Acyl Acceptor Research Articles

Effect of feedstocks and chain length of alcohols on whole-cell-catalyzed generation of alkyl esters

ABSTRACTThe dried biomass of a whole-cell catalyst was used for the generation of alkyl esters from cottonseed oil and acid oil with different alcohols (methanol to decanol) as acyl acceptors. The conversion of alkyl esters increased from methanol (12%) to pentanol (96%), followed by decreased from hexanol (86%) to decanol (64%) in case of acid oil. The extent of transesterification was significantly higher (P < 0.01) in case of acid oil, with most of the acyl acceptors as compared to cottonseed oil. The study demonstrates the use of dried biomass of fungus as catalyst and an effective feedstock such as acid oil, for the generation of various industrially important alkyl esters.

Synthesis of methyl esters and triacetin from macaw oil (Acrocomia aculeata) and methyl acetate over γ-alumina

Glycerol excess in the market and high cost of common feedstock have encouraged the investigations of new technologies for biodiesel production. In this context, the use of macaw oil (Acrocomia aculeata) for biodiesel production through interesterification and esterification reactions with methyl acetate as acyl acceptor was evaluated, using heterogeneous catalyst γ-alumina (γ-Al2O3). Experiments were performed at different reaction temperatures (225–300 °C), catalyst contents (0–10% relation to oil mass), oil to methyl acetate molar ratios (1:10–1:40) and reaction times in a batch reactor to determine the effect of these variables in FAME (fatty acid methyl esters) and triacetin production. Catalyst γ-Al2O3 improved FAME and triacetin contents in comparison with no catalyzed reaction. Reaction at 300 °C, with 2% of catalyst in relation to oil mass and molar ratio of 1:20, in 60 min provided the best results. The catalyst could be recovered and reused for at least 6 cycles without significant activity loss. The results allowed to point out that the process showed to be promising for industrial biodiesel production with no glycerol and was financial attractive, since it used a low cost feedstock.

Improved lipase-catalyzed methanolysis for biodiesel production by combining in-situ removal of by-product glycerol

During lipase-catalyzed biodiesel production, it has been demonstrated that by-product glycerol had negative effect on the catalytic performance of lipase especially in the repeated uses. This work presents a novel process by introducing dimethyl carbonate (DMC) into lipase-mediated methanolysis in due time, where DMC reacts with by-product glycerol and release methanol simultaneously, which not only realizes the in-situ removal of glycerol, but also achieves higher biodiesel yield at shorter reaction time compared to only use DMC as the acyl acceptor. At the optimized condition a biodiesel yield of 95.3% could be achieved with glycerol concentration only 0.049%. Improved reusability of lipase was given and there was a negligible loss in lipase’s activity after its being consecutively used for 6 batches. This process for in-situ removal of glycerol during lipase-catalyzed methanolysis was prospective for practical application in biodiesel industry.

Studies on the catalytic behavior of a membrane-bound lipolytic enzyme from the microalgae Nannochloropsis oceanica CCMP1779

The catalytic behavior of a membrane-bound lipolytic enzyme (MBL-Enzyme) from the microalgae Nannochloropsis oceanica CCMP1779 was investigated. The biocatalyst showed maximum activity at 50 °C and pH 7.0, and was stable at pH 7.0 and temperatures from 40 to 60 °C. Half-lives at 60 °C, 70 °C and 80 °C were found 866.38, 150.67 and 85.57 min respectively. Thermal deactivation energy was 68.87 kJ mol−1. The enzyme’s enthalpy (ΔΗ*), entropy (ΔS*) and Gibb’s free energy (ΔG*) were in the range of 65.86–66.27 kJ mol−1, 132.38–140.64 J mol−1 K−1 and 107.80–115.81 kJ mol−1, respectively. Among p-nitrophenyl esters of fatty acids tested, MBL-Enzyme exhibited the highest hydrolytic activity against p-nitrophenyl palmitate (pNPP). The Km and Vmax values were found 0.051 mM and of 0.054 mmole pNP mg protein−1 min−1, respectively with pNPP as substrate. The presence of Mn2+ increased lipolytic activity by 68.25%, while Fe3+ and Cu2+ ions had the strongest inhibitory effect. MBL-Enzyme was stable in the presence of water miscible (66% of the initial activity in ethanol) and water immiscible (71% of the initial activity in n-octane) solvents. Myristic acid was found to be the most efficient acyl donor in esterification reactions with ethanol. Methanol was the best acyl acceptor among the primary alcohols tested.

Interesterification of rapeseed oil catalysed by a low surface area tin (II) oxide heterogeneous catalyst

The interesterification of rapeseed oil was performed in a batch reactor using for the first time low surface area massive tin(II) oxide as heterogeneous catalyst and methyl acetate as acyl acceptor. The effect of reaction temperature, methyl acetate to oil molar ratio and catalyst loading on the performances of the process were investigated.Yields in fatty acid methyl esters (FAMEs) and triacetin (TA) up to 90% and 70% respectively, were achieved after 4 h of reaction time at 483 K in the presence of 0.69 mol of SnO per mole of rapeseed oil using a methyl acetate to oil molar ratio of 40.Quite interestingly, the catalyst performances improved when water was added to the reactions system. Moreover the same catalyst sample was used three consecutive times without observing any depletion of the catalytic activity.Collected results indicate that SnO is a promising heterogeneous catalyst for the interesterification of triglycerides with methyl acetate.

The Rising of Acylsugar Diversity: Metabolic Innovation in Tomato Trichomes through BAHD Enzyme Promiscuity and Pathway Evolution

In cultivated (Solanum lycopersicum) and wild tomatoes, glandular trichomes produce structurally diverse acylsugars. These insect protective metabolites are mixtures of aliphatic esters of sucrose and glucose, with acylchains varying in length from C2 to C11. We are analyzing acylsugar biosynthesis in the cultivated tomato, along with the biochemical and evolutionary mechanisms that generate acylsugar diversity across wild tomatoes (1, 2). Previous studies identified four BAHD family Acylsucrose Acyltransferase (ASAT) enzymes. These catalyze consecutive reactions from sucrose and acyl‐CoA substrates to produce the full set of cultivated tomato acylsucroses in vitro (3). Recently we identified a modified ‘upside down’ acylsugar pathway in the wild tomatoes S. pennellii and S. habrochaites that produces unusual types of acylsucroses, namely triacylsucroses with all three chains on the pyranose ring (P‐type) (4). This is in contrast with the furanose ring acylated acylsucroses (F‐type) produced in cultivated tomatoes and a variety of wild tomato species. Biochemical analysis demonstrates that the acyl acceptor substrate specificities were changed in two S. pennellii ASAT orthologous enzymes, which reversed their enzymatic activity order, leading to the ‘flipped pathway’. A phylogeny‐based structure‐function analysis of the ASAT2 and ASAT3 enzymes led to identification of a small number of amino acid changes that switch their activities between the F‐ and P‐ acylsucrose pathways (4, 5). Investigation of these pathways in other tomato species allowed inference of the evolutionary events giving rise to divergence of F‐ and P‐type acylsucroses across tomato species. This work demonstrates multiple evolutionary mechanisms in biochemical pathway evolution, including gene duplication and loss, amino acid substitution, and how the emergence of enzyme promiscuity restructured a specialized metabolic pathway and led to metabolic product innovation.Support or Funding InformationThis work was funded by National Science Foundation grants IOS‐1025636 and IOS‐PGRP‐1546617 to A.D.J. and R.L.L. Abigail Miller was supported by NSF REU grant DBI‐1358474 in the summer of 2014 and an American Society of Plant Biologists Summer Undergraduate Research Award during the summer of 2015. A.D.J. acknowledges support from the USDA National Institute of Food and Agriculture, Hatch project MICL‐02143.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enzymatic Production of Biodiesel: Strategies to Overcome Methanol Inactivation.

Lipase-catalyzed transesterification of triglycerides and alcohols to obtain biodiesel is an environmentally friendly and sustainable route for fuels production since, besides proceeding in mild reaction conditions, it allows for the use of low-cost feedstocks that contain water and free fatty acids, for example non-edible oils and waste oils. This review article reports recent advances in the field and focus in particular on a major issue in the enzymatic process, the inactivation of most lipases caused by methanol, the preferred acyl acceptor used for alcoholysis. The recent results about immobilization of enzymes on nano-materials and the use of whole-cell biocatalysts, as well as the use of cell-surface display technologies and metabolic engineering strategies for microbial production of biodiesel are described. It is discussed also insight into the effects of methanol on lipases obtained by modeling approaches and report on studies aimed at mining novel alcohol stable enzymes or at improving robustness in existing ones by protein engineering.

15(V/K) kinetic isotope effect and steady-state kinetic analysis for the transglutaminase 2 catalyzed deamidation and transamidation reactions

The Ca2+-dependent deamidation and transamidation activities of transglutaminase 2 (TG2) are important to numerous physiological and pathological processes. Herein, we have examined the steady-state kinetics and 15(V/K) kinetic isotope effects (KIEs) for the TG2-catalyzed deamidation and transamidation of N-Benzyloxycarbonyl-l-Glutaminylglycine (Z-Gln-Gly) using putrescine as the acyl acceptor substrate. Kinetic parameters determined from initial velocity plots are consistent with previously proposed mechanisms. Significant differences in the 15(V/K) KIEs on NH3 release determined for the deamidation (0.2%) and the transamidation (2.3%) of Z-Gln-Gly suggest the rate-limiting steps of TG2 active site acylation are dependent on the presence of the acyl acceptor. We propose a plausible mechanistic explanation where substrate-induced conformational changes may play a role in promoting catalysis.

Enzymatic production of biodiesel from insect fat using methyl acetate as an acyl acceptor: Optimization by using response surface methodology

Black soldier fly larvae (BSFL) are oleaginous insects that can assimilate organic waste for fat accumulation, and thus serve as an alternative feedstock for biodiesel production. In lipase-catalyzed transesterification, enzymes are deactivated by excess methanol. To address this obstacle, methyl acetate is suggested as an alternative acyl acceptor to methanol. In this study, methyl acetate was first used in the enzymatic production of biodiesel with BSFL as a triglyceride source. The interesterification of BSFL fat with methyl acetate was catalyzed using Novozym 435 as an efficient immobilized lipase. Response surface methodology was used to optimize the reaction and establish a reliable mathematical model for prediction. A maximum biodiesel yield of 96.97% was reached at a reaction time of 12 h, molar ratio of methyl acetate to fat of 14.64: 1, enzyme loading of 17.58%, and temperature of 39.5 °C. Under these optimal reaction conditions, Novozym 435 could be reused for up to 20 cycles without loss in enzyme activity. The properties of BSFL biodiesel were also investigated and all met the European standard EN 14214. This study indicates that the enzymatic interesterification of BSFL fat with methyl acetate is a promising and ecofriendly method for green fuel production.

Determinants of pH profile and acyl chain selectivity in lysosomal phospholipase A2 [S

Lysosomal phospholipase A2 (LPLA2) is characterized by broad substrate recognition, peak activity at acidic pH, and the transacylation of lipophilic alcohols, especially N-acetyl-sphingosine. Prior structural analysis of LPLA2 revealed the presence of an atypical acidic residue, Asp13, in the otherwise hydrophobic active site cleft. We hypothesized that Asp13 contributed to the pH profile and/or substrate preference of LPLA2 for unsaturated acyl chains. To test this hypothesis, we substituted Asp13 for alanine, cysteine, or phenylalanine; then, we monitored the formation of 1-O-acyl-N-acetylsphingosine to measure the hydrolysis of sn-1 versus sn-2 acyl groups on a variety of glycerophospholipids. Substitutions with Asp13 yielded significant enzyme activity at neutral pH (7.4) and perturbed the selectivity for mono- and double-unsaturated acyl chains. However, this position played no apparent role in selecting for either the acyl acceptor or the head group of the glycerophospholipid. Our modeling indicates that Asp13 and its substitutions contribute to the pH activity profile of LPLA2 and to acyl chain selectivity by forming part of a hydrophobic track occupied by the scissile acyl chain.

Evolution of a flipped pathway creates metabolic innovation in tomato trichomes through BAHD enzyme promiscuity

Plants produce hundreds of thousands of structurally diverse specialized metabolites via multistep biosynthetic networks, including compounds of ecological and therapeutic importance. These pathways are restricted to specific plant groups, and are excellent systems for understanding metabolic evolution. Tomato and other plants in the nightshade family synthesize protective acylated sugars in the tip cells of glandular trichomes on stems and leaves. We describe a metabolic innovation in wild tomato species that contributes to acylsucrose structural diversity. A small number of amino acid changes in two acylsucrose acyltransferases alter their acyl acceptor preferences, resulting in reversal of their order of reaction and increased product diversity. This study demonstrates how small numbers of amino acid changes in multiple pathway enzymes can lead to diversification of specialized metabolites in plants. It also highlights the power of a combined genetic, genomic and in vitro biochemical approach to identify the evolutionary mechanisms leading to metabolic novelty.

Phosphatidylserine decarboxylase CT699, lysophospholipid acyltransferase CT775, and acyl-ACP synthase CT776 provide membrane lipid diversity to Chlamydia trachomatis

De novo lipid synthesis and scavenging of fatty acids (FA) are processes essential for the formation of the membrane of the human pathogen Chlamydia trachomatis (C.t.). Host FA are assimilated via esterification by the bacterial acyl-acyl carrier protein (ACP) synthase AasC but inhibitors of the host acyl-CoA synthetase enymes ACSL also impaired growth of C.t. in human cells. In E. coli, activity of AasC was sensitive to triacsin C and rosiglitazone G. The absence of a triacsin C-insensitive pathway and the increased inhibition by rosiglitazone G confirmed the sensitivity of the bacterial acyl-ACP synthase to these drugs in infected human cells. We found no evidence that the human ACSL enzymes are required for lipid formation by C.t. The broad substrate specificity of acyltransferase CT775 provides C.t. with the capacity to incorporate straight-chain and bacterial specific branched-chain fatty acids. CT775 accepts both acyl-ACP and acyl-CoA as acyl donors and, 1- or 2-acyl isomers of lysophosphoplipids as acyl acceptors. The enzyme responsible for remodeling of human phosphatidylserine to bacterial phosphatidylethanolamine was identified as CT699. These findings provide evidence that the pathogen has the ability to extend the lipid diversity of its membrane.

Environmentally-Benign Dimethyl Carbonate-Mediated Production of Chemicals and Biofuels from Renewable Bio-Oil

Due to the increasing emission of carbon dioxide (CO2), the development of fuels and chemicals based on renewable resources has attracted much attention. Bio-oil, as a carbon rich material, has been considered as a feedstock for biodiesel production. In conventional methanol-mediated transesterification of bio-oil for biodiesel production, significant amounts of glycerol are being generated as a byproduct. In order to overcome these issues, dimethyl carbonate (DMC) has been recently used as an alternative acyl acceptor to avoid the generation of glycerol. DMC is an environmentally-benign chemical reagent and reactive solvent due to safety, health, and environmental benefits. Moreover, DMC can be produced from CO2. Co-production of biodiesel and chemicals such as glycerol carbonate is possible as the concept of zero-waste utilization of bio-oil. Value-added chemicals can be synthesized using DMC as a reagent. This paper provides a review on the physical and chemical properties of DMC as a solvent, as well as the production methods for DMC. DMC-mediated production of various chemicals and fuels in both chemical and enzymatic processes are discussed together with their pros and cons.

Functional Characterization of a Hydroxyacid/Alcohol Hydroxycinnamoyl Transferase Produced by the Liverwort Marchantia emarginata.

The aerial organs of most terrestrial plants are covered by a hydrophobic protective cuticle. The main constituent of the cuticle is the lipid polyester cutin, which is composed of aliphatic and aromatic domains. The aliphatic component is a polyester between fatty acid/alcohol and hydroxycinnamoyl acid. The BAHD/HxxxD family enzymes are central to the synthesis of these polyesters. The nature of this class of enzymes in bryophytes has not been explored to date. Here, a gene encoding a fatty ω-hydroxyacid/fatty alcohol hydroxycinnamoyl transferase (HFT) has been isolated from the liverwort Marchantia emarginata and has been functionally characterized. Experiments based on recombinant protein showed that the enzyme uses ω-hydroxy fatty acids or primary alcohols as its acyl acceptor and various hydroxycinnamoyl-CoAs—preferentially feruloyl-CoA and caffeoyl-CoA—as acyl donors at least in vitro. The transient expression of a MeHFT-GFP fusion transgene in the Nicotiana benthamiana leaf demonstrated that MeHFT is directed to the cytoplasm, suggesting that the feruloylation of cutin monomers takes place there.

A Flexible Polyphosphate‐Driven Regeneration System for Coenzyme A Dependent Catalysis

AbstractCoenzyme A (CoA) is a common cofactor in biochemical reactions, and CoA‐dependent enzymes catalyze essential steps in anabolism and catabolism. This complex molecule also plays an important role in the synthesis of many high‐value products, such as synthetic antibiotics, vitamins, pheromones, and biopolymers. Nevertheless, the synthetic potential for biocatalytic processes cannot be fully exploited owing to the lack of an efficient regeneration system. Here, we report an acyl‐CoA regeneration system with integrated adenosine triphosphate (ATP) regeneration that is based on inexpensive polyphosphate as the single energy source. In the four‐enzyme cascade, two cofactors, acyl‐CoA and ATP, are each regenerated up to 2000 times. The applicability for different acyl donors and acceptors is shown by HPLC analysis. Owing to its flexibility toward virtually all relevant substrates, the system has the potential to make CoA‐dependent reactions more accessible for chemical synthesis in vitro.

Highly Efficient Deacidification of High-Acid Rice Bran Oil Using Methanol as a Novel Acyl Acceptor.

A highly efficient process for reducing the fatty acid (FA) content of high-acid rice bran oil (RBO) was developed by immobilized partial glycerides-selective lipase SMG1-F278N-catalyzed esterification/transesterification using methanol as a novel acyl acceptor. Molecular docking simulation indicated that methanol was much closer to the catalytic serine (Ser-171) compared with ethanol and glycerol, which might be one of the reasons for its high efficiency in the deacidification of high-acid RBO. Additionally, the reaction parameters were optimized to minimize the FA content of high-acid RBO. Under the optimal conditions (substrate molar ratio of methanol to FAs of 1.8:1, enzyme loading of 40U/g, and at 30°C), FA content decreased from 25.14 to 0.03% after 6h of reaction. Immobilized SMG1-F278N exhibited excellent methanol tolerance and retained almost 100% of its initial activity after being used for ten batches. After purification by molecular distillation, the final product contained 97.86% triacylglycerol, 2.10% diacylglycerol, and 0.04% FA. The acid value of the final product was 0.09mg KOH/g, which reached the grade one standard of edible oil. Overall, methanol was a superior acyl acceptor for the deacidification of high-acid RBO and the high reusability of immobilized SMG1-F278N indicates an economically attractive process.

Phenylpropanoid profiling reveals a class of hydroxycinnamoyl glucaric acid conjugates in Isatis tinctoria leaves

The brassicaceous herb, Isatis tinctoria, is an ancient medicinal plant whose rosette leaf extracts have anti-inflammatory and anti-allergic activity. Brassicaceae are known to accumulate a variety of phenylpropanoids in their rosette leaves acting as antioxidants and a UV-B shield, and these compounds often have pharmacological potential. Nevertheless, knowledge about the phenylpropanoid content of I. tinctoria leaves remains limited to the characterization of a number of flavonoids. In this research, we profiled the methanol extracts of I. tinctoria fresh leaf extracts by liquid chromatography – mass spectrometry (LC-MS) and focused on the phenylpropanoid derivatives. We report the structural characterization of 99 compounds including 18 flavonoids, 21 mono- or oligolignols, 2 benzenoids, and a wide spectrum of 58 hydroxycinnamic acid esters. Besides the sinapate esters of malate, glucose and gentiobiose, which are typical of brassicaceous plants, these conjugates comprised a large variety of glucaric acid esters that have not previously been reported in plants. Feeding with 13C6-glucaric acid showed that glucaric acid is an acyl acceptor of an as yet unknown acyltransferase activity in I. tinctoria rosette leaves. The large amount of hydroxycinnamic acid derivatives changes radically our view of the woad metabolite profile and potentially contributes to the pharmacological activity of I. tinctoria leaf extracts.

The Arabidopsis thaliana lysophospholipid acyltransferase At1g78690p acylates lysocardiolipins

The Arabidopsis thaliana lysophospholipid acyltransferase At1g78690 acylates a variety of lysophospholipids such as lyso phosphatidylglycerol, lyso phosphatidylethanolamine and lyso phosphatidylserine. Despite di-acylate phosphatidylglycerol being a substrate, overexpression of At1g78690 in Escherichia coli leads to the accumulation of acyl-PG. Here we show that cardiolipin also accumulates in cells overexpressing At1g78690. To help try and explain this observation, we show, using a liquid chromatography mass spectrometry (LC-MS) based assay, that At1g78690 utilizes both mono- and di-lyso cardiolipin as an acyl acceptor. Because At1g78690 shares high homology (∼40%) with the cardiolipin remodeling enzyme tafazzin, we also tested whether At1g78690 was able to catalyze a tafazzin-like transacylation reaction. Di-linoleoyl phosphatidylcholine was used as the acyl donor and mono-lyso cardiolipin was used as the acyl acceptor in a reaction and the reaction was monitored by LC-MS. No transfer of the linoleoyl chains was detected in an At1g78690 dependent manner suggesting that, despite the strong homology, these enzymes catalyze unique reactions.

Site-Specific Conjugation to Native and Engineered Lysines in Human Immunoglobulins by Microbial Transglutaminase.

The use of microbial transglutaminase (MTG) to produce site-specific antibody-drug conjugates (ADCs) has thus far focused on the transamidation of engineered acyl donor glutamine residues in an antibody based on the hypothesis that the lower specificity of MTG for acyl acceptor lysines may result in the transamidation of multiple native lysine residues, thereby yielding heterogeneous products. We investigated the utilization of native IgG lysines as acyl acceptor sites for glutamine-based acyl donor substrates. Of the approximately 80 lysines in multiple recombinant IgG monoclonal antibodies (mAbs), none were transamidated. Because recombinant mAbs lack the C-terminal Lys447 due to cleavage by carboxypeptidase B in the production cell host, we explored whether blocking the cleavage of Lys447 by the addition of a C-terminal amino acid could result in transamidation of Lys447 by a variety of acyl donor substrates. MTG efficiently transamidated Lys447 in the presence of any nonacidic, nonproline amino acid residue at position 448. Lysine scanning mutagenesis throughout the antibody further revealed several transamidation sites in both the heavy- and light-chain constant regions. Additionally, scanning mutagenesis of the hinge region in a Fab' fragment revealed sites of transamidation that were not reactive in the context of the full-length mAb. Here, we demonstrate the utility of single lysine substitutions and the C-terminal Lys447 for engineering efficient acyl acceptor sites suitable for site-specific conjugation to a range of glutamine-based acyl donor substrates.

Abstract 65: RESPECT (REsidue-SPEcific Conjugation Technology): A platform technology utilizing native cysteine and lysine residues for the generation of homogeneous antibody-drug conjugates

Abstract The random conjugation of toxins, dyes, peptides, or other payloads to monoclonal antibodies often targets free thiol groups generated by partial reduction methods or lysine residues using succinimide- or isothiocyanate-based chemistry. There remains a need for conjugation technologies targeting specific amino acid residues as a way to produce a homogeneous antibody-drug conjugate (ADC) product with a defined drug-to-antibody ratio (DAR). Our REsidue-SPEcific Conjugation Technology (RESPECT) utilizes two methods by which payloads can be conjugated to specific residues in an antibody. Our cysteine-specific conjugation method exploits a unique intrachain disulfide bond in the light chain of rabbit antibodies between residues 80 and 171 of the variable and constant domains, respectively. Our humanization strategy allows retention of the cysteine at position 80 with a free thiol group that is both amenable for residue-specific conjugation and compatible with optimal antibody biophysical properties including antigen binding and structural stability. This platform has been optimized via antibody engineering strategy stems from in silico modeling, extensive mutagenesis, and crystallographic studies, which have allowed defining the contribution of neighboring residues to the retention of a reactive thiol group as well as the desired humanized antibody’s properties. Our C-terminal lysine-specific linkage method employs the transglutaminase enzyme that catalyzes the formation of a stable isopeptide bond between the γ-carboxyamide group (acyl donor) of a glutamine and the ϵ-amino group (acyl acceptor) of a lysine. While we found no acyl acceptor sites in recombinant wild-type IgG, all antibodies investigated lacked the C-terminal Lys447 due to cleavage by carboxypeptidase B in the antibody production cell line. Blocking the cleavage of Lys447 by addition of a C-terminal amino acid resulted in transamidation of Lys447 by a variety of acyl donor substrates in the presence of any non-acidic, non-proline amino acid residue at position 448. Antibody-drug conjugates (ADCs) prepared using our RESPECT technology targeting the tumor associated-mesothelin protein produced uniform drug-to-antibody ratios (DAR) and were shown to be highly potent and specific in vitro and effective in vivo in reduction of tumor growth in a highly aggressive mesothelin-expressing xenograft tumor model. Citation Format: Earl Albone, Jared Spidel, Xin Cheng, Young Chul Park, Sara Jacob, Arielle Verdi, Andrew Milinichik, Ben Vaessen, J. Bradford Kline, Luigi Grasso. RESPECT (REsidue-SPEcific Conjugation Technology): A platform technology utilizing native cysteine and lysine residues for the generation of homogeneous antibody-drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 65. doi:10.1158/1538-7445.AM2017-65