Reactive Lysine Research Articles

Sculpting a Uniquely Reactive Cysteine Residue for Site-Specific Antibody Conjugation.

Catalytic antibody 38C2 and its humanized version h38C2 harbor a uniquely reactive lysine at the bottom of a 11 Å deep pocket that permits site-specific conjugation of β-diketone-, β-lactam-, and heteroaryl methylsulfonyl-functionalized small and large molecules. Various dual variable domain formats pair a tumor-targeting antibody with h38C2 to enable precise, fast, and stable assembly of antibody-drug conjugates (ADCs). Here, we expand the scope of this ADC assembly strategy by mutating h38C2's reactive lysine to a cysteine. X-ray crystallography of this point mutant, h38C2_K99C, confirmed a deeply buried unpaired cysteine. Probing h38C2_K99C with maleimide, monobromomaleimide, and dibromomaleimide derivatives of a fluorophore revealed highly disparate conjugation efficiencies and stabilities. Dibromomaleimide emerged as a suitable electrophile for the precise, fast, efficient, and stable assembly of ADCs with the h38C2_K99C module. Mass spectrometry indicated the presence of a thio-monobromomaleimide linkage which was further supported by in silico docking studies. Using a dibromomaleimide derivative of the highly potent tubulin polymerization inhibitor monomethyl auristatin F, h38C2_K99C-based ADCs were found to be as potent as h38C2-based ADCs and afford a new assembly route for ADCs with single and dual payloads.

The Integrated Stress Response and Eukaryotic Initiation Factor 2a Regulate Methylglyoxal-induced Pain Hypersensitivity

Diabetic neuropathic pain (DNP) is a debilitating condition. Current therapies are ineffective at treating DNP. We have previously demonstrated that methylglyoxal (MGO), a reactive metabolite of glycolysis that is elevated in the plasma of diabetic patients, pain hypersensitivity by engaging the integrated stress response (ISR) in mice. Induction of the ISR alters protein synthesis by inhibiting eukaryotic initiation factor 2α (eIF2α) and relying on eIF2A-mediated translation. We hypothesize that eIF2A regulates protein synthesis that contributes to pain hypersensitivity under conditions of ISR, especially after MGO application. We employed the recently characterized eIF2A-/- mouse to investigate how MGO treatment induces the ISR, facilitates eIF2A-mediated translation, and consequently causes pain hypersensitivity. We also subjected mice to tunicamycin (TUN), a well-known ISR inducer, and spared nerve injury (SNI). With donated human dorsal root ganglia (DRG) and spinal nerves (SpN), we aimed to translate our finding in rodents to humans. Intraplantar and intraperitoneal administrations of MGO induced pain hypersensitivity in a dose-dependent manner in male and female WT animals but not in eIF2A-/- mice. Following TUN and SNI, the eIF2A-/-¬¬ mice were protected against ISR-dependent pain (tunicamycin) but not against ISR-independent pain (SNI). MGO treatment of human DRG and SpN explants, and cultured human DRG neurons elevated p-eIF2α and eIF2A levels suggesting that MGO induces ISR not only in mice, but also in humans. Carboxyethyl lysine (CEL), a byproduct of reactive MGO and lysine residues, is increased in MGO treated cells and DRG and spinal nerve explants but not in tunicamycin treated explants, despite sharing elevated p-eIF2α and eIF2A levels. We demonstrate that eIF2A is necessary for the development of ISR-induced pain hypersensitivity. We further suggest that MGO and its byproduct, CEL, can be used as a biomarker to stratify DNP patients that would respond to ISR inhibitors like ISRIB. Diabetic neuropathic pain (DNP) is a debilitating condition. Current therapies are ineffective at treating DNP. We have previously demonstrated that methylglyoxal (MGO), a reactive metabolite of glycolysis that is elevated in the plasma of diabetic patients, pain hypersensitivity by engaging the integrated stress response (ISR) in mice. Induction of the ISR alters protein synthesis by inhibiting eukaryotic initiation factor 2α (eIF2α) and relying on eIF2A-mediated translation. We hypothesize that eIF2A regulates protein synthesis that contributes to pain hypersensitivity under conditions of ISR, especially after MGO application. We employed the recently characterized eIF2A-/- mouse to investigate how MGO treatment induces the ISR, facilitates eIF2A-mediated translation, and consequently causes pain hypersensitivity. We also subjected mice to tunicamycin (TUN), a well-known ISR inducer, and spared nerve injury (SNI). With donated human dorsal root ganglia (DRG) and spinal nerves (SpN), we aimed to translate our finding in rodents to humans. Intraplantar and intraperitoneal administrations of MGO induced pain hypersensitivity in a dose-dependent manner in male and female WT animals but not in eIF2A-/- mice. Following TUN and SNI, the eIF2A-/-¬¬ mice were protected against ISR-dependent pain (tunicamycin) but not against ISR-independent pain (SNI). MGO treatment of human DRG and SpN explants, and cultured human DRG neurons elevated p-eIF2α and eIF2A levels suggesting that MGO induces ISR not only in mice, but also in humans. Carboxyethyl lysine (CEL), a byproduct of reactive MGO and lysine residues, is increased in MGO treated cells and DRG and spinal nerve explants but not in tunicamycin treated explants, despite sharing elevated p-eIF2α and eIF2A levels. We demonstrate that eIF2A is necessary for the development of ISR-induced pain hypersensitivity. We further suggest that MGO and its byproduct, CEL, can be used as a biomarker to stratify DNP patients that would respond to ISR inhibitors like ISRIB.

An Engineered Arginine Residue of Unusual pH-Sensitive Reactivity Facilitates Site-Selective Antibody Conjugation.

Monoclonal antibody h38C2 is a humanized catalytic antibody that has been used to generate various immunoconjugate species such as chemically programmed antibodies, antibody-drug conjugates, and antibody-siRNA conjugates. Highly efficient and specific conjugation of h38C2 occurs at its uniquely reactive lysine (Lys) residue buried inside the antibody's catalytic pocket. We recently reported the rational mutation of this Lys residue at position 99 in the heavy chain variable domain to an arginine (Arg) residue. The Lys99Arg mutation can be site-selectively conjugated with molecules containing a hapten-like triazolyl-phenylglyoxal (TPG) unit. Here we show that this conjugation is facilitated by the unusual pH-sensitive reactivity of the Arg99 residue, consistent with an indirectly measured pKa of 5.2. The Arg99/TPG conjugation holds promise to further expand the versatility of the h38C2 conjugation platform, such as for the generation of antibody conjugates with dual payloads.

Site-Specific Antibody-Drug Conjugates in Triple Variable Domain Fab Format.

The interest in replacing the conventional immunoglobulin G (IgG) format of monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs) with alternative antibody and antibody-like scaffolds reflects a need to expand their therapeutic utility and potency while retaining their exquisite specificity, affinity, and low intrinsic toxicity. For example, in the therapy of solid malignancies, the limited tumor tissue penetration and distribution of ADCs in IgG format mitigates a uniform distribution of the cytotoxic payload. Here, we report triple variable domain Fab (TVD–Fab) as a new format that affords the site-specific and stable generation of monovalent ADCs without the Fc domain and a drug-to-antibody ratio (DAR) of 2. TVD–Fabs harbor three variable fragment (Fv) domains: one for tumor targeting and two for the fast, efficient, precise, and stable conjugation of two cargos via uniquely reactive lysine residues. The biochemical and in vitro cytotoxicity properties of a HER2-targeting TVD–Fab before and after conjugation to a tubulin inhibitor were validated. In vivo, the TVD–Fab antibody carrier revealed a circulatory half-life of 13.3 ± 2.5 h and deeper tumor tissue distribution compared to our previously reported dual variable domain (DVD)–IgG1 format. Taken together, the TVD–Fab format merits further investigations as an antibody carrier of site-specific ADCs targeting solid malignancies.

Generation and validation of structurally defined antibody-siRNA conjugates.

Gene silencing by RNA interference (RNAi) has emerged as a powerful treatment strategy across a potentially broad range of diseases. Tailoring siRNAs to silence genes vital for cancer cell growth and function could be an effective treatment, but there are several challenges which must be overcome to enable their use as a therapeutic modality, among which efficient and selective delivery to cancer cells remains paramount. Attempts to use antibodies for siRNA delivery have been reported but these strategies use either nonspecific conjugation resulting in mixtures, or site-specific methods that require multiple steps, introduction of mutations, or use of enzymes. Here, we report a method to generate antibody–siRNA (1:2) conjugates (ARCs) that are structurally defined and easy to assemble. This ARC platform is based on engineered dual variable domain (DVD) antibodies containing a natural uniquely reactive lysine residue for site-specific conjugation to β-lactam linker-functionalized siRNA. The conjugation is efficient, does not compromise the affinity of the parental antibody, and utilizes chemically stabilized siRNA. For proof-of-concept, we generated DVD-ARCs targeting various cell surface antigens on multiple myeloma cells for the selective delivery of siRNA targeting β-catenin (CTNNB1). A set of BCMA-targeting DVD-ARCs at concentrations as low as 10 nM revealed significant CTNNB1 mRNA and protein knockdown.

Site-Specific Lysine Arylation as an Alternative Bioconjugation Strategy for Chemically Programmed Antibodies and Antibody–Drug Conjugates

By exploiting a uniquely reactive lysine residue (Lys99) for site-specific attachment of small molecules, the humanized catalytic antibody h38C2 has been used as bioconjugation module in the assembly of chemically programmed antibodies and antibody-drug conjugates. Treatment of h38C2 with β-lactam-functionalized small molecules has been previously shown to result in covalent conjugation by selective formation of a stable amide bond with the ε-amino group of the Lys99 residue. Here we report that heteroaryl methylsulfonyl (MS-PODA)-functionalized small molecules represent an alternative bioconjugation strategy through highly efficient, site-specific, and stable arylation of the Lys99 residue. A set of chemically programmed antibodies and antibody-drug conjugates assembled by Lys99 arylation provided proof-of-concept for the therapeutic utility of this alternative bioconjugation strategy. While being equally effective as β-lactam-functionalized ligands for bioconjugation with catalytic antibody h38C2, the MS-PODA moiety offers distinct synthetic advantages, making it highly attractive.

Conjugation Site Analysis of Lysine-Conjugated ADCs.

Lysine-conjugated antibody-drug conjugates (ADCs) are formed by attaching cytotoxic drugs to reactive lysine residues of monoclonal antibodies (mAbs) through chemical linkers. During production, the payloads are conjugated nonspecifically to lysine residues in mAbs, resulting in a heterogeneous mixture of ADCs with both different number and conjugation sites of drug payloads per mAb. On account of the drug conjugation sites and levels that both have significant influences on physical and pharmaceutical properties of ADCs, a reliable and straightforward approach for conjugation site analysis for ADCs is highly demanded. Herein, we used a lysine-conjugated ADC, Trastuzumab-MCC-DM1 (T-DM1), as a model ADC, and described an integrative strategy that combines the signature ion fingerprinting method for rapid and reliable filtering of DM1-conjugated peptides, and the normalized area quantitation approach for accurately gauging the conjugation levels for each identified site. This approach is believed to be readily applicable to other maytansinoid derivatives-modified ADCs, and more importantly, universally applicable to lysine-conjugated ADCs for both the recognition of conjugation sites and the measurement of conjugation levels.

SUBSTRATE BINDING INHIBITS γ-SECRETASE CLEAVAGE OF AMYLOID PRECURSOR PROTEIN

Alzheimer's disease (AD) has become a severe public health challenge with no disease-modifying treatment available. The inhibition of amyloid precursor protein (APP) processing by γ-secretase is a prominent strategy, while clinical trials of γ-secretase inhibitors largely failed due to serious side effects. A Compound C1 was discovered by NMR binding based on a previously solved NMR structure of the transmembrane domain of APP (APPTM) in micelle. MALDI-TOF-MS and LC-ESI-MS were employed to access the C1 modification pattern and mechanism on APPTM. A gel-based cleavage assay and AlphaLISA assay were used to test if C1-modified substrate can inhibit γ-secretase cleavage of APP. The effect of C1 on the Aβ production was also detected in HEK 293 cells. C1 covalently modifies the C-terminal juxtamembrane lysines (K53 K54 K55) side chain of APPTM. C1-modified substrate inhibits γ-secretase cleavage with an IC50 value of 1.9 μM for the inhibition on Aβ42 production and 3.9 μM for Aβ40. In agreement with the smaller IC50 for Aβ42 in AlphaLISA assay, C1 reduces the level of Aβ42 more than that of Aβ40 in HEK 293 cells, resulting in a reduced Aβ42/Aβ40 ratio. By mutagenesis, K55 was found to be the most reactive lysine in APPTM and plays crucial role in intramembrane proteolysis. Our work demonstrates that targeting the substrate of γ-secretase to reduce the production of Aβ is viable and novel strategy. This is to our knowledge the first example of small molecule that inhibits γ-secretase cleavage and Aβ production by interacting with APPTM. Our results point to a new direction in AD drug discovery for reducing amyloid load as disease-modifying therapy.

Site-Selective Antibody Functionalization via Orthogonally Reactive Arginine and Lysine Residues

Homogeneous antibody-drug conjugates (ADCs) that use a highly reactive buried lysine (Lys) residue embedded in a dual variable domain (DVD)-IgG1 format can be assembled with high precision and efficiency under mild conditions. Here we show that replacing the Lys with an arginine (Arg) residue affords an orthogonal ADC assembly that is site-selective and stable. X-ray crystallography confirmed the location of the reactive Arg residue at the bottom of a deep pocket. As the Lys-to-Arg mutation is confined to a single residue in the heavy chain of the DVD-IgG1, heterodimeric assemblies that combine a buried Lys in one arm, a buried Arg in the other arm, and identical light chains, are readily assembled. Furthermore, the orthogonal conjugation chemistry enables the loading of heterodimeric DVD-IgG1s with two different cargos in a one-pot reaction and thus affords a convenient platform for dual-warhead ADCs and other multifaceted antibody conjugates.

Merits and pitfalls of conventional and covalent docking in identifying new hydroxyl aryl aldehyde like compounds as human IRE1 inhibitors

IRE1 is an endoplasmic reticulum (ER) bound transmembrane bifunctional kinase and endoribonuclease protein crucial for the unfolded protein response (UPR) signaling pathway. Upon ER stress, IRE1 homodimerizes, oligomerizes and autophosphorylates resulting in endoribonuclease activity responsible for excision of a 26 nucleotide intron from the X-box binding protein 1 (XBP1) mRNA. This unique splicing mechanism results in activation of the XBP1s transcription factor to specifically restore ER stress. Small molecules targeting the reactive lysine residue (Lys907) in IRE1α’s RNase domain have been shown to inhibit the cleavage of XBP1 mRNA. Crystal structures of murine IRE1 in complex with covalently bound hydroxyl aryl aldehyde (HAA) inhibitors show that these molecules form hydrophobic interactions with His910 and Phe889, a hydrogen bond with Tyr892 and an indispensable Schiff-base with Lys907. The availability of such data prompted interest in exploring structure-based drug design as a strategy to develop new covalently binding ligands. We extensively evaluated conventional and covalent docking for drug discovery targeting the catalytic site of the RNase domain. The results indicate that neither computational approach is fully successful in the current case, and we highlight herein the potential and limitations of the methods for the design of novel IRE1 RNase binders.

Identification of [6-Hydroxy-2-(hydroxymethyl)-5-oxo-5,6-dihydro-2 H-pyran-3-yl]-cysteine (HHPC) as a Cysteine-specific Modification Formed from 3,4-Dideoxyglucosone-3-ene (3,4-DGE).

Glucose degradation products (GDPs) are formed from glucose and other reducing sugars during heat treatment, for example, in heat-sterilized peritoneal dialysis fluids or foods. Because of their reactive mono- and dicarbonyl structure, they react readily with proteins, resulting in the formation of advanced glycation end products (AGEs), loss of protein functionality, and cytotoxicity. Among the GDPs, 3,4-dideoxyglucosone-3-ene (3,4-DGE) exerts the strongest effects despite its relatively low concentration levels. The goal of the present study was therefore to identify the structure of specific protein modifications deriving from 3,4-DGE. A nonapeptide containing the reactive amino acids lysine, arginine, and cysteine was incubated with 3,4-DGE and the dominant GDPs 3-deoxyglucosone (3-DG) and 3-deoxygalactosone (3-DGal) in concentrations as present in peritoneal dialysis fluids (235 μM 3-DG, 100 μM 3-Gal, and 11 μM 3,4-DGE). Glycation rate and product formation were determined by ultra-HPLC-MS/MS (UHPLC-MS/MS). 3,4-DGE showed the strongest glycation activity. After 2 h of incubation, 3,4-DGE had modified 57% of the nonapeptide, whereas 3-DG had modified only 2% and 3-DGal had modified 29% of the peptide. A stable 3,4-DGE-derived cysteine modification was isolated. Its structure was determined by comprehensive NMR and MS experiments to be [6-hydroxy-2-(hydroxymethyl)-5-oxo-5,6-dihydro-2 H-pyran-3-yl]-cysteine (HHPC), which represents a novel cysteine-AGE derived from 3,4-DGE. The results indicate that 3,4-DGE might contribute to a severe loss of protein functionality by forming cysteine-specific AGEs, such as HHPC.

Lysine Bioconjugation on Native Albumin with a Sulfonyl Acrylate Reagent.

This protocol details a novel bioconjugation strategy that uses a methanesulfonyl acrylate reagent that is directed to the most reactive lysine on human serum albumin, which enables the construction of chemically defined and stable bioconjugates. The reaction proceeds rapidly and a regioselective modification is achieved using a single molar equivalent of the reagent under biocompatible conditions (37°C, pH8.0). Importantly, the bioconjugate retains both the secondary structural content and function of the unmodified protein. During the reaction of the amino group of lysine and the sulfonyl acrylate reagent, methanesulfinic acid is released after the conjugate addition, which then generates an electrophilic acrylate moiety on the protein. This acrylate can be further used for site-specific protein labeling using a synthetic molecule bearing a reactive amine under biocompatible conditions (21°C, pH8.0).

Engineering Dual Variable Domains for the Generation of Site-Specific Antibody-Drug Conjugates.

Site-specific antibody-drug conjugate (ADC) technologies are highly desirable for the production of therapeutics with well-defined biochemical and pharmacological characteristics. We have developed a strategy to produce site-specific ADCs using a highly reactive lysine residue embedded in a dual-variable-domain (DVD) format. Here we provide protocols for the engineering, expression, and purification of the DVDs used for this strategy. We also provide a protocol for DVD-drug conjugation and describe methods for their biochemical characterization, including a catalytic assay to monitor conjugation efficiency.

Engineering SpyCatcher Variants with Proteolytic Sites for Less‐Trace Ligation

Summary of main observation and conclusionThe SpyTag/SpyCatcher reaction is a powerful tool for bioconjugation, but it leaves a complex of considerable size after ligation. To facilitate removal of the catalytic fragment, proteolytic recognition sites (such as DDDDK, AVLQ, and WELQ) were directly engineered into the first or second loop of SpyCatcher at locations after the reactive lysine to give a set of cleavable SpyCatcher variants. Among them, SpyCatcherDDDDK exhibits excellent reactivity with SpyTag and could still be cleaved proteolytically by enterokinase after ligation. Notably, SpyCatcherDDDDK is disordered in solution and forms an ordered complex upon reaction with SpyTag with a second order rate constant of 99.2 ± 0.1 M–1·s–1, which is comparable to, if not faster than, most click reactions. The results demonstrate the high sequence plasticity of SpyCatcher and suggest that covalent bond formation may confer robustness on the folded structure against extensive mutation. These variants add to the expanding toolbox of genetically‐encoded peptide‐protein chemistry with diverse features.

Effects of different souring methods on the protein quality and iron and zinc bioaccessibilities of non‐alcoholic beverages from sorghum and amaranth

SummarySouring by lactic acid fermentation and lactic acid acidification as well as inclusion of amaranth were explored as ways of improving the protein quality and iron and zinc bioaccessibilities of non‐alcoholic sorghum‐based beverages. The bioaccessible iron and zinc increased by 128–372%, 24–194%, respectively, in the fermented and chemically acidified beverages compared to the beverages without fermentation or acidification. The protein digestibility, reactive lysine, and bioaccessible iron in sorghum‐amaranth beverages increased by 14–58%, 24–52% and 34–64%, respectively, compared with the 100% sorghum beverages. Both fermentation and acidification with lactic acid have the potentials for improving the nutritional quality of cereal‐based foods as a means of combating protein malnutrition and iron and zinc deficiencies.

Modification of erythropoietin structure by N-homocysteinylation affects its antiapoptotic and proliferative functions.

Many patients under therapy with recombinant human erythropoietin (rhuEPO) show resistance to the treatment, an effect likely associated with the accumulation of tissue factors, especially in renal and cardiovascular diseases. Hyperhomocysteinemia due to high serum levels of homocysteine has been suggested among the risk factors in those pathologies. Its main effect is the N-homocysteinylation of proteins due to the interaction between the highly reactive homocysteine thiolactone (HTL) and lysine residues. The aim of this study was to evaluate the effect of N-homocysteinylation on the erythropoietic and antiapoptotic abilities of EPO, which can be a consequence of structural changes in the modified protein. We found that both cellular functions were altered in the presence of HTL-EPO. A decreased net positive charge of HTL-EPO was detected by capillary zone electrophoresis, while analysis of polyacrylamide gel electropherograms suggested formation of aggregates. Far-UV spectra, obtained by Circular Dichroism Spectroscopy, indicated a switch of the protein's secondary structure from α-helix to β-sheet structures. Results of Congo red and Thioflavin T assays confirm the formation of repetitive β-sheet structures, which may account for aggregates. Accordingly, Dynamic Light Scattering analysis showed a markedly larger radius of the HTL-EPO structures, supporting the formation of soluble oligomers. These structural changes might interfere with the conformational adaptations necessary for efficient ligand-receptor interaction, thus affecting the proliferative and antiapoptotic functions of EPO. The present findings may contribute to explain the resistance exhibited by patients with cardio-renal syndrome to treatment with rhuEPO, as a consequence of structural modifications due to protein N-homocysteinylation.

Reagent enables new protein modification

Chemists modify protein sidechains to modulate the biomolecules’ functions. Researchers are usually stuck modifying cysteines because the amino acid’s sulfide side chain makes it the most reactive amino acid. But cysteines are relatively rare, representing ~1.9% of the amino acids in human proteins. Cysteines can be installed using genetic engineering, but researchers would like to modify fully functional native proteins. An international team of researchers led by Goncalo J. L. Bernardes of the University of Cambridge and the Institute of Molecular Medicine Lisbon and Gonzalo Jimenez-Oses of the University of Rioja has now discovered a sulfonyl acrylate reagent that should give researchers more options for modifying proteins. The reagent selectively targets only the most reactive lysine in a protein—not other lysines or buried cysteines (J. Am. Chem. Soc. 2018, DOI: 10.1021/jacs.7b12874). Lysines are nucleophilic and much more abundant than cysteines, representing ~5.9% of the amino acids in human protei...

Pelleting and extrusion can ameliorate negative effects of toasting of rapeseed meal on protein digestibility in growing pigs

Toasting time (TT) of rapeseed meal (RSM), the diet processing (DP) method and the interaction between both on the apparent CP digestion along the gastrointestinal tract and the apparent ileal digestibility (AID) of amino acids of growing pigs were investigated. The experiment consisted of a 3×3 factorial design of TT of RSM (0, 60 and 120 min) and DP method (mash, pelleting and extrusion). In total, 81 boars with a starting BW of 20 kg were euthanized 4 h after their last feeding. The gastrointestinal tract was dissected and the small intestine divided in three sections of similar length. Samples were collected from the stomach, 1.5 m from the ends of each of the three sections of the small intestine, and the rectum. The apparent digestibility (AD) of CP for each of the small intestine sections was used to calculate the rate of CP digestion. Increasing the TT of RSM resulted in lower protein solubility, lower lysine/reactive lysine contents and higher protein denaturation, indicative of the occurrence of protein aggregation and Maillard reactions. There were significant effects (P⩽0.01) of TT on the AD of CP in the different sections of the gastrointestinal tract. The rate of CP digestion of the 0 min toasted RSM diets was 23% and 35% higher than that of the 60 and 120 min toasted RSM diets, respectively. There was a significant interaction (P=0.04) between TT and DP for the AID of CP. Although pelleting of the 0 and 60 min toasted RSM diets did not change the AID of CP with respect to the mash diets, pelleting of the 120 min toasted RSM diet increased the AID of CP by 9.3% units. Extrusion increased the AID of CP of the 0 and 60 min toasted RSM diets by 3.4% and 4.3% units with respect to the mash diets, whereas extrusion of the 120 min toasted RSM diet increased the AID of CP by 6.9% units. Similar positive effects of pelleting and extrusion were obtained for the AID of lysine and reactive lysine, especially in the diets with higher TT. In conclusion, processing (pelleting and extrusion) of RSM containing diets can ameliorate the negative effects of RSM toasting on protein and amino acid digestibility; these effects were larger for the RSM toasted for longer times.

Predicting the nutritional quality of feed ingredients for pigs using near-infrared spectroscopy (NIRS) and chemical analysis

Capacity to routinely, accurately and cost-effectively measure variation in the nutritional quality of feed ingredients before diet formulation represents a fundamental pillar of sustainable pork production worldwide. Factors driving sustainable pork production include pork price, feed cost, utilisation of co-products and downgraded raw materials and variation in pork production, with all being related to the definition and ultimate nutritional quality of feed ingredients. The present paper defines rapid measures of nutritional quality in feed ingredients for pigs and demonstrates the range that can exist in these parameters, specifically digestible energy of cereal grains and the reactive-lysine concentration of oilseed meals. It provides an overview of the development of near-infrared spectroscopy (NIRS) calibrations for key nutritional-quality parameters and how they are being applied by the pork industry. Adjunct ways to measure nutritional quality of feed ingredients for pigs such as the glucose-release index and how these can be used in conjunction with NIRS are reviewed. The paper reports advanced correlation analysis between chemical components and digestible-energy concentration of cereals, and how these could be used for screening of NIRS outliers, and discusses future opportunities for application of nutritional-quality analysis using NIRS calibrations, including feed intake and portable solutions. Using advanced NIRS calibrations for digestible energy in cereals and reactive lysine in oilseed meals, pork producers will ensure that they make best use of limited resources and, as a consequence, pork will remain a nutritionally accretive food source for increasingly discerning consumers worldwide.

Casein-based scaffold for artificial cellulosome design

Abstract Cellulosomal systems are known as highly efficient biocatalysts in the degradation of lignocellulosic biomass in nature, but they remain unsuitable for industrial applications. In seeking alternatives to natural cellulosomes, casein was chosen as a scaffold for cellulase clustering. Casein is recognized as an excellent substrate for microbial transglutaminase (MTG) because it contains naturally reactive glutamine and lysine residues. A substrate peptide containing an MTG-reactive lysine residue was inserted into the C-terminus of the endoglucanase Cel5A and Cel6A from Thermobifida fusca using genetic engineering. The engineered cellulases, EG(Cel5A) and EG(Cel6A), were conjugated onto casein in different ratios by an MTG-mediated site-specific protein crosslinking reaction. Overall, a more than two-fold increase was observed when EG(Cel5A) was conjugated onto N,N-dimethylcasein, but a small or no change was observed for EG(Cel6A).