment which can be used to evaluate an immobilized enzyme column's suitability prior to performing an experiment, to track performance over a column's lifetime, to optimize protease immobilization protocols specifically for the quench conditions of a particular experiment, and to optimize the digestion conditions.

Chromatography at -30 °C for Reduced Back-Exchange, Reduced Carryover, and Improved Dynamic Range for Hydrogen-Deuterium Exchange Mass Spectrometry.

For hydrogen–deuterium exchange mass spectrometry (HDX-MS) to have an increased role in quality control of biopharmaceuticals, H for D back-exchange occurring during protein analyses should be minimized to promote greater reproducibility. Standard HDX-MS analysis systems that digest proteins and separate peptides at pH 2.7 and 0 °C can lose >30% of the deuterium marker within 15 min of sample injection. This report describes the architecture and performance of a dual-enzyme, HDX-MS instrument that conducts liquid chromatography (LC) separations at subzero temperature, thereby reducing back-exchange and supporting longer LC separations with improved chromatographic resolution. LC separations of perdeuterated, fully reduced, iodoacetamide-treated BSA protein digest standard peptides were performed at 0, −10, −20, and −30 °C in ethylene glycol (EG)/H2O mixtures. Analyses conducted at −20 and −30 °C produced similar results. After subtracting for deuterium retained in arginine side chains, the average peptide eluted during a 40 min gradient contained ≈16% more deuterium than peptides eluted with a conventional 8 min gradient at 0 °C. A subset of peptides exhibited ≈26% more deuterium. Although chromatographic peaks shift with EG concentration and temperature, the apparatus elutes unbroadened LC peaks. Electrospray ion intensity does not decline with increasing EG fraction. To minimize bias from sample carryover, the fluidic circuits allow flush and backflush cleaning of all enzyme and LC columns. The system can perform LC separations and clean enzyme columns simultaneously. Temperature zones are controlled ±0.058 °C. The potential of increased sensitivity by mixing acetonitrile with the analytical column effluent was also examined.

Rapid determination of o-phenylenediamine of water using an enzyme thermistor

ABSTRACTIn this paper, we describe an enzyme thermistor with a flow injection analysis system for the determination of o-phenylenediamine (OPD) in water samples. O-phenylenediamine solutions of different concentrations and actual water samples were tested, and their OPD values were determined by measuring the heat generated when the samples passed through an enzyme column containing horseradish peroxidase and hydrogen peroxide. The biosensor exhibited a large linear range (1 to 1000 µM) and a low detection limit (1.99 µM). The thermal sensor was successfully used for detecting the OPD values of actual water samples. The OPD values of water samples from various sources were correlated very well with those obtained by the reference method; the relative standards were less than 5%. The sensor is environmentally friendly, economical and highly stable, and exhibits good reproducibility and accuracy. In addition, its response time is short, and there is no danger of hazardous emissions or external contamination. Finally, the samples to be tested do not have to be pretreated. These results suggest that the enzyme thermistor is suitable for the continuous monitoring of the OPD values of actual wastewater samples.

Preparation of Diacylglycerol-enriched Rice Bran Oil by Lipase-catalyzed Deacidification in Packed-bed Reactors by Continuous Dehydration.

Diacylglycerol-enriched rice bran oil (RBO-DAG) was produced by deacidification of high-acid rice bran oil (RBO) with glycerol (Gly) using Lipozyme RM IM by continuous dehydration by combination of two enzyme columns (column 1 and 3, used for deacidification) with one molecular sieves column (column 2, used for dehydration). The conditions for three columns were respectively optimized. Response surface methodology (RSM) was used to optimize the conditions of column 1. The content of DAG and conversion of free fatty acid (FFA) were used as indicators and the effects of the enzyme load (8-12 g), flow rate (0.3-0.6 mL/min), substrate molar ratio (4-6) and reaction temperature (55-75°C) were investigated. The content of DAG and conversion of FFA were significantly correlated to the flow rate and substrate molar ratio. Most desirable conditions of the reaction with respect to the maximal DAG content and FFA conversion was attained under the residence time of 40 min, substrate molar ratio of 5.52 (Gly: RBO) and temperature of 66°C. The conditions for column 2 were investigated by varying molecular sieves load and flow rate, and the maximal dehydration rate of 85.22% was obtained under the optimal conditions. For column 3, the optimum conditions were obtained as: flow rate, 0.2mL/min; temperature, 65°C, and the content of DAG and FFA were 38.99% and 3.04%, respectively under these conditions. The catalytic activity of the lipase was stable in twelve continuous operations with 83.22% of its original ability, demonstrating its potential in the continuous packed-bed reactors (PBRs) system. These results showed that packed-bed reactors combined with continuous deacidification and dehydration in one system had great value in industrial production for high-acid RBO with the improved conversion rate.

Continuous, packed-bed, enzymatic bioreactor production and stability of feruloyl soy glycerides

The synthesis of feruloyl soy glycerides was demonstrated on a pilot-scale (1metric ton/year) in a continuous, four-column series, packed-bed, enzymatic bioreactor (herein referred to as the bioreactor). Ethyl ferulate and soybean oil were combined and converted at 3.5kg/d over Candida antarctica lipase B immobilized on an acrylic resin (Novozym 435) at 60°C. The ethyl ferulate/soybean oil substrate was fed through the four, packed-bed, enzyme columns in series with each column fitted with a recirculation pump, which improved conversion and throughput compared to smaller, batch bioreactor systems. The synthesis was carried out over 4.5 months with a single charge of enzyme to produce over 450kg of feruloyl soy glycerides at 65% conversion. The stability of the feruloyl soy glycerides was determined by comparing the total ferulate concentration (UV absorbance), free radical scavenging (antioxidant) capacity, total acid value (hydrolysis), and Lovibond color of samples produced in 2009 to samples produced in 2015. The feruloyl soy glycerides showed no appreciable loss of UV absorbing (λmax 325nm) or rapid antioxidant capacity (50% reduction of initial radical concentration <5min) after six years of storage. Feruloyl soy glycerides were shown to be hygroscopic over six years, but did not show any appreciable difference in total acid number due to hydrolysis or change in color. The successful pilot-scale synthesis and long term efficacy and stability of feruloyl soy glycerides was critical for the adoption of feruloyl soy glycerides as broad range UV absorbing and antoxidant ingredient by the cosmetic and skin care industries.

Immobilized Enzyme Column Combined with HPLC and Column Switching Method for the Analysis of Complicated Matrix Such As Body Fluids

Copyright: © 2014 Shintani H. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Enzymes are bio-catalysts which affect transformation of substrates to products with high specificity. The usage of enzymes in domestic and industrial applications is well known and has been well documented since the early history of civilization. With the advances in understanding of enzymology, usage of enzymes in industrial and biotechnological processes and molecular medicine has proliferated.

Structured and Specialty Lipids in Continuous Packed Column Reactors: Comparison of Production Using One and Two Enzyme Beds

AbstractIn this study, speciality lipids based on fish oil (FO) and capric acid (CA) were produced in packed bed bioreactors using immobilized Lipozyme IM from Rhizomucor miehei in a solvent‐free environment. Our goal was to compare the product quality and yield among reactors consisting of one or two enzyme columns. Response surface methodology (RSM) was used to optimize process variables for maximum incorporation (Inc) of CA for each reactor configuration. The studied process variables were substrate molar ratio (1:1–3:1 CA/FO), temperature (35–55 °C) and flow rate (0.5–1.5 mL/min). All experiments were conducted based on a face‐centered cube design. The maximum predicted Inc of CA into FO (31.7 mol%) using one column was obtained when substrate molar ratio, temperature and flow rate of substrates were 2.70:1 (CA/FO), 55 °C and 0.5 mL/min. The corresponding optimal Inc of CA into FO (22.7 mol%) using two columns was predicted at 2.95:1 (CA/FO), 55 °C and 0.86 mL/min. Analysis of variance (ANOVA) showed that Inc of CA into the one‐bed design was significantly influenced by all experimental conditions, with substrate molar ratio having the greatest impact. In the two‐bed design, only temperature and flow rate had an effect; the molar ratio of substrates was not significant. Coefficients of determination were low for both designs, indicating a poor fit of our data to the model. However, the main purpose of this study was to assess the effect of process factors on Inc of CA into FO, rather than generate a model useful for prediction of responses under conditions not examined with this design. Therefore, the significant ANOVA results are much more important, stressing the real relationship between parameters and response, than the low coefficients of determination.

Quantitation of the Cancer Marker LASA Using Immobilized Enzymes in a Semiautomated System

Cancer patients are found to have elevated serum levels of lipid-associated sialic acid (LASA). LASA measurement provides a valuable test for the diagnosis of human cancer. The currently used measurement methods are tedious and nonselective. Here we report a method based on immobilized enzyme minicolumns in a flow system for the analysis of this clinically important analyte. Three enzymes comprising two minicolumns are introduced online and the analyte in a precisely injected volume of 20 microL is made to pass through these immobilized enzyme columns. The final product of the reaction is detected amperometrically. Neuraminidase and NANA-aldolase are coimmobilized on controlled pore glass, followed by a column of pyruvate oxidase. The method was applied to the quantitation of LASA in the sera of normal individuals and patients with cancer of the esophagus. We observed no loss of activity of the immobilized enzymes after analyzing about 300 samples. The sample throughput was 30 samples per hour.

Quantitation of the cancer marker LASA using immobilized enzymes in a semiautomated system

Cancer patients are found to have elevated serum levels of lipid-associated sialic acid (LASA). LASA measurement provides a valuable test for the diagnosis of human cancer. The currently used measurement methods are tedious and nonselective. Here we report a method based on immobilized enzyme minicolumns in a flow system for the analysis of this clinically important analyte. Three enzymes comprising two minicolumns are introduced online and the analyte in a precisely injected volume of 20 microL is made to pass through these immobilized enzyme columns. The final product of the reaction is detected amperometrically. Neuraminidase and NANA-aldolase are coimmobilized on controlled pore glass, followed by a column of pyruvate oxidase. The method was applied to the quantitation of LASA in the sera of normal individuals and patients with cancer of the esophagus. We observed no loss of activity of the immobilized enzymes after analyzing about 300 samples. The sample throughput was 30 samples per hour.

The evolution of enzymatic interesterification in the oils and fats industry

AbstractImmobilised lipases have now become accepted as a mainstream technology for fat modification. This paper presents the development of this technology in particular for the production of trans‐free fats. One of the factors influencing this development has been concern for health, which has made trans fats a major issue for food manufacturers and consumers. Enzymatic interesterification is a relatively new method for producing trans‐free alternatives for fats used in conventional margarines and shortenings. The development of this technology is examined from the perspective of an eventual industrial application rather than operation only at the laboratory scale. This paper also covers the practical means of operating immobilised enzyme columns and gives examples of how formulations can be adapted to match existing specifications. There are environmental benefits when choosing enzyme technology in comparison with chemical‐based routes. Life cycle assessments have been used to quantify the differences in environmental impact of this new technology. The final process is both capable of providing fats with the correct melting properties but without trans fats and of reducing the environmental impact of fat processing. Finally, the future developments that are anticipated in the applications of this technology are considered.

Sensitive Determination of Reactive Oxygen Species by Chemiluminescence Methods and Their Application to Biological Samples and Health Foods

Sensitive and selective methods, based on chemiluminescence reactions, were introduced for determination of reactive oxygen species (ROS) and their applications to biological samples and health foods. First, a sensitive method for determination of H(2)O(2) by peroxyoxalate chemiluminescence (PO-CL) was developed. This method could be applied to determine small amounts of H(2)O(2) in cola drinks and bacterial contamination of food items. Secondly, the combination of immobilized enzyme column reactor, or ultraviolet irradiation system, with the PO-CL detection method was able to determine clinical substrates (i.e. choline-containing phospholipids, polyamines and D-amino acids) and organic peroxides. Also, an evaluation method of the quenching effect of luminol chemiluminescence against ROS was developed. The sensitive, rapid and precise measurement of the quenching effect against ROS such as superoxide, singlet oxygen, hydroxyl radical, peroxynitrite and hypochlorous ion was achieved. The proposed method could be applied to rosemary extracts, natural colorants and grape seed extracts.

Determination of aromatic compounds by high‐performance liquid chromatography with on‐line photoreactor and peroxyoxalate chemiluminescence detection

This paper describes a novel high-performance liquid chromatographic (HPLC) method for the determination of aromatic compounds with peroxyoxalate chemiluminescence (PO-CL ) detection following on-line UV irradiation. Aromatic compounds were UV irradiated (254 nm, 15 W) to generate hydrogen peroxide, which was determined via PO-CL detection using a mixture of bis(2,4,6-trichlorophenyl)oxalate (aryloxalate) and 2,4,6,8-tetrathiomorpholinopyrimido[5,4-d]pyrimidine (fluorophore) as a post-column CL reagent. Generation of hydrogen peroxide from aromatic compounds was confirmed using a flow injection analysis (FIA) system incorporating an enzyme column reactor immobilized with catalase. The conditions for UV irradiation were optimized using benzene and monosubstituted benzenes (phenol, benzaldehyde, nitrobenzene and N,N-dimethylaniline) by an HPLC system to evaluate the analytical performance of the proposed system. The detection limits for benzene and monosubstituted benzenes were in the range 2.1-124 pmol/injection at signal:noise (S:N) ratio = 3. Monocyclic and polycyclic hydrocarbons were also employed to investigate their CL properties. The possibility of PO-CL detection for a wide variety of aromatic compounds was shown for the first time.

High infusion pressure in conjunction with vitreous surgery alters the morphology and function of the retina of rabbits

To investigate the effects of high infusion pressure in conjunction with pars plana vitrectomy (PPV) on retinal morphology and function in rabbits. Pars plana vitrectomy was performed under urethane (0.8 mg/kg) anaesthesia in the right eye of albino rabbits following phacoemulsification and aspiration (PEA). The left eyes were not touched. After PEA, the animals were divided into two groups. In six eyes, intraocular pressure (IOP) was increased to 80 mmHg for 30 mins (high-pressure group) and in five eyes IOP was maintained at 40 mmHg for 30 mins (low-pressure group). The IOPs were regulated by the height of the bottle of balanced salt solution (BSS) and monitored with a pressure transducer. After the pressure elevation, vitreous fluid was collected to measure the glutamate concentration. Then, PPV was performed for 15 mins in both groups under an infusion pressure of 40 mmHg. In five additional rabbits, PEA alone was performed in the right eye, and vitreous fluid was collected (PEA group). Functional alterations were assessed by recording visual evoked potentials (VEPs) and electroretinograms (ERGs). Ten days after the IOP changes, the animals were killed with intravenous pentobarbital sodium and the eyes were prepared for histological analysis. Damage to retinal ganglion cells (RGCs) was quantified by counting the number of cells in the ganglion cell layer (GCL). The contralateral eyes in the high-pressure group served as controls (n = 6). The mean implicit time (IT) of the VEPs in the high-pressure group was significantly longer than that before the IOP elevation, by 114-124% (p < 0.05, paired t-test), and also than that of control eyes (p < 0.05, anova followed by t-test). No significant changes in the VEPs were detected in either the low-pressure group or the PEA group. There were significantly fewer cells in the GCL in the high-pressure group (24.7/mm) than in the control animals (41.4/mm; p < 0.05, Dunnett's test). The number of cells in the GCL in the low-pressure and PEA groups did not significantly differ to that in the controls. The amplitudes of the ERG a- and b-waves were not significantly changed (p > 0.05, paired t-test). These results suggest that high infusion pressure in conjunction with PPV leads to morphological and functional changes in the retina. The absence of ERG changes and presence of VEP changes suggest that these changes were due to damage to RGCs, which supports the morphological observations.

Sample clean-up with sol–gel enzyme and immunoaffinity columns for the determination of bisphenol A in human urine

The paper describes the development of a simple and highly selective analytical method for the determination of free and total bisphenol A in urine samples. Free bisphenol A levels can be determined after sample clean-up using sol–gel immunoaffinity columns containing anti-bisphenol A antibodies. In determining total bisphenol A levels, the sample pre-treatment procedure consists of sample preparation using an on-line combination of two sol–gel columns, an enzyme column containing glucuronidase and arylsulfatase, and an immunoaffinity column. Bisphenol A can then be quantified by high-performance liquid chromatography and fluorescence detection. The mean recovery was found to be 78% with a standard deviation of 3.4%, the LOD (S/N = 3) was 0.2 ng/ml. The method was applied to determine free and total urinary BPA levels of healthy adults and dialysis patients.

Selective determination of quinones by high-performance liquid chromatography with on-line post column ultraviolet irradiation and peroxyoxalate chemiluminescence detection

A new HPLC method was developed for the simultaneous determination of quinones with peroxyoxalate chemiluminescence (PO-CL) detection following on-line UV irradiation. Quinones [i.e., 1,2-naphthoquinone, 1,4-naphthoquinone, 9,10-anthraquinone, 9,10-phenanthrenequinone] were UV irradiated (254 nm, 15 W) to generate hydrogen peroxide and a fluorescent product that were determined via PO-CL detection. Generation of hydrogen peroxide from quinones with on-line UV irradiation was confirmed using flow injection analysis (FIA) system whereby incorporating an enzyme column reactor immobilized with catalase. Moreover, the structure of the produced fluorophore was confirmed using LC–MS, IR, and 1H NMR. Afterwards, the conditions for UV irradiation and PO-CL detection were optimized. The separation of four quinones by HPLC was accomplished isocratically on an ODS column within 25 min. The detection limits (signal-to-noise ratio = 3) were 6.0 pmol/injection for 1,2-naphthoquinone, 4.4 pmol/injection for 1,4-naphthoquinone, 0.2 pmol/injection for 9,10-anthraquinone, and 0.45 pmol/injection for 9,10-phenanthrenequinone.

Enzymatic transformation of aliphatic polyesters into cyclic oligomers using enzyme packed column under continuous flow of supercritical carbon dioxide with toluene

The enzymatic degradation of chemically synthesized typical biodegradable plastics, such as poly(R,S-3-hydroxybutanoate), poly(3-caprolactone) and poly(butylene adipate), into reactive cyclic oligomers under the continuous flow of supercritical or subcritical carbon dioxide (scCO2) with toluene through an enzyme column was studied with reference to chemical recycling. It was confirmed that all the tested polymers were quantitatively transformed into the corresponding cyclic oligomers by passage through the column packed with immobilized lipase from Candida antarctica (Novozym 435) at 40 8C under the continuous flow of scCO2 with some organic solvent. Compared to the pure organic solvents, by the addition of scCO2 as a mobile phase degradation of the polymer was significantly promoted with respect to the reaction time, temperature and polymer concentration for complete transformation of the polyesters into oligomers through the enzyme column. This phenomenon is ascribed to the high diffusivity, low viscosity and better mass-transfer properties of scCO2 as the reaction media. The degradation activity of the enzyme column was maintained for at least 6 months at 40 8C.

Analysis of Phytoestrogens in Foods Using Sol-Gel Enzyme Columns for Sample Preparation

The paper describes the development and application of a new method in phytoestrogen analysis using sol-gel cellulase columns for sample preparation. Enzyme columns were produced by entrapping commercially available cellulase by slightly modifying a simple sol-gel procedure previously optimised for immobilising antibodies. Quantitative hydrolysis of both lignan and isoflavone glycosides was achieved within 5 min. Immobilisation of the enzyme enabled the elimination of enzyme-born impurities prior to analysis. Sol-gel cellulase columns could be used for at least 15 cleavage cycles over the investigated period of two months.

Cover Picture: Macromol. Biosci. 10/2004

Cover: The conceptual scheme shows the lipase‐catalyzed green chemical recycling of biodegradable polyesters and the equipment for the continuous enzymatic degradation of biodegradable polyesters, using the enzyme column. The typical chemically‐synthesized biodegradable plastics, such as poly(butylene adipate) and poly(ε‐caprolactone), were degraded and transformed quantitatively into a repolymerizable cyclic oligomer passing through the enzyme packed column under continuous flow. And it was confirmed that the degradation activity of the enzyme column was maintained for at least six months at 40 °C.Further details can be found in the Full Paper by Y. Osanai, K. Toshima, and S. Matsumura* on page 936.

Transformation of Biodegradable Polyesters into Cyclic Oligomers under Continuous Flow Using an Enzyme‐Packed Column

The enzymatic degradation of typical chemically synthesized biodegradable plastics, such as poly(butylene adipate) and poly(epsilon-caprolactone), to a repolymerizable cyclic oligomer under continuous flow using an enzyme column was studied, directed towards green chemical recycling. It was confirmed that both polymers were quantitatively transformed into the corresponding cyclic oligomers by passage through the column packed with immobilized lipase from Candida antarctica (Novozym 435) at 40 degrees C using a toluene solution. Compared to the batch treatment in a flask, a short reaction time and a low reaction temperature occurred for complete transformations of the polyesters by continuous flow through the enzyme column. The degradation activity of the enzyme column was maintained for at least six months at 40 degrees C. Conceptual scheme of the continuous degradation equipment using the enzyme column.

Fluorometric determination of urea in alcoholic beverages by using an acid urease column-FIA system

An acid urease column was applied to a fluorometric flow-injection analysis (FIA) system as a recognition element for determination of urea in rice wines. The acid urease has specific properties of showing its catalytic activity in low pH range and tolerance to ethanol in comparison to those of a urease from jack-beans. The enzymes were covalently immobilized onto porous glass beads with controlled pore size and then, packed into a small polymer column. The flow-type of the biosensing system was assembled with a sample injection valve, the immobilized enzyme column, and a flow-through quartz cell attached to a fluorescent spectrophotometer. Citrate buffer (50 mM, pH 5.0) as the carrier solution was continuously pumped through the system. Sample solutions were introduced into the system via a rotary injection valve. A standard urea solution was measured through monitoring variations in fluorescent intensity attributable to fluorescent isoindole derivatives formed by coupling with ammonia molecules released in the enzymatic hydrolysis of urea and orthophthalaldehyde reagents. The fluorescent intensity was measured under the conditions of λ ex = 415 nm and λ em = 485 nm. A wide, linear relationship was obtained between the concentration of urea (1.0–100 μM) and the variation in fluorescent intensity. The monitoring did not suffer from ethanol and various amino acids contained in rice wines. Real samples pretreated with ion exchange resins for removal of endogenous ammonia were introduced into the FIA system and urea in the samples was determined. These results were compared with those obtained with use of an F-kit method. The proposed FIA system should present sensitive, selective and convenient analysis of urea in alcoholic beverages.