Post-enzymatic hydrolysis heat treatment as an essential unit operation for collagen solubilization from poultry by-products.

Enzymatic protein hydrolysis (EPH) is an invaluable process to increase the value of food processing by-products. In the current work the aim was to study the role of standard thermal inactivation in collagen solubilization during EPH of poultry by-products. Hundred and eighty hydrolysates were produced using two proteases (stem Bromelain and Endocut-02) and two collagen-rich poultry by-products (turkey tendons and carcasses). Thermal inactivation was performed with and without the sediment to study the effect of heat on collagen solubilization. A large difference in molecular weight distribution profiles was observed when comparing hydrolysate time series of the two proteases. In addition, it was shown that 15 min heat treatment, conventionally used for inactivating proteases, is essential in solubilizing collagen fragments, which significantly contributes to increasing the protein yield of the entire process. The study thus demonstrated the possibility of producing tailored products of different quality by exploiting standard heat inactivation in EPH.

Exploring Effects of Protease Choice and Protease Combinations in Enzymatic Protein Hydrolysis of Poultry By-Products.

A study of the effects of single and combined protease hydrolysis on myofibrillar versus collagenous proteins of poultry by-products has been conducted. The aim was to contribute with knowledge for increased value creation of all constituents of these complex by-products. A rational approach was implemented for selecting proteases exhibiting the most different activity towards the major protein-rich constituents of mechanically deboned chicken residue (MDCR). An initial activity screening of 18 proteases on chicken meat, turkey tendons and MDCR was conducted. Based on weight yield, size exclusion chromatography (SEC) and SDS-PAGE, stem Bromelain and Endocut-02 were selected. Studies on hydrolysis of four different poultry by-products at 40 °C, evaluated by protein yield, SEC, and SDS-PAGE, indicate that the proteases' selectivity difference can be utilized in tailor-making hydrolysates, enriched in either meat- and collagen-derived peptides or gelatin. Three modes of stem Bromelain and Endocut-02 combinations during hydrolysis of MDCR were performed and compared with single protease hydrolysis. All modes of the protease combinations resulted in a similar approximately 15% increase in product yield, with products exhibiting similar SEC and SDS-PAGE profiles. This shows that irrespective of the modes of combination, the use of more than one enzyme in hydrolysis of collagen-rich material can provide means to increase the total protein yield and ultimately contribute to increased value creation of poultry by-products.

Fourier-transform infrared spectroscopy for monitoring proteolytic reactions using dry-films treated with trifluoroacetic acid.

In this study we explore the potential of using Fourier-transform infrared (FTIR) spectra of trifluoroacetate-protein and peptide complexes for monitoring proteolytic reactions. The idea of treating dry-films of protein hydrolysates with trifluoroacetic acid (TFA) prior to FTIR analysis is based on the unique properties of TFA. By adding a large excess of TFA to protein hydrolysate samples, the possible protonation sites of the proteins and peptides will be saturated. In addition, TFA has a low boiling point when protonated as well as complex-forming abilities. When forming TFA-treated dry-films of protein hydrolysates, the excess TFA will evaporate and the deprotonated acid (CF3COO-) will interact as a counter ion with the positive charges on the sample materials. In the study, spectral changes in TFA-treated dry-films of protein hydrolysates from a pure protein and poultry by-products, were compared to the FTIR fingerprints of untreated dry-films. The results show that time-dependent information related to proteolytic reactions and, consequently, on the characteristics of the protein hydrolysates can be obtained. With additional developments, FTIR on dry-films treated with TFA may be regarded as a potential future tool for the analysis of all types of proteolytic reactions in the laboratory as well as in industry.

Average molecular weight, degree of hydrolysis and dry-film FTIR fingerprint of milk protein hydrolysates: Intercorrelation and application in process monitoring.

Fourier-transform infrared (FTIR) spectroscopy was applied to predict the degree of hydrolysis (DH%) and weight-average molecular weight (Mw) in milk protein hydrolysates. Both DH% and Mw are important quality parameters of protein hydrolysates. Measuring these parameters and following their development during proteolytic reactions is therefore essential for process control and optimization in industry. In the present study the intercorrelation and the complimentary nature of these parameters were investigated and a partial least squares regression (PLSR) model was developed for the prediction of DH% from molecular weight distributions. Finally, we developed PLSR models based on dry-film FTIR spectroscopy for the prediction of both DH% and Mw. Here spectral changes in the amide region were found to be important for the two calibration models, underlining the advantage of dry-film FTIR measurement. This shows that dry-film infrared spectroscopy is a promising tool for dual prediction of DH% and Mw.

FTIR-based hierarchical modeling for prediction of average molecular weights of protein hydrolysates.

In the presented study, Fourier-transform infrared (FTIR) spectroscopy is used to predict the average molecular weight of protein hydrolysates produced from protein-rich by-products from food industry using commercial enzymes. Enzymatic protein hydrolysis is a well-established method for production of protein-rich formulations, recognized for its potential to valorize food-processing by-products. The monitoring of such processes is still a significant challenge as the existing classical analytical methods are not easily applicable to industrial setups. In this study, we are reporting a generic FTIR-based approach for monitoring the average molecular weights of proteins during enzymatic hydrolysis of by-products from the food industry. A total of 885 hydrolysate samples from enzymatic protein hydrolysis reactions of poultry and fish by-products using different enzymes were studied. FTIR spectra acquired from dry-films of the hydrolysates were used to build partial least squares regression (PLSR) models. The most accurate predictions were obtained using a hierarchical PLSR approach involving supervised classification of the FTIR spectra according to raw material quality and enzyme used in the hydrolysis process, and subsequent local regression models tuned to specific enzyme-raw material combinations. The results clearly underline the potential of using FTIR for monitoring protein sizes during enzymatic protein hydrolysis in industrial settings, while also paving the way for measurements of protein sizes in other applications.

Fourier-transform infrared spectroscopy for characterization of protein chain reductions in enzymatic reactions.

The potential of dry-film Fourier-transform infrared (FTIR) measurements as a monitoring tool for enzymatic hydrolysis of protein-based substrates is explored in this study. As a proof-of-concept, the enzymatic digestion of bovine serum albumin using Alcalase was monitored. To evaluate the analytical approach on complex substrates with industrial relevance, salmon- and chicken-based substrates were digested for 80 minutes using Alcalase and a total of 12 FTIR spectra were acquired during the course of the hydrolysis. The observed changes in the IR spectral features as a function of hydrolysis time were found to be in agreement with the breakdown of the amide backbone and formation of amino and carboxylate terminals. Some of the most consistent markers for hydrolysis time were the bands at 1516 cm-1 (-NH3+) and ∼1400 cm-1 (-COO-). Moreover, principal component analysis (PCA) of the FTIR spectra was used to demonstrate the systematic relationship of the hydrolysis time with key variables (wavelengths) in the protein backbone region (800-1800 cm-1). Scores in the first principal component versus the hydrolysis time have been shown to provide an overview of the process dynamics related to protein structural changes. The herein presented results suggest that dry-film FTIR measurements have potential as a rapid tool for monitoring industrial protein hydrolysis processes.

Valorization of Proteins from Co- and By-Products from the Fish and Meat Industry.

Large volumes of protein-rich residual raw materials, such as heads, bones, carcasses, blood, skin, viscera, hooves and feathers, are created as a result of processing of animals from fisheries, aquaculture, livestock and poultry sectors. These residuals contain proteins and other essential nutrients with potentially bioactive properties, eligible for recycling and upgrading for higher-value products, e.g. for human, pet food and feed purposes. Here, we aim to cover all the important aspects of achieving optimal utilization of proteins in such residual raw materials, identifying those eligible for human consumption as co-products and for feed applications as by-products. Strict legislation regulates the utilization of various animal-based co- and by-products, representing a major hurdle if not addressed properly. Thorough understanding and optimization of all parts of the production chain, including conservation and processing, are important prerequisites for successful upgrading and industrial implementation of such products. This review includes industrially applied technologies such as freezing/cooling, acid preservation, salting, rendering and protein hydrolysis. In this regard, it is important to achieve stable production and quality through all the steps in the manufacturing chain, preferably supported by at- or online quality control points in the actual processing step. If aiming for the human market, knowledge of consumer trends and awareness are important for production and successful introduction of new products and ingredients.

Genetic fusion of single-chain variable fragments to partial spider silk improves target detection in micro- and nanoarrays.

Immobilizing biomolecules with retained functionality and stability on solid supports is crucial for generation of sensitive immunoassays. However, upon use of conventional immobilization strategies, a major portion of the biomolecules (e.g. antibodies) frequently tends to lose their bioactivity. In this study, we describe a procedure to immobilize human single-chain variable fragment (scFv) via genetic fusion to partial spider silk, which have a high tendency to adhere to solid supports. Two scFvs, directed towards serum proteins, were genetically fused to partial spider silk proteins and expressed as silk fusion proteins in E. coli. Antigen binding ability of scFvs attached to a partial silk protein denoted RC was investigated using microarray analysis, whereas scFvs fused to the NC silk variant were examined using nanoarrays. Results from micro- and nanoarrays confirmed the functionality of scFvs attached to both RC and NC silk, and also for binding of targets in crude serum. Furthermore, the same amount of added scFv gives higher signal intensity when immobilized via partial spider silk compared to when immobilized alone. Together, the results suggest that usage of scFv-silk fusion proteins in immunoassays could improve target detection, in the long run enabling novel biomarkers to be detected in crude serum proteomes.

Recombinant spider silk genetically functionalized with affinity domains.

Functionalization of biocompatible materials for presentation of active protein domains is an area of growing interest. Herein, we describe a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains of broad biotechnological use. Four affinity domains of different origin and structure; the IgG-binding domains Z and C2, the albumin-binding domain ABD, and the biotin-binding domain M4, were all successfully produced as soluble silk fusion proteins under nondenaturing purification conditions. Silk films and fibers produced from the fusion proteins were demonstrated to be chemically and thermally stable. Still, the bioactive domains are concluded to be folded and accessible, since their respective targets could be selectively captured from complex samples, including rabbit serum and human plasma. Interestingly, materials produced from mixtures of two different silk fusion proteins displayed combined binding properties, suggesting that tailor-made materials with desired stoichiometry and surface distributions of several binding domains can be produced. Further, use of the IgG binding ability as a general mean for presentation of desired biomolecules could be demonstrated for a human vascular endothelial growth factor (hVEGF) model system, via a first capture of anti-VEGF IgG to silk containing the Z-domain, followed by incubation with hVEGF. Taken together, this study demonstrates the potential of recombinant silk, genetically functionalized with affinity domains, for construction of biomaterials capable of presentation of almost any desired biomolecule.

One-step enzyme extraction and immobilization for biocatalysis applications.

An extraction/immobilization method for HIs(6) -tagged enzymes for use in synthesis applications is presented. By modifying silica oxide beads to be able to accommodate metal ions, the enzyme was tethered to the beads after adsorption of Co(II). The beads were successfully used for direct extraction of C. antarctica lipase B (CalB) from a periplasmic preparation with a minimum of 58% activity yield, creating a quick one-step extraction-immobilization protocol. This method, named HisSi Immobilization, was evaluated with five different enzymes [Candida antarctica lipase B (CalB), Bacillus subtilis lipase A (BslA), Bacillus subtilis esterase (BS2), Pseudomonas fluorescence esterase (PFE), and Solanum tuberosum epoxide hydrolase 1 (StEH1)]. Immobilized CalB was effectively employed in organic solvent (cyclohexane and acetonitrile) in a transacylation reaction and in aqueous buffer for ester hydrolysis. For the remaining enzymes some activity in organic solvent could be shown, whereas the non-immobilized enzymes were found inactive. The protocol presented in this work provides a facile immobilization method by utilization of the common His(6) -tag, offering specific and defined means of binding a protein in a specific location, which is applicable for a wide range of enzymes.

Deep eutectic solvents (DESs) are viable cosolvents for enzyme-catalyzed epoxide hydrolysis.

A special group of ionic liquids, deep eutectic solvents (DESs) have been tested as cosolvents in enzyme-catalyzed hydrolysis of a chiral (1,2)-trans-2-methylstyrene oxide. The choline chloride:ethane diol (ET), choline chloride:glycerol (GLY) and choline:chloride:urea (REL) DESs were included in the reaction mixtures with epoxide and the potato epoxide hydrolase StEH1. The effect of the DESs on enzyme function was primarily elevations of K(M) (up to 20-fold) and with lesser effects on turnover numbers (twofold variation). The regioselectivity in hydrolysis of the (1R,2R)-2-trans-methylstyrene oxide was altered in the presence of GLY or ET to favor epoxide ring opening at the benzylic carbon (R=2.33), enhancing the regioselectivity observed in buffer-only systems (R=1.35). The DES solutions dissolved 1.5-fold higher epoxide concentrations as compared to phosphate buffer. The total conversion of high concentration (40 g/l) of (1S,2S)-MeSO was not negatively affected by addition of 40% GLY.

Temperature and pH dependence of enzyme-catalyzed hydrolysis of trans-methylstyrene oxide. A unifying kinetic model for observed hysteresis, cooperativity, and regioselectivity.

The underlying enzyme kinetics behind the regioselective promiscuity shown by epoxide hydrolases toward certain epoxides has been studied. The effects of temperature and pH on regioselectivity were investigated by analyzing the stereochemistry of hydrolysis products of (1R,2R)-trans-2-methylstyrene oxide between 14-46 degrees C and pH 6.0-9.0, either catalyzed by the potato epoxide hydrolase StEH1 or in the absence of enzyme. In the enzyme-catalyzed reaction, a switch of preferred epoxide carbon that is subjected to nucleophilic attack is observed at pH values above 8. The enzyme also displays cooperativity in substrate saturation plots when assayed at temperatures < or = 30 degrees C and at intermediate pH. The cooperativity is lost at higher assay temperatures. Cooperativity can originate from a kinetic mechanism involving hysteresis and will be dependent on the relationship between k(cat) and the rate of interconversion between two different Michaelis complexes. In the case of the studied reactions, the proposed different Michaelis complexes are enzyme-substrate complexes in which the epoxide substrate is bound in different binding modes, allowing for separate pathways toward product formation. The assumption of separated, but interacting, reaction pathways is supported by that formation of the two product enantiomers also displays distinct pH dependencies of k(cat)/K(M). The thermodynamic parameters describing the differences in activation enthalpy and entropy suggest that (1) regioselectivity is primarily dictated by differences in activation entropy with positive values of both DeltaDeltaH(++) and DeltaDeltaS(++) and (2) the hysteretic behavior is linked to an interconversion between Michaelis complexes with rates increasing with temperature. From the collected data, we propose that hysteresis, regioselectivity, and, when applicable, hysteretic cooperativity are closely linked properties, explained by the kinetic mechanism earlier introduced by our group.

Mutations in salt-bridging residues at the interface of the core and lid domains of epoxide hydrolase StEH1 affect regioselectivity, protein stability and hysteresis.

Epoxide hydrolase, StEH1, shows hysteretic behavior in the catalyzed hydrolysis of trans-2-methylstyrene oxide (2-MeSO)(1). Linkage between protein structure dynamics and catalytic function was probed in mutant enzymes in which surface-located salt-bridging residues were substituted. Salt-bridges at the interface of the alpha/beta-hydrolase fold core and lid domains, as well as between residues in the lid domain, between Lys(179)-Asp(202), Glu(215)-Arg(41) and Arg(236)-Glu(165) were disrupted by mutations, K179Q, E215Q, R236K and R236Q. All mutants displayed enzyme activity with styrene oxide (SO) and 2-MeSO when assayed at 30 degrees C. Disruption of salt-bridges altered the rates for isomerization between distinct Michaelis complexes, with (1R,2R)-2-MeSO as substrate, presumably as a result of increased dynamics of involved protein segments. Another indication of increased flexibility was a lowered thermostability in all mutants. We propose that the alterations to regioselectivity in these mutants derive from an increased mobility in protein segments otherwise stabilized by salt bridging interactions.

Structure-function relationships of epoxide hydrolases and their potential use in biocatalysis.

BACKGROUND: Chiral epoxides and diols are important synthons for manufacturing fine chemicals and pharmaceuticals. The epoxide hydrolases (EC 3.3.2.-) catalyze the hydrolytic ring opening of epoxides producing the corresponding vicinal diol. Several isoenzymes display catalytic properties that position them as promising biocatalytic tools for the generation of enantiopure epoxides and diols. SCOPE OF REVIEW: This review focuses on the present data on enzyme structure and function in connection to biocatalytic applications. Available data on biocatalysis employed for purposes of stereospecific ring opening, to produce chiral vicinal diols, and kinetic resolution regimes, to achieve enantiopure epoxides, are discussed and related to results gained from structure-activity studies on the enzyme catalysts. More recent examples of the concept of directed evolution of enzyme function are also presented. MAJOR CONCLUSIONS: The present understanding of structure-activity relationships in epoxide hydrolases regarding chemical catalysis is strong. With the ongoing research, a more detailed view of the factors that influence substrate specificities and stereospecificities is expected to arise. The already present use of epoxide hydrolases in synthetic applications is expected to expand as new enzymes are being isolated and characterized. Refined methodologies for directed evolution of desired catalytic and physicochemical properties may further boost the development of novel and useful biocatalysts. GENERAL SIGNIFICANCE: The catalytic power of enzymes provides new possibilities for efficient, specific and sustainable technologies to be developed for production of useful chemicals.

Substrate-dependent hysteretic behavior in StEH1-catalyzed hydrolysis of styrene oxide derivatives.

The substrate selectivity and enantioselectivity of Solanum tuberosum epoxide hydrolase 1 (StEH1) have been explored by steady-state and pre-steady-state measurements on a series of styrene oxide derivatives. A preference for the (S)- or (S,S)-enantiomers of styrene oxide, 2-methylstyrene oxide and trans-stilbene oxide was established, with E-values of 43, 160 and 2.9, respectively. Monitoring of the pre-steady-state phase of the reaction with (S,S)-2-methylstyrene oxide revealed two observed rates for alkylenzyme formation. The slower of these rates showed a negative substrate concentration dependence, as did the rate of alkylenzyme formation in the reaction with the (R,R)-enantiomer. Such kinetic behavior is indicative of an additional, off-pathway step in the mechanism, referred to as hysteresis. On the basis of these data, a kinetic mechanism that explains the kinetic behavior with all tested substrates transformed by this enzyme is proposed. Regioselectivity of StEH1 in the catalyzed hydrolysis of 2-methylstyrene oxide was determined by (13)C-NMR spectroscopy of (18)O-labeled diol products. The (S,S)-enantiomer is attacked exclusively at the C-1 epoxide carbon, whereas the (R,R)-enantiomer is attacked at either position at a ratio of 65 : 35 in favor of the C-1 carbon. On the basis of the results, we conclude that differences in efficiency in stabilization of the alkylenzyme intermediates by StEH1 are important for enantioselectivity with styrene oxide or trans-stilbene oxide as substrate. With 2-methylstyrene oxide, slow conformational changes in the enzyme also influence the catalytic efficiency.