Fabrication and characterization of soy protein isolation-ferulic acid antioxidant hydrogels.

BACKGROUND: Soy protein gel products are prone to direct oxidation by reactive oxygen during processing and transportation, thus reducing their functional properties and nutritional values. A covalent complex was prepared with soy protein isolate (SPI) and ferulic acid (FA) catalyzed by laccase (LC). The complex was further treated with microbial transglutaminase (TGase) to form hydrogels. The structural changes of the covalent complex (SPI-FA) and the properties and antioxidant stability of hydrogel were investigated. RESULTS: The SPI-FA complexes were demonstrated to be covalently bound by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and they had the least hydrophobic and free sulfhydryl groups at a 1.0 mg mL-1 FA concentration. The α-helix of complexes increased from 11.50% to 27.39%, and random coil dropped from 26.06% to 14.44%. The addition of FA caused SPI fluorescence quenching and redshift. The hydrogel was formed after the complex was induced with TGase, and its hardness and water holding capacity was increased by 50.61% and 26.21%, respectively. Scanning electron microscopy showed that a layered and ordered gel structure was formed. After in vitro digestion, the complex hydrogels maintained stable antioxidant activity, and the free radical scavenging rates of DPPH and ABTS reached 87.65% and 84.45%, respectively. CONCLUSION: SPI-FA covalent complexes were prepared under laccase catalysis, and complex hydrogels were formed by TGase. Hydrogels have stable antioxidant activity, which provides application prospects for the antioxidant development of food. © 2023 Society of Chemical Industry.

Recombinant Streptomyces netropsis transglutaminase expressed in Komagataella phaffii (Pichia pastoris) and applied in plant-based chicken nugget.

Transglutaminase (TG, EC 2.3.2.13) is widely used to modify functional properties in food systems, which can catalyze cross-linking reaction of proteins. In this work, microbial transglutaminase (MTG) from Streptomyces netropsis was heterologously expressed in the methylotrophic yeast Komagataella phaffii (Pichia pastoris). The specific activity of recombinant microbial transglutaminase (RMTG) was 26.17 ± 1.26 U/mg, and the optimum pH and temperature were measured as 7.0 and 50 °C, respectively. Bovine serum albumin (BSA) was used as a substrate to evaluate the effect of cross-linking reaction, and we found that RMTG had significant (p < 0.05) cross-linking effect for more than 30 min reactions. RMTG was further utilized in the investigation of plant-based chicken nuggets. Results showed that the hardness, springiness and chewiness of nuggets increased, and the adhesiveness decreased after RMTG treatment, which can prove that RMTG has the potential to improve the texture properties of plant-based chicken nuggets.

Liposomal Amphotericin B Formulation Displaying Lipid-Modified Chitin-Binding Domains with Enhanced Antifungal Activity.

Fungal infections affect more than one billion people worldwide and cause more than one million deaths per year. Amphotericin B (AmB), a polyene antifungal drug, has been used as the gold standard for many years because of its broad antifungal spectrum, high activity, and low tendency of drug resistance. However, the side effects of AmB, such as nephrotoxicity and hepatotoxicity, have hampered its widespread use, leading to the development of a liposome-type AmB formulation, AmBisome. Herein, we report a simple but highly effective strategy to enhance the antifungal activity of AmBisome with a lipid-modified protein. The chitin-binding domain (LysM) of the antifungal chitinase, Pteris ryukyuensis chitinase A (PrChiA), a small 5.3 kDa protein that binds to fungal cell wall chitin, was engineered to have a glutamine-containing peptide tag at the C-terminus for the microbial transglutaminase (MTG)-catalyzed crosslinking reaction (LysM-Q). LysM-Q was site-specifically modified with a lysine-containing lipid peptide substrate of MTG with a palmitoyl moiety (Pal-K). The resulting palmitoylated LysM (LysM-Pal) exhibited negligible cytotoxicity to mammalian cells and can be easily anchored to yield LysM-presenting AmBisome (LysM-AmBisome). LysM-AmBisome exhibited a dramatic enhancement of antifungal activity toward Trichoderma viride and Cryptococcus neoformans, demonstrating the marked impact of displaying a cell-wall binder protein on the targeting ability of antifungal liposomal formulations. Our simple strategy with enzymatic protein lipidation provides a potent approach to upgrade other types of lipid-based drug formulations.

Rational design of a disulfide bridge increases the thermostability of microbial transglutaminase.

Microbial transglutaminase (MTG) has numerous industrial applications in the food and pharmaceutical sectors. Unfortunately, the thermostability of MTG is too low to tolerate the desired conditions used in many of these commercial processes. In a previous study, we used protein engineering to improve the thermostability of MTG. Specifically, we generated a T7C/E58C mutant of MTG from Streptomyces mobaraensis that displayed enhanced resistance to thermal inactivation. In this study, a rational structure-based approach was adopted to introduce a disulfide bridge to further increase the thermostability of MTG. In all, four new mutants, each containing a novel disulfide bond, were engineered. Of these four mutants, D3C/G283C showed the most promising thermostability with a significantly higher ∆T50 (defined as the temperature of incubation at which 50% of the initial activity remains) of + 9 °C by comparison to wild-type MTG. Indeed, D3C/G283C combined enhanced thermostability with a 2.1-fold increased half-life at 65 °C compared with the wild-type enzyme. By structure-based rational design, we were able to create an MTG variant which might be useful for expanding the scope of application in food. KEY POINTS: • Microbial transglutaminase (MTG) is an enzyme used in many food applications • The applicability of MTG to various industrial processes other than the food sector is being investigated • Improvement of thermostability was confirmed for the disulfide bridge mutant D3C/G283C.

Construction, expression, purification, characterization, and structural analysis of microbial transglutaminase variants.

Microbial transglutaminase (MTG, EC 2.3.2.13) derived from Streptomyces mobaraensis is widely used in the food and pharmaceutical industry because of its ability to synthesize isopeptide bonds between the proteinogenic side chains of glutamine and lysine. The half-life (t1/2 ) of the activated wild-type enzyme at 60°C is 2 min. To improve the activity and thermostability of MTG for higher temperature application, three variants (Mut1, Mut2, and Mut3) were obtained by combining key amino acid mutations on the basis of previous research results. The best variant Mut2 with a specific combination of five of seven substitutions (S2P-S23V-Y24N-R215A-H289Y) shows a 10-fold increased half-life at 60°C (t1/2  = 27.6 min), and a 2.4-fold increased specific enzyme activity (39.3 U/mg). As measured by circular dichroism, the curve of Mut2 was basically the same as that of MTG-WT. The structural simulation of Mut2 shows that the overall structure is discoid with a crack, but the crack openings are wider than that of MTG-WT. Furthermore, structural analysis of Mut2 showed that there were seven hydrogen bonds and one π-anion interaction between Mut2 and its adjacent amino acids, and the number of hydrogen bonds was one more than that of MTG-WT (six hydrogen bonds).

Tissue Transglutaminase but Not Microbial Transglutaminase Is Inhibited by Exogenous Oxidative Substances in Celiac Disease.

Enzymatic modification of gliadin peptides by human transglutaminase 2 (TG2) is a central step in celiac disease (CD) pathogenesis. Microbial transglutaminase (mTG) mimics the enzymatic function of TG2 and might play a role in CD. TG2 is inhibited by endogenous oxidative endoplasmic reticulum-resident protein 57 (ERp57), but data about mTG are lacking. We investigated the localization of ERp57 in duodenal biopsies and examined inhibition of TG2, and mTG by competitive, and oxidative molecules. Localization of ERp57 was investigated in duodenal biopsies from CD, and control patients by electron microcopy. Inhibition of TG2 and mTG was analyzed on an in vitro level using a photometric assay. ERp57 was observed within the lamina propria and its abundance within the endoplasmic reticulum (ER) was reduced in CD patients. TG2 was oxidatively inhibited by up to 95% by PX12 (p < 0.001) and L-cystine (p < 0.001), whereas mTG remained unaffected. The reduced presence of ERp57 within the ER of CD biopsies suggests a regulatory function of this protein within CD pathogenesis. PX12 and L-cystine oxidatively inhibit TG2 and might serve as treatment options in CD. mTG is poorly regulated and could contribute to the accumulation of immunogenic peptides within the gut with potential pathogenic effects.

Effect of introducing a disulfide bridge on the thermostability of microbial transglutaminase from Streptomyces mobaraensis.

Microbial transglutaminase (MTG) has been used extensively in academic research and the food industry through cross-linking or posttranslational modification of proteins. In our previous paper, the activity-increased MTG mutants were obtained by means of rational mutagenesis and random mutagenesis coupled with the newly developed screening system. In addition, the improvement of heat resistance of MTG is needed to expand further its industrial applications. Here, a structure-based rational enzyme engineering approach was applied to improve the thermostability of MTG by introducing an artificial disulfide bridge. As a result of narrowing down candidates using a rational approach, we successfully engineered a disulfide bridge into the N-terminal region of MTG by substituting Thr-7 and Glu-58 with cysteine. The T7C/E58C mutant was observed to have a de novo disulfide bridge and showed an increased melting temperature (Tm value) of 4.3 °C with retained enzymatic activity. To address the benefit-gained reason, we focused on the Cß temperature factor of the amino-acid residues that might form a disulfide bridge in MTG. Introducing the disulfide bridge had no remarkable effect on the mutant aiming to stabilize the high temperature factor. On the other hand, the mutation was effective on the relatively stable region. The introduction of a disulfide bridge may therefore be effective to stabilize further the relatively stable part. This finding is considered to be useful for the rational design of mutants aiming at heat resistance of proteins.Key Points• Microbial transglutaminase (MTG) is used as a binder in the food industry.• MTG has the potential for use in the manufacturing of various commercial materials.• Enhanced thermostability was observed for the disulfide bridge mutant, T7C/G58C.

Transamidation Down-Regulates Intestinal Immunity of Recombinant α-Gliadin in HLA-DQ8 Transgenic Mice.

Enzymatic transamidation of gliadins by microbial transglutaminase (mTG) inhibits interferon-γ (IFN-γ) secretion by intestinal T cell lines in patients with celiac disease (CD). To gain insight into the cellular mechanisms underlying the down-regulatory effects of transamidation, we tested a single recombinant α-gliadin (r-gliadin) harbouring two immunodominant peptides, p13 (aa. 120-139) and p23 (aa. 220-239), in HLA-DQ8 transgenic mice, a model of gluten sensitivity. Mice were intranasally immunised with r-gliadin or r-gliadin transamidated by mTG (K-r-gliadin) along with cholera toxin, and the response of mesenteric lymph node cells was analysed by cytokine multiplex assay. An in vitro challenge with r-gliadin was characterised by secretion of specific cytokines featuring both innate immunity and the Th1/Th2/Th17 pattern of the adaptive response. Notably, transamidation specifically down-regulated the Th1 response. Structural studies performed on K-r-gliadin confirmed that specific glutamine residues in p13 and p23, previously found to be deamidated by tissue transglutaminase, were also transamidated by mTG. In silico analysis, simulating p13 and p23 peptide binding to HLA-DQ8 showed that these glutamines, in the form of glutamate, could interact by means of salt bridges with peculiar amino acids of the alpha chain of HLA-DQ8, suggesting that their transamidation may influence the HLA-restricted recognition of these peptides. Thus, the structural findings provided a rationale to explain the down-regulation of the r-gliadin-specific Th1 response following transamidation.

Optimization of soy protein isolate, microbial transglutaminase and glucono-δ-lactone in gluten-free rice noodles.

BACKGROUND: Rice flour does not contain gluten and lacks cohesion and extensibility, which is responsible for the poor texture of rice noodles. Different technologies have been used to mitigate this challenge, including hydrothermal treatments of rice flour, direct addition of protein in noodles, use of additives such as hydrocolloids and alginates, and microbial transglutaminase (MTG). Recently, the inclusion of soy protein isolate (SPI), MTG, and glucono-δ-lactone (GDL) in the rice noodles system yielded rice noodles with improved texture and more compact microstructure, hence the need to optimize the addition of SPI, MTG, and GDL to make quality rice noodles. RESULTS: Numerical optimization showed that rice noodles prepared with SPI, 68.32 (g kg-1 of rice flour), MTG, 5.06 (g kg-1 of rice flour) and GDL, 5.0 (g kg-1 of rice flour) gave the best response variables; hardness (53.19 N), springiness (0.76), chewiness (20.28 J), tensile strength (60.35 kPa), and cooking time (5.15 min). The pH, sensory, and microstructure results showed that the optimized rice noodles had a more compact microstructure with fewer hollows, optimum pH for MTG action, and overall sensory panelists also showed the highest preference for the optimized formulation, compared to other samples selected from the numerical optimization and desirability tests. CONCLUSION: Optimization of the levels of SPI, MTG, and GDL yielded quality noodles with improved textural, mechanical, sensory, and microstructural properties. This was partly due to the favourable pH value of the optimized noodles that provided the most suitable conditions for MTG crosslinking and balanced electrostatic interaction of proteins. © 2020 Society of Chemical Industry.

Rheological and textural properties of goat's milk set-type yoghurt as affected by heat treatment, transglutaminase addition and storage.

BACKGROUND: Production of goat's milk set-style yoghurt encounters challenges in achieving the texture characteristic for this type of product, primarily due to protein composition of this milk. This study evaluated the effects of using microbial transglutaminase (mTGase) concomitantly with starter culture in the production of goat's milk yoghurt - a method that has not been employed with this milk type until now- indicating the potential of the enzyme to change yoghurt's textural properties. Goat's milk set yoghurts were produced from milk heated at 72 °C/30 s and 90 °C/5 min, without (G72 and G90) and with mTGase (G72TG and G90TG) and starter culture addition. Protein profiles of goat's milks and yoghurts were also examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Yoghurts were evaluated for rheological properties, texture, microbiological and sensory profile over 2 weeks to study the influence of mTGase, pasteurization and storage. RESULTS: The enzyme caused significant increases of storage moduli at the end of fermentation: 8.32 ± 0.27 Pa (G90TG) and 2.89 ± 0.18 Pa (G72TG) vs. 6.13 ± 0.07 Pa (G90) and 1.27 ± 0.18 Pa (G72) without enzyme. Lower loss tangent values indicated the enhanced elastic character of the gels with enzyme. Enzyme increased yoghurt's firmness from 49.69 ± 2.61 g (G90) to 60.81 ± 5.29 g (G90TG) after 1 day and from 58.21 ± 0.53 g (G90) to 80.45 ± 0.59 g (G90TG) after 15 days' storage. Enzyme improved starter bacteria survivability during storage of G72TG yoghurt. CONCLUSION: mTGase can be used simultaneously with the starter culture to improve the rheological properties and texture of goat's milk yoghurt, without deteriorating effect on its flavour. © 2021 Society of Chemical Industry.

Antibodies against neo-epitope of microbial and human transglutaminase complexes as biomarkers of childhood celiac disease.

Tissue transglutaminase (tTG) and microbial transglutaminase (mTG) cross-link gliadins to form complexes that expose immunogenic neo-epitopes to produce tTG and mTG-neo-epitope antibodies. The aim of this study was to test the diagnostic performance of antibodies against non-complexed and complexed forms of transglutaminases, to correlate their activities to the intestinal damage and to explore age group dependency in celiac disease (CD). A total of 296 children with untreated CD and 215 non-celiac disease controls were checked by in-house enzyme-linked immunosorbent assays detecting immunoglobulin (Ig)A, IgG or combined detection of IgA and IgG (check) against tTG, AESKULISA® tTG New Generation (tTG-neo) and mTG-neo (RUO), IgA and IgG antibodies against deamidated gliadin peptide (DGP) and human IgA anti-endomysium antibodies (EMA) using AESKUSLIDES® EMA. Intestinal pathology was graded according the revised Marsh criteria, and age dependencies of the antibody activities were analysed. Using cut-offs estimated from receiver operating characteristic (ROC) curves, the highest area under curve (AUC) of the TG assays was 0·963 for tTG-neo check, followed by tTG check (0·962) when the diagnosis was based on enteric mucosal histology. tTG-neo check was the most effective to reflect the intestinal abnormalities in CD (r = 0·795, P < 0·0001). High levels of anti-mTG-neo IgG and anti-tTG-neo IgG appeared in the earlier age groups, as compared to anti-tTG IgG (P < 0·001). Considering antibody diagnostic performance based on AUC, enteric damage reflection and predictability at an early age, the anti-neo tTG check was the most effective diagnostic biomarker for pediatric CD. The mTG neo check might represent a new marker for CD screening, diagnosis and predictability.

PolyTag: A peptide tag that affords scaffold-less covalent protein assembly catalyzed by microbial transglutaminase.

Assembling proteins in close vicinity to each other provides an opportunity to gain unique function because collaborative and even synergistic functionalities can be expected in an assembled form. There have been a variety of strategies to synthesize functional protein assemblies but site-specific covalent assembly of monomeric protein units without impairing their intrinsic function remains challenging. Herein we report a powerful strategy to design protein assemblies by using microbial transglutaminase (MTG). A serendipitous discovery of self-crosslinking of enhanced green fluorescent protein (EGFP) fused with StrepTag I at the C-terminus revealed that EGFP was assembled through the crosslinking of the Lys (K) residue in the C-terminus of EGFP and the Gln (Q) residue in StrepTag I (AWRHPQFGG). Site-directed mutagenesis of the residues next to the K and Q yielded EGFP assemblies with higher molecular weights. An optimized peptide tag comprised of both K and Q residues (HKRWRHYQRGG) enabled the assembly of different types of proteins of interest (POI) when it was fused to either the N- or C-terminus. The peptide tag that enabled the self-polymerization of the functional POI without a scaffold was designated as a 'PolyTag'.

Microbial transglutaminase: A biotechnological tool to manage gluten intolerance.

Celiac disease (CD) is a chronic immune-mediated disease in which gluten ingestion leads to damage of the small intestinal mucosa in genetically susceptible individuals. The enteropathy is mainly induced by the production of IFN-γ from intestinal CD4+T cells that recognise gliadin peptides following deamidation by tissue transglutaminase. The only available therapy is a strict, lifelong gluten-free diet (GFD). This diet is strongly demanding for patients, which justifies the search for alternative strategies. The enzyme approach is one promising strategy to address this issue. In particular, transamidation of wheat gliadin by microbial transglutaminase (mTG) was fully effective at inhibiting gliadin-specific IFN-γ secretion in intestinal T cells from CD patients. Furthermore, transamidated gliadin induced higher levels of the anti-inflammatory IL-10 than native gliadin in different in vitro models. These data suggest that a more balanced immune response could be induced by mTG-treated gliadin in the small intestine of celiac patients. Furthermore, the highlighted biological property of mTG-treated gliadin could be exploited to induce tolerance to native gliadin in at-risk individuals.

Enzymatic activity and thermoresistance of improved microbial transglutaminase variants.

Microbial transglutaminase (MTG, EC 2.3.2.13) of Streptomyces mobaraensis is widely used in industry for its ability to synthesize isopeptide bonds between the proteinogenic side chains of glutamine and lysine. The activated wild-type enzyme irreversibly denatures at 60 °C with a pseudo-first-order kinetics and a half-life time (t1/2) of 2 min. To increase the thermoresistance of MTG for higher temperature applications, we generated 31 variants based on previous results obtained by random mutagenesis, DNA shuffling and saturation mutagenesis. The best variant TG16 with a specific combination of five of seven substitutions (S2P, S23Y, S24 N, H289Y, K294L) shows a 19-fold increased half-life at 60 °C (t1/2 = 38 min). As measured by differential scanning fluorimetry, the transition point of thermal unfolding was increased by 7.9 °C. Also for the thermoresistant variants, it was shown that inactivation process follows a pseudo-first-order reaction which is accompanied by irreversible aggregation and intramolecular self-crosslinking of the enzyme. Although the mutations are mostly located on the surface of the enzyme, kinetic constants determined with the standard substrate CBZ-Gln-Gly-OH revealed a decrease in KM from 8.6 mM (± 0.1) to 3.5 mM (± 0.1) for the recombinant wild-type MTG and TG16, respectively. The improved performance of TG16 at higher temperatures is exemplary demonstrated with the crosslinking of the substrate protein ß-casein at 60 °C. Using molecular dynamics simulations, it was shown that the increased thermoresistance is caused by a higher backbone rigidity as well as increased hydrophobic interactions and newly formed hydrogen bridges.

Recombinant production of active microbial transglutaminase in E. coli by using self-cleavable zymogen with mutated propeptide.

Microbial transglutaminase from Streptomyces mobaraensis (MTG) has been widely used in food industry and also in research and medical applications, since it can site-specifically modify proteins by the cross-linking reaction of glutamine residue and the primary amino group. The recombinant expression system of MTG in E. coli provides better accessibility for the researchers and thus can promote further utilization of MTG. Herein, we report production of active and soluble MTG in E. coli by using a chimeric protein of tobacco etch virus (TEV) protease and MTG zymogen. A chimera of TEV protease and MTG zymogen with native propeptide resulted in active MTG contaminated with cleaved propeptide due to the strong interaction between the propeptide and catalytic domain of MTG. Introduction of mutations of K9R and Y11A to the propeptide facilitated dissociation of the cleaved propeptide from the catalytic domain of MTG and active MTG without any contamination of the propeptide was obtained. The specific activity of the active MTG was 22.7 ± 2.6 U/mg. The successful expression and purification of active MTG by using the chimera protein of TEV protease and MTG zymogen with mutations in the propeptide can advance the use of MTG and the researches using MTG mediated cross-linking reactions.

Microbial transglutaminase alters the immunogenic potential and cross-reactivity of horse and cow milk proteins.

Horse milk is a valuable raw material and a very attractive alternative for scientific research to address the issue of cow milk (CM) allergy due to its protein profile. A decrease in immunoreactive properties can be achieved by thermal, enzymatic, and hydrolytic processing. Therefore, the aim of this study was to explore the possibility of reducing the immunoreactivity of horse milk proteins by microbial transglutaminase (TG) polymerization. To determine how TG linking alters immunoreactivity under simulated digestion of the examined milk, analyses were performed before, during, and after digestion. The dose-dependent (1, 10, and 100 U) effects of microbial TG on horse and cow milk were analyzed. A consecutive 3-stage digestion was simulated with salivary, gastric, and intestinal fluids. The effects of digestion were analyzed by SDS-PAGE, particle size analysis, and size-exclusion chromatography. Immunoreactivity was assessed using competitive ELISA (ß-lactoglobulin and α-casein) and immunodot (sera from 7 patients aged 3 to 13 years who are allergic to CM proteins). Horse milk contained almost half of the amount of total proteins in CM. The dose 1 U/g of total milk protein changed the immunoreactivity of both cow and horse milk. With increasing TG doses, α-casein immunoreactivity increased, and ß-lactoglobulin decreased. After total digestion, horse milk was characterized by 2.4-fold lower average IgE and 4.8-fold lower IgG reactivity than CM. We found that TG alters the IgE and IgG reactivity of CM after in vitro digestion. Horse milk was less reactive to IgE and IgG than was CM, with animal and patient sera. The effect of TG on immunoreactivity depends on enzyme quantity and milk protein type. The diet based on modified horse milk proteins could be an alternative for some patients with CM protein allergy; however, confirmation through clinical trials is needed.

Processed Food Additive Microbial Transglutaminase and Its Cross-Linked Gliadin Complexes Are Potential Public Health Concerns in Celiac Disease.

Microbial transglutaminase (mTG) is a survival factor for microbes, but yeasts, fungi, and plants also produce transglutaminase. mTG is a cross-linker that is heavily consumed as a protein glue in multiple processed food industries. According to the manufacturers' claims, microbial transglutaminase and its cross-linked products are safe, i.e., nonallergenic, nonimmunogenic, and nonpathogenic. The regulatory authorities declare it as "generally recognized as safe" for public users. However, scientific observations are accumulating concerning its undesirable effects on human health. Functionally, mTG imitates its family member, tissue transglutaminase, which is the autoantigen of celiac disease. Both these transglutaminases mediate cross-linked complexes, which are immunogenic in celiac patients. The enzyme enhances intestinal permeability, suppresses mechanical (mucus) and immunological (anti phagocytic) enteric protective barriers, stimulates luminal bacterial growth, and augments the uptake of gliadin peptide. mTG and gliadin molecules are cotranscytosed through the enterocytes and deposited subepithelially. Moreover, mucosal dendritic cell surface transglutaminase induces gliadin endocytosis, and the enzyme-treated wheat products are immunoreactive in CD patients. The present review summarizes and updates the potentially detrimental effects of mTG, aiming to stimulate scientific and regulatory debates on its safety, to protect the public from the enzyme's unwanted effects.

Greatest Hits-Innovative Technologies for High Throughput Identification of Bispecific Antibodies.

Recent years have shown a tremendous increase and diversification in antibody-based therapeutics with advances in production techniques and formats. The plethora of currently investigated bi- to multi-specific antibody architectures can be harnessed to elicit a broad variety of specific modes of actions in oncology and immunology, spanning from enhanced selectivity to effector cell recruitment, all of which cannot be addressed by monospecific antibodies. Despite continuously growing efforts and methodologies, the identification of an optimal bispecific antibody as the best possible combination of two parental monospecific binders, however, remains challenging, due to tedious cloning and production, often resulting in undesired extended development times and increased expenses. Although automated high throughput screening approaches have matured for pharmaceutical small molecule development, it was only recently that protein bioconjugation technologies have been developed for the facile generation of bispecific antibodies in a 'plug and play' manner. In this review, we provide an overview of the most relevant methodologies for bispecific screening purposes-the DuoBody concept, paired light chain single cell production approaches, Sortase A and Transglutaminase, the SpyTag/SpyCatcher system, and inteins-and elaborate on the benefits as well as drawbacks of the different technologies.

A recent update on the use of microbial transglutaminase for the generation of biotherapeutics.

The recent scientific progresses on the use of enzyme-mediated reactions in organic, non-aqueous and aqueous media have significantly supported the growing demand of new biotechnological and/or pharmacological products. Today, a plethora of microbial enzymes, used as biocatalysts, are available. Among these, microbial transglutaminases (MTGs) are broadly used for their ability to catalyse the formation of an isopeptide bond between the γ-amide group of glutamines and the ε-amino group of lysine. Due to their promiscuity towards primary amine-containing substrates and the more stringent specificity for glutamine-containing peptide sequences, several combined approaches can be tailored for different settings, making MTGs very attractive catalysts for generating protein-protein and protein small molecule's conjugates. The present review offers a recent update on the modifications attainable by MTG-catalysed bioreactions as reported between 2014 and 2019. In particular, we present a detailed and comparative overview on the MTG-based methods for proteins and antibodies engineering, with a particular outlook on the synthesis of homogeneous antibody-drug conjugates.

Expanding the Versatility of Microbial Transglutaminase Using α-Effect Nucleophiles as Noncanonical Substrates.

The substrate promiscuity of microbial transglutaminase (mTG) has been exploited in various applications in biotechnology, in particular for the attachment of alkyl amines to glutamine-containing peptides and proteins. Here, we expand the substrate repertoire to include hydrazines, hydrazides, and alkoxyamines, resulting in the formation of isopeptide bonds with varied susceptibilities to hydrolysis or exchange by mTG. Furthermore, we demonstrate that simple unsubstituted hydrazine and dihydrazides can be used to install reactive hydrazide handles onto the side chain of internal glutamine residues. The distinct hydrazide handles can be further coupled with carbonyls, including ortho-carbonylphenylboronic acids, to form site-specific and functional bioconjugates with tunable hydrolytic stability. The extension of the substrate scope of mTG beyond canonical amines thus substantially broadens the versatility of the enzyme, providing a new approach to facilitate novel applications.