Free Trypsin Research Articles

Biomolecular Condensation of Trypsin Prevents Autolysis and Promotes Ca2+-Mediated Activation of Esterase Activity.

The presence of Ca2+ ions is known to facilitate the activity of trypsin-like serine proteases via structural stabilization against thermal denaturation and autolysis. Herein, we report a new and hidden regulatory role of Ca2+ in the catalytic pathways of trypsin and α-chymotrypsin under physiological conditions. We discovered that macromolecular crowding promotes spontaneous homotypic condensation of trypsin via liquid-liquid phase separation to yield membraneless condensates over a broad range of concentrations, pH, and temperature, which are stabilized by multivalent hydrophobic interactions. Interestingly, we found that Ca2+ binding in the calcium binding loop reversibly regulates the condensation of trypsin and α-chymotrypsin. Spontaneous condensation effectively prevents autolysis of trypsin and preserves its native-like esterase activity for a prolonged period of time. It has also been found that phase-separated trypsin responds to Ca2+-dependent activation of its esterase activity even after 14 days of storage while free trypsin failed to do so. The present study highlights an important physiological aspect by which cells can spatiotemporally regulate the biocatalytic efficacy of trypsin-like serine proteases via Ca2+-signaling.

Hierarchical biocatalytic membranes embedded with trypsin–inorganic hybrid nanoflowers for effective β-lactoglobulin hydrolysis

β-lactoglobulin (β-LG) is a significant allergen in milk. Enzymatic hydrolysis of β-LG produces peptides with low allergenicity and improved functionality. The biocatalytic membranes combine enzyme function and membrane separation in one step, but is still challenging for proteolysis. Herein, the biocatalytic membranes embedded with trypsin-inorganic hybrid nanoflowers are developed by a facile and green method for β-LG hydrolysis. Ethylene vinyl alcohol copolymer (EVAL) nanofibers are fabricated as 3D scaffolding on the membrane surface and trypsin-copper phosphate nanoflowers are in-situ grown in the porous structure of membranes. The nanoflowers are uniformly entrapped in the EVAL nanofiber layer and preferentially grow in the EVAL nanofibers layer, leading to the hierarchical structure of membranes. The trypsin loading of the nanoflowers membrane is approximate to 282.1 µg/cm2. Lower Michaelis-Menten constants Km prove the better affinity of Cu3(PO4)2-TR nanoflower membrane with substrates during filtration than that in static mode, even than free trypsin. The hierarchical structure of the membranes ensures both the enhanced trypsin-substrate contacts and low mass transfer resistance during the β-LG filtration. This work not only presents a novel concept of enzyme immobilized biocatalytic membranes, but also offers a sustainable solution for high-performance proteolysis.

Ultrafast protein digestion using an immobilized enzyme reactor following high-resolution mass spectrometry analysis for rapid identification of abrin toxin.

Abrin toxin, highly dangerous with an estimated human lethal dose of 0.1-1 μg per kg body weight, has attracted much attention regarding criminal and terroristic misuse over the past decade. Therefore, developing a rapid detection method for abrin toxin is of great significance in the field of biosecurity. In this study, based on the specific dissociation method of an immobilized enzyme reactor, the trypsin immobilized reactor Fe3O4@CTS-GA-Try was prepared to replace free trypsin, and the immobilized enzyme digestion process was systematically investigated and optimized by using bovine serum albumin as the simulant of abrin. After 5 min one-step denaturation and reduction, a satisfactory peptide number and coverage were yielded with only 15 s assisted by an ultrasound probe to identify model proteins. Subsequently, abrin was rapidly digested using the established method, resulting in a stable and highly reproducible characteristic peptide number of 39, which can be analyzed by nanoelectrospray ionization coupled with high-resolution mass spectrometry. With the acquisition mode of full MS scan coupled with PRM, not only MS spectroscopy of total abrin peptides but also the corresponding MS/MS spectroscopy of specific abrin peptides can achieve the characteristic detection of abrin toxin and its different isoforms in less than 10 minutes, with high repeatability. This assay provides a universal platform and has great potential for the development of on-site detection and rapid mass spectrometric analysis techniques for macromolecular protein toxins and can further be applied to the integrated detection of chemical and biological agents.

Trypsin-Based Chemoenzymatic Assay for Detection of Pollutants and Safety Assessment of Food Additives

Chemoenzymatic assay systems are widely used to detect toxicants in various samples, including food and environment specimens. These methods are based on the ability of various types of toxicant to specifically inhibit/activate the functions of individual enzymes or enzyme systems. The present study examines the possibility of using the proteolytic enzyme trypsin as a specific marker to detect protease inhibitors in different samples. The study shows that trypsin activity is not affected by various heavy metals, pesticides, or quinones at levels considerably greater than their maximum allowable concentrations (MACs) in water bodies. At the same time, the IC50 value for the food preservative potassium sorbate (E202) is 15 mg/L, which is substantially lower than its acceptable daily intake (ADI). The quenching of trypsin fluorescence in the presence of potassium sorbate suggests that inhibition could occur due to the binding of the preservative to the enzyme in the region adjacent to the active center. The trypsin was immobilized in starch gel to ensure its stability in the enzyme inhibition based assay. Single-use reagents were prepared as dry starch disks that could be stored over long periods. Their sensitivity to copper (II) chloride, potassium sorbate, and chromium (III) chloride was similar to the sensitivity of the free trypsin.

Metal-Organic Materials (MOMs) Enhance Proteolytic Selectivity, Efficiency, and Reusability of Trypsin: A Time-Resolved Study on Proteolysis.

Proteases are involved in essential biological functions in nature and have become drug targets recently. In spite of the promising progress, two challenges, (i) the intrinsic instability and (ii) the difficulty in monitoring the catalytic process in real time, still hinder the further understanding and engineering of protease functionalities. These challenges are caused by the lack of proper materials/approaches to stabilize proteases and monitor proteolytic products (truncated polypeptides) in real time in a highly heterogeneous reaction mixture. This work combines metal-organic materials (MOMs), site-directed spin labeling-electron paramagnetic resonance (SDSL-EPR) spectroscopy, and mass spectrometry (MS) to overcome both barriers. A model protease, trypsin, which cleaves the peptide bonds at lysine or arginine residues, was immobilized on a Ca-MOM via aqueous-phase, one-pot cocrystallization, which allows for trypsin protection and ease of separation from its proteolytic products. Time-resolved EPR and MS were employed to monitor the populations, rotational motion, and sequences of the cleaved peptide truncations of a model protein substrate as the reaction proceeded. Our data suggest a significant (at least 5-10 times) enhancement in the catalytic efficiency (kcat/km) of trypsin@Ca-MOM and excellent reusability as compared to free trypsin in solution. Surprisingly, entrapping trypsin in Ca-MOMs results in cleavage site/region selectivity against the protein substrate, as compared to the near nonselective cleavage of all lysine and arginine residues of the substrate in solution. Remarkably, immobilizing trypsin allows for the separation and, thus, MS study on the sequences of truncated peptides in real time, leading to a time-resolved "movie" of trypsin proteolysis. This work demonstrates the use of MOMs and cocrystallization to enhance the selectivity, catalytic efficiency, and stability of trypsin, suggesting the possibility of tuning the catalytic performance of a general protease using MOMs.

Modified magnetite nanoparticles synthesized using cetyltrimethylammonium bromide and their application to immobilize trypsin

This paper presents the study of the properties of immobilized trypsin from the bovine pancreas. Fe3O4 nanoparticles as magnetically detachable carriers were synthesized using surfactant cetyltrimethylammonium bromide (CTAB). CTAB allows for obtaining non-agglomerated nanosized particles with superparamagnetic properties. The support surface was functionalized with amino groups using (3–aminopropyl)triethoxysilane and next glutaraldehyde. Immobilization of trypsin was carried out by covalent binding of trypsin with modified magnetite. The activities of native and immobilized trypsin were measured using 1% casein. The effect of reuse at nine cycles on the activity of the immobilized trypsin was tested. The activity of immobilized trypsin on magnetite was studied during storage at 4 °C for 28 days. It was found that in optimal conditions at a temperature of 55 °C and pH 7.6. The Michaelis–Menten constant Km was 5.59 ± 0.37 mM and the maximum reaction rate vmax was 10.74 ± 1.85 mM min−1 for immobilized trypsin, compared to a Km of 4.63 ± 0.34 mM and a vmax of 8.74 ± 0.88 mM min−1 for free trypsin at a temperature of 37 °C. High activity of the immobilized trypsin during reusability, presents that immobilized trypsin might find potential application in practice.

Trypsin/Zn3(PO4)2 Hybrid Nanoflowers: Controlled Synthesis and Excellent Performance as an Immobilized Enzyme.

Immobilized enzymes are a significant technological approach to retain enzyme activity and reduce enzyme catalytic cost. In this work, trypsin-incorporated Zn3(PO4)2 hybrid nanoflowers were prepared via mild precipitation and coordination reactions. The controllable preparation of hybrid nanoflowers was achieved by systematically investigating the effects of the raw-material ratio, material concentration and reaction temperature on product morphology and physicochemical properties. The enzyme content of hybrid nanoflowers was about 6.5%, and the maximum specific surface area reached 68.35 m2/g. The hybrid nanoflowers exhibit excellent catalytic activity and environmental tolerance compared to free trypsin, which was attributed to the orderly accumulation of nanosheets and proper anchoring formation. Further, the enzyme activity retention rate was still higher than 80% after 12 repeated uses. Therefore, trypsin/Zn3(PO4)2 hybrid nanoflowers—which combine functionalities of excellent heat resistance, storage stability and reusability—exhibit potential industrial application prospects.

Nutritional prospective of Sesbania species: an underutilized wild legume from Northern Western Ghats, Maharashtra, India

Nutrition plays very important role in longevity of life. Processed food has created various health disorders at a very alarming rate. Wild genotypes have potent nutritive characteristics which are relatively less explored. In the current investigation, two species of Sesbania were evaluated for their potent nutritional value. Sesbania grandiflora and Sesbania bispinosa seeds contained 37.78% and 30.2% crude proteins, 4.24% and 2.06% albumins, 7.20% and 5.16% globulins, 1.75% and 8.34% free amino acids, 34.91% and 59.08% total carbohydrates and 5.2% and 5.02% total lipids respectively. DPPH Radical scavenging assay revealed notable antioxidant potential with 62.27% and 65.45% inhibition respectively. Antinutrient analysis indicated the presence of total free phenols (0.29% and 0.63%), tannins (0.28% and 0.34%), phytic acid (1.70% and 1.59%) and trypsin inhibitor activity (41.8 and 54.4 TIU/mg) respectively. Both Sesbania species had excellent nutritional potential as well as antioxidant activity and a low antinutrient profile. Based on the findings, Sesbania species may be investigated further as a potential alternative food source in the near future, particularly in economically disadvantaged areas, and may pave the way toward the wider aim of food security.

Evaluation of interaction between Ponceau 4R (P4R) and trypsin using kinetic, spectroscopic, and molecular dynamics simulation methods

Azo-dyes, a diverse group of synthetic dyes, are commonly used for coloring food products. However, these dyes are persistent in nature with the potential to cause harmful effects on the human body. Hence, it seems that studies are required about the effects of azo dyes on the structure and function of biomacromolecules. Ponceau 4R (P4R) is an azo-dye found in foods, beverages, and pharmaceuticals, but its potential risk on gastrointestinal enzymes such as trypsin is poorly characterized. Thus, this study focused on analyzing the binding behavior between trypsin and P4R by spectroscopic, kinetic, and theoretical methods. Intrinsic fluorescence revealed that there was an interaction between P4R and trypsin, and P4R could quench the trypsin fluorescence by a static model. The resonance light scattering (RLS) method was used to confirm the formation of the P4R-trypsin complex. The thermodynamic data indicated that the predominant interactions between P4R and trypsin were Van der Waals and hydrogen bonds, consistent with docking results. The distance between P4R and trypsin after the interaction was calculated based on fluorescence resonance energy transfer (FRET) theory and found to be 3.36 nm. Synchronous fluorescence, three-dimensional fluorescence, UV–Visible spectrophotometry, and circular dichroism (CD) analyses indicated that the conformation of trypsin was changed upon binding with P4R, which could affect the trypsin function. Kinetic studies revealed that P4R inhibited trypsin activity in a non-competitive model, and docking analysis confirmed that P4R was not bound to the active site of the trypsin. The molecular dynamics (MD) simulation studies showed that the root mean square deviation (RMSD) in the trypsin-P4R complex (0.270 ± 0.02 nm) was larger than that of the free trypsin system (0.221 ± 0.01 nm), which agreed with CD and thermal stability experiments. Thus, P4R was shown to be a potent inhibitor and quencher of trypsin.

Zinc oxide nanoparticles-impregnated chitosan surfaces for covalent immobilization of trypsin: Stability & kinetic studies

Trypsin (Try, EC. 3.4.21.4) was effectively immobilized on the surface of glutaraldehyde(GA)-activated ZnO/Chitosan nanocomposite through covalent attachment via Schiff-base linkages. Size, structure, surface morphology, & percentage elemental composition of the prepared ZnO nanoparticles and chitosan-coated ZnO nanocomposite were studied by UV–Visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), X-Ray diffraction analysis (XRD), transmission electron microscopy (TEM), Scanning electron microscopy (SEM), and Energy-Dispersive X-Ray Microanalysis (EDAX) techniques. Optimal immobilization conditions (incubation time (16 h), enzyme concentration (1.8 mg/ml), and pH (7.8)) were investigated to obtain the maximum expressed activity of the immobilized trypsin. Immobilized & solubilized trypsin exhibited the optimum catalytic activity at pH 8.5, 60 °C, and pH 7.8, 45 °C respectively. Kinetic parameters (Km, Vmax) of immobilized (27.12 μM, 8.82 μM/min) & free trypsin (25.76 μM, 4.16 μM/min) were determined, indicating that efficiency of trypsin improves after immobilization. Immobilized trypsin preserved 67% of initial activity at 50 °C during 2 h of incubation & sustained nearly 50% of catalytic activity until the 9th repeated cycle of utilization. Moreover, immobilized trypsin retained 50% of enzymatic activity after 90 days of storage at 4 °C. Hence, the current findings suggest that ZnO/Chitosan-GA-Trypsin would be a promising biocatalyst for large-scale biotechnological applications.

Self-assembly synthes is of trypsin-immobilized monolithic microreactor for fast and efficient proteolysis

Fast and highly efficient digestion of proteins is essential for high-throughput proteomic analysis. Herein, a facile approach was developed for self-assembly preparation of trypsin-immobilized capillary monolithic column and its application as an immobilized enzyme microreactor (IMER) for fast and highly efficient proteolysis was described. The performance of the trypsin-immobilized monolithic enzyme microreactor was evaluated by in-situ digestion of model proteins. The results showed that the trypsin-immobilized monolithic enzyme microreactor had much higher tryptic digestion efficiency than the free trypsin in solution, where the coverage of peptide sequences by mass spectrometry (MS)-based analysis could bear comparison with the free one, while the digestion time was dramatically shortened from 12 h to 16 s. Furthermore, the trypsin-immobilized monolithic enzyme microreactor also exhibited good practicability to complex human serum sample, in which the total of 45 peptides from human serum albumin (HSA) matched with sequence coverage of 75% were precisely identified. The successful application demonstrated the promising potential of the trypsin-immobilized capillary monolithic column as the IMER in high-throughput proteomic analysis.

Room‐Temperature Synthesis of Trypsin‐Inorganic Hybrid Nanocomposites for Fast and Efficient Protein Digestion

AbstractFast and high‐efficiency protein digestion is essential for high‐throughput proteome analysis. Herein, a new kind of trypsin‐inorganic hybrid nanocomposites were room‐temperature synthesized based on self‐assembly stragety, where trypsin and nickel phosphate (Ni3(PO4)2) was used as organic and inorganic ligands, respectively. The general applicability of the synthetic method was also confirmed by using a series of other metal phosphates including Mg2+, Ba2+, Zn2+, and Co2+. The as‐prepared trypsin‐inorganic hybrid nanocomposites with flower‐like morphology showed higher enzymatic acticity (206 % enhancement) and faster catalytic kinetics than the free trypsin. Taking the merits togethers as mentioned above, the hybrid nanocomposites could be employed as an immmobilized enzyme reactor (IMER) for fast and high‐efficiency protein digestion. The peptide mass fingerprint analysis of the model proteins demonstrated that the coverage rate of the sequences treated by IMER could bear comparsion with that of the free one, but the digestion time was shorten from 12 h to 1 min. In addition, the IMER showed a good applicability for complex human serum sample, showing a promising potential in proteomics research.

NUTRITIONAL AND ANTINUTRITIONAL ASSESSMENT OF SOME UNDERUTILIZED CORMS, RHIZOMES AND TUBERS

<p><strong>Background. </strong>With ever-increasing populace pressure and rapid depletion of natural resources, it has become exceptionally important to diversify the present time agriculture with the cultivation of some wild varieties of tubers, rhizomes and corms in order to meet various human nutrient needs. However, information regarding the nutritional and antinutritional composition is meager. <strong>Objective.</strong> To study the chemical composition and antinutritional factors of the wild edible corms, rhizomes and tubers consumed by the Palliyars and Kanikkar tribes living in South Eastern Slopes of Western Ghats, Tamil Nadu<strong> Methodology</strong>. The wild edible corms of <em>Alocasia macrorrhiza</em>, <em>Amorphophallus paeoniifolius</em> var <em>campanulatus</em>,<em> Amorphophallus sylvaticus</em>,<em> Colocasia esculenta</em>,<em> Xanthosoma sagittifolium</em>, <em>Xanthosoma violaceum</em>, rhizomes of <em>Canna indica</em>,<em> Maranta arundinacea</em> and tubers of <em>Asparagus racemosus</em>,<em> Nymphaea pubescens</em> and<em> Nymphaea rubra</em> were analysed for proximate and mineral composition, starch, vitamins like niacin, ascorbic acid and antinutritional factors like total free phenolics, tannins, hydrogen cyanide, total oxalate, amylase and trypsin inhibitor activities were quantified. Also, <em>in vitro</em> protein and starch digestibility were assessed. <strong>Results.</strong> The rhizome of <em>Maranta arundinacea</em> and tubers of <em>Nymphaea pubescens </em>contain high quantity of crude proteins. The tubers of <em>Asparagus racemosus</em> contained higher amount of crude lipids. The corms of <em>Alocasia macrorrhiza, Colocasia esculenta, Xanthosoma sagittifolium</em> and <em>Xanthosoma violaceum</em> appeared to have a higher level of potassium content compared to recommended Dietary allowances (RDA) for infants, children and adults. The corms of <em>Alocasia macrorrhiza, Amorphophallus sylvaticus, Xanthosoma violaceum</em> and rhizomes of <em>Maranta arundinacea</em> were found to contain more starch. All the investigated samples had low <em>in vitro</em> protein digestibility. <strong>Implications.</strong> The present result highlights the potentiality of these underground plant parts as source of unconventional foods. Being wild, they also are easily accessible and cheaper. Studies on nutritional value of wild plant food are of extensive importance since it may help to recognize long forgotten food resources. <strong>Conclusion. </strong>Most of the wild edible corms, rhizomes and tubers were found to be a good source of protein, lipid, total dietary fiber, starch, vitamins and minerals. All the investigated samples exhibited variations in the levels of the total free phenolics, tannins, hydrogen cyanide, total oxalate, amylase and trypsin inhibitors</p>

Covalent immobilization of trypsin on polyvinyl alcohol-coated magnetic nanoparticles activated with glutaraldehyde

Magnetic nanoparticles were coated with polyvinyl alcohol and activated with glutaraldehyde for trypsin immobilization. The prepared magnetic nanoparticles were characterized by transmission electron microscopy, fourier transform infrared spectroscopy, thermal gravimetric analysis, zeta potential meter and vibrating sample magnetometer. Free and immobilized trypsin showed optimum activity at pH 6.0, 30 °C and pH 7.0, 40 °C, respectively. Immobilized trypsin was more stable than the free enzyme at 40 °C. After immobilization, Km of the immobilized trypsin increased, however, Vmax value was almost the same with free trypsin. According to the results, the immobilized trypsin retained 50 % of its initial activity, whereas free trypsin retained 19 % of its initial activity after 12-days at 4 °C. Immobilized trypsin sustained 56 % of its initial activity after eight times of successive reuse. The performance of the immobilized trypsin was evaluated by digestion of cytochrome c. The peptide fragments in digest solution were determined by using MALDI-TOF mass spectrometry. Immobilized trypsin showed effective proteolytic activity in shorter time (15 min) than free trypsin (24 h). Hence, immobilized trypsin on the polyvinyl alcohol coated magnetic nanoparticles could be promising biocatalyst for large-scale proteomics studies and practical applications.

Immobilization of Trypsin from Porcine Pancreas onto Chitosan Nonwoven by Covalent Bonding.

The present study deals with the potential application of chitosan nonwoven for biomedical textiles based on enzyme immobilization. For this, chitosan nonwoven was first cross-linked with glutaraldehyde to introduce aldehyde groups at optimal conditions. To immobilize the enzyme trypsin onto glutaraldehyde-pre-activated chitosan nonwoven, several parameters such as pH, enzyme concentration, and reaction times were investigated. In addition, the pH, thermal stability, storage stability, and reusability of immobilized trypsin were examined. We found that the optimal immobilization conditions for trypsin were pH 8.5, enzyme concentration of 8% (owf), and treatment time of 30 min. Trypsin was immobilized at 25 °C efficiently. The immobilized trypsin showed lower pH stability and better thermal stability than free trypsin. The immobilized trypsin showed 50% of its initial activity after being used 15 times and 80% of that after 20 days of storage at 4 °C. SEM analysis also confirmed that trypsin was immobilized on chitosan nonwoven.

Spectroscopic study and application of the interaction mechanism of meloxicam with trypsin

In order to explore the interaction between meloxicam and trypsin, the interaction mechanism between meloxicam and trypsin was studied by fluorescence spectrum, UV-vis absorption spectrum, circular dichroism spectrum, and molecular docking simulation under the experimental condition of pH = 7.40. The results of spectral experiments showed that meloxicam could effectively quench the internal fluorescence of trypsin in the form of static quenching, formed a stable complex at 1:1, and changed the conformation of trypsin. The results of thermodynamic constant showed that ΔG < 0, indicating that the reaction is spontaneous, ΔH < 0, ΔS > 0 indicates that the main force type of the binding system was hydrophobic interaction and hydrogen bonding. Molecular docking technique showed that the best binding site between meloxicam and trypsin was near the catalytic active center of trypsin, and the interaction between them changed the microenvironment of amino acid residues in the catalytic active center of trypsin. The mathematical model of drug and protein showed that when the concentration ratio of meloxicam to trypsin was 1:1, the protein binding rate of the binding system was 5.15%. The concentration ratio of meloxicam to trypsin was 30: 1, and the protein binding rate was 45.4%. The results showed that when the drug concentration was high, the binding effect of the system had a great influence on the concentration of free trypsin.

β-Lactoglobulin hydrolysis by a flow-through monolithic immobilized trypsin reactor in ethanol/aqueous solvents

• Reducing agents showed varied effects on immobilization yield and activity. • Activity of immobilized trypsin increased by 20% in 20% ethanol/water. • 20% ethanol increased the hydrolysis of β-Lg by immobilized trypsin. • Releases of peptides locating inner β-Lg increased in 20% ethanol than in water. Monolith based immobilized trypsin reactors (MITRs) were developed using different immobilization protocols. Compared with the use of reducing agent NaCNBH 3 , the application of 2-PB led to around 33% decrease in immobilization yield, while improved the specific activity of immobilized trypsin. The developed flow-through MITR was used to hydrolyze β-Lactoglobulin (β-Lg), and the feasibility of integrating ethanol in reaction media was evaluated. Raising ethanol concentrations from 0 to 50% increased the size of β-Lg molecules from 1.74 ± 0.32 nm to 5.67 ± 2.78 nm, and doubled the viscosity of β-Lg solutions. To avoid high backpressure and potential clog on MITR, β-Lg was hydrolyzed with the addition of ethanol up to 20%, where the activity of immobilized trypsin increased by 20% than that in aqueous media. Similar to free trypsin, the addition of ethanol contributed to an increase in DH and a faster depletion of intact protein for MITR. The presence of ethanol decreased the releases of peptides locating on C- termini, i.e., f(149–162) and f(142–148) for free and immobilized trypsin. However, the release of peptides locating inner β-Lg increased, e.g., a 5 times higher amount of f(125–138) was detected for MITR in 20% ethanol.

A rapid immobilized trypsin digestion combined with liquid chromatography – Tandem mass spectrometry for the detection of milk allergens in baked food

Cow's milk allergy is the most common food allergy, and caseins are the major allergens in milk. A quantitative proteomics method based on liquid chromatography coupled to mass spectrometry has been widely used in allergen analysis. However, the long period of digestion has limited its use in routine analysis. PHMN-Trypsin (trypsin immobilized on hairy polymer-chain hybrid magnetic nanoparticles), a new type of immobilized trypsin, was used to shorten the digestion time and enhance the digestion efficiency in this study. We developed a rapid digestion method using PHMN-Trypsin to detect milk allergens in baked food by ultrahigh-performance liquid chromatography – tandem mass spectrometry (UPLC-MS/MS). The PHMN-Trypsin digestion was completed within 15 min, with higher or equal sequence coverage compared to 12–16 h of conventional free trypsin digestion. αs1-casein, αs2-casein, β-casein, and κ-casein were monitored as the allergen proteins. For each target allergen, two or three specific and signature peptides were selected for the UPLC-MS/MS strategy in view of the species specificity, mass response intensity as well as the stability. The limits of quantification of the four proteins in baked foods were 0.38–0.83 μg/g, and their recoveries ranged from 65.2% to 86.1%, with relative standard deviations of less than 11.0%. The method was applied to investigate allergens in commercial baked foods, and the results indicate that this method would be helpful in food allergen screening.

Influence of salts on hydrolysis of β-lactoglobulin by free and immobilised trypsin

Immobilised trypsin is an alternative to free trypsin for producing protein hydrolysates with increased functionalities. However, the influence of hydrolytic conditions on this process remains unclear. The influence of salts on β-lactoglobulin (β-Lg) hydrolysis by free and immobilised trypsin was compared. For both forms of trypsin, 0.1 m Tris accelerated the release of most final peptides except f (71–75), and had no significant effects on the hydrolysis of intact β-Lg. Increasing NaCl concentrations from 0 to 0.02 m increased the degree of hydrolysis (DH) by 22.4% for free trypsin versus 62.1% for immobilised trypsin. The presence of 0.1 or 0.5 m NaCl hindered the release of peptides associated with the breakdown of intact protein. This led to 2–4 fold decreases in depleting intact β-Lg and DH, except immobilised trypsin at 0.1 m NaCl (DH increased by 44.3% versus without NaCl). Potential mechanisms underlying the effects of salts are discussed.

On-line protein digestion by immobilized enzyme microreactor capillary electrophoresis-mass spectrometry

In this study, we present the use of microreactors packed with immobilized trypsin particles for the rapid and efficient bottom-up analysis of proteins by on-line immobilized enzyme microreactor capillary electrophoresis mass spectrometry (IMER-CE-MS). The results obtained digesting β-lactoglogulin (β-LG) off-line with free trypsin in solution and with immobilized trypsin particles were taken as a reference for the optimization of the on-line protein digestion. Under the optimized conditions, on-line digestion, separation and characterization of the protein digests were possible in less than 30 min. The limit of detection for complete sequence coverage was around 10 µg mL−1 (~500 µM) of β-LG, the repeatability was comparable to the off-line digestion methods and the microreactor could be reused until thirty times. The good performance of IMER-CE-MS was also demonstrated for several other proteins as α-casein (α-CSN), β-casein (β-CSN), and κ-casein (κ-CSN), as well as for a complex protein mixture (an Escherichia coli whole cell lysate).