Acyl Acceptor Research Articles

Performance of Liquid Eversa on Fatty Acid Ethyl Esters Production by Simultaneous Esterification/Transesterification of Low-to-High Acidity Feedstocks

Liquid Eversa was evaluated in hydrolysis of acylglycerols from soybean oil deodorizer distillate (SODD), as well as simultaneous esterification/transesterification of SODD with low-to-high free fatty acids (FFAs) content using ethanol as acyl acceptor. Hydrolysis of SODD at mild temperature (37 °C) and without pH control (water:SODD mass ratio of 4:1) increased its FFAs content from 17.2 wt.% to 72.5 wt.% after 48 h reaction. A cold saponification of SODD allowed a saponification phase (SODD-SP) to be recovered with 93 wt.% saponification index and 2.25 wt.% FFAs content, which was used to find the experimental conditions for simultaneous esterification/transesterification reactions by experimental design. Temperature of 35 °C, enzyme concentration of 8.36 wt.%, and molar ratio of 3.64:1 (ethanol:SODD-SP) were found as the best conditions for fatty acid ethyl esters (FAEEs) production from SODD-SP (86.56 wt.% ester yield after 23 h reaction). Under the same reaction conditions, crude SODD (17.2 wt.% FFAs) and hydrolyzed SODD (72.5 wt.% FFAs) yielded products containing around 80 wt.% FAEEs. Caustic treatment could increase the ester content to around 90 wt.% and reduce the FFAs content to less than 1 wt.%. Our results show the good performance of liquid Eversa in aqueous (hydrolysis reactions) and organic (esterification/transesterification reactions) media.

Hemp (Cannabis sativa) seed oilcake as a promising by-product for developing protein-based films: Effect of transglutaminase-induced crosslinking

Protein concentrates were obtained from hemp seed oilcakes (OCs) and investigated as potential waste-derived source of biodegradable films at different protein and glycerol concentrations and at different pH values. These studies indicated that hemp protein (HP) film forming solutions gave rise to higher performance films when cast at pH 12 in the presence of 50% glycerol (w/w protein) used as plasticizer. Since HPs were demonstrated to act as both acyl donor and acceptor substrates of microbial transglutaminase (mTGase), they have been used as raw material to obtain films also after enzyme treatment. Film morphological characterization demonstrated that mTGase treatment was effective to produce more homogeneous and smoother films, influencing in turn positively their properties. In fact, mTGase-crosslinked films were shown to be more resistant, still flexible and exhibited a higher heat-sealing strength. In addition, the enzymatic treatment of HPs originated bio-plastics with a higher gas permeability and a greater hydrophobicity. These findings suggest the possibility to exploit the mTGase-crosslinked proteins derived from hemp OC as a promising source to produce bio-based materials useful as packaging systems for protecting food products from physical contamination and, thus, for extending their shelf-life.

Biodiesel production and comparison using commercial lipase and chemical catalyst from Cassia auriculata oil

In this present investigation, Cassia auriculata was explored as a feedstock for production of biodiesel. Transesterification reaction was performed by both enzyme (lipase) and chemical (potassium hydroxide) catalyst with diverse acyl acceptors such as methanol, ethanol, propanol, n-propanol, butanol, n- butanol, and finally their biodiesel yield were also recorded. Process optimization was performed by both one factor at a time method and response surface method. The maximal biodiesel yield of 92% (weight/weight) was obtained at the following optimal conditions: Oil:Methanol molar ratio of 1:6 (moles/moles), the lipase concentration of 2% (weight/weight), at 35 ​°C and 120 ​min. The highest biodiesel yield from Cassia auriculata oil was occurred with excess methanol that aids the equilibrium shift in the forward direction. The kinetics of the transesterification reaction was investigated under optimal conditions and the activation energy was found to be 14.91 ​kJ/mol. Simultaneously Gas Chromatography – Mass Spectroscopy was also carried out for the biodiesel produced from Cassia auriculata and the same has been reported. The GC analysis declares the existence of fatty acid esters like hexadecanoic acid methyl ester, methyl stearate and the peak with retention time 12.8 ​min signifies the evidence of hexadecanoic acid methyl ester with 28% of yield content. This investigation also evaluated the biodiesel quality produced from lipase-transesterified Cassia auriculata oil based on fuel properties. Biodiesel properties Flash Point (FC), Pour Point (PP) and kinematic viscosity were compared with American (ASTM 6751) and European (EN 14214) Standards. Cassia auriculata oil had PP 6.7 ​°C and Kinematic viscosity (813 ​kg/m3) that agreed with both the standards. Thus this study showed that Cassia auriculata oil could be a better fuel alternative with further improvement of fuel properties.

Design of functional glycerol-based deep eutectic solvents as reaction media for enzymatic deacidification of high-acid rice bran oil

The effectiveness of deacidification technologies for high-acid rice bran oil (HRBO) are still one of the main challenges in the industrialization process of rice bran oil (RBO). A novel function of glycerol-based deep eutectic solvents (DESs) as an acyl acceptor in combination with lipases for enzymatic esterification of deacidification of HRBO was described in this study. The maximum deacidification efficiency of 92.38% was obtained under the optimized reaction conditions. Molecular docking simulation indicated that the minimum binding energy of Novozym 435 was lower than those of Lipozyme TL IM and Lipozyme RM IM, which might be one of the underlying mechanisms for its better performance. Additionally, γ-oryzanols, tocopherols and tocotrienols were retained in deacidified RBO. We are convinced that the reusability of biocatalyst and DESs systems provide an alternative strategy to design a facile, environmentally-friendliness DESs towards economic feasibility of applications and processes for oil-refined industries.

Rice bran oil deacidification by immobilized Aspergillus Niger lipase catalyzed esterification with D-isoascorbic acid

The main challenge associated with the upgrading of crude rice bran oil (RBO) rests with their high content of free fatty acids (FFA). In this study, we describe a green and sustainable process for the deacidification of high-acid RBO using d-isoascorbic acid as a novel acyl acceptor in combination with a newly prepared immobilized Aspergillus Niger lipase (ANL). The process contributed a high deacidification efficiency of 97.53 % with a desirable d-isoascorbic acid ester (DIAE) content of 36.10 %. The immobilized ANL could be used consecutively for at least 10 batches with only 7.86 % of activity loss. Scale-up reaction was implemented to verify the amenability of the procedure, and a low acid value of 1.46 mg KOH/g conformed to the quality criteria of edible RBO was then obtained. Physicochemical indices analysis indicated that this procedure was amicable to bioactive phytochemicals in RBO. In addition, the Rancimat test suggested that this process greatly improved the shelf life of deacidified RBO due to the formation of DIAE. This is the first time that d-isoascorbic acid has been used in the refining of RBO. Overall, we report an economically and efficiently viable process for the upgrading of high-acid RBO.

Stabilization and operational selectivity alteration of Lipozyme 435 by its coating with polyethyleneimine: Comparison of the biocatalyst performance in the synthesis of xylose fatty esters

Differently modified Lipozyme 435 (L435) (immobilized lipase B from Candida antarctica) preparations were used as biocatalysts in the esterification reaction to synthesize sugar fatty acid esters (SFAEs) from xylose (acyl acceptor) and lauric/palmitic acids (acyl donors) in methyl ethyl ketone (MEK) solvent. The L435 treatment with polyethyleneimine (PEI) (2; 25; and 750 KDa) prevented the enzyme leakage in the crude sugar ester reaction product. The 2 KDa PEI coating of this enzyme preparation produced the highest enzyme stability in MEK, buffer solutions (pHs 5 and 7), and methanol aqueous phosphate buffer at pH 7. Using an excess of the acyl donor (1:5 xylose: fatty acid molar ratio), high xylose conversions (70–84%) were obtained after 24 h-reaction using both, non-modified and PEI (2 KDa) coated L435, but the PEI treated biocatalyst afforded a higher xylose modification degree. After 5 reuse cycles with the L435 coated with PEI 2 KDa, the xylose conversions only decreased 10%, while with the non-treated biocatalyst they decreased by 37%. The formation of SFAEs was confirmed by mass spectrometry, which showed the presence of xylose mono-, di-, and triesters. They exhibited emulsion capacities close to that of a commercial sucrose monolaurate.

Non-catalytic and glycerol-free biodiesel production from rice bran oil fatty acid distillate in a microreactor

Obtained from rice bran oil refining process, rice bran oil fatty acid distillate (RBOFAD) is a low-value, non-edible, and unwanted by-product, which can be used as a promising alternative raw material for biodiesel production. However, the conventional biodiesel process cannot handle this material due to the high content of free fatty acid (FFA). A novel supercritical process for biodiesel synthesis using dimethyl carbonate (DMC) as acyl acceptor in a microreactor was proposed in this work for the raw material with high FFA. High quality of biodiesel and value-added by-product (glyoxal) were obtained in our process. In this process, the biodiesel content of 80.9% was achieved for the case of RBOFAD as feedstock, compared to only 43.6% for the case of refined rice bran oil as feedstock. The influence of operating conditions on biodiesel and glyoxal content including reaction temperature, residence time, and DMC-to-RBOFAD molar ratio was investigated and optimized via response surface methodology. The biodiesel content of 97.1% was achieved at the optimal conditions (reaction temperature of 360 °C, residence time of 35 min, and RBOFAD-to-oil molar ratio of 11:1). The required pressure and the amount of DMC were significantly reduced compared to other processes. Most of the biodiesel properties met the international standards except for some impurities (mono- and diglycerides). These results provided the new insight for the development of biodiesel production from low-grade feedstocks.

Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli.

Bacteria resist to the turgor pressure of the cytoplasm through a net-like macromolecule, the peptidoglycan, made of glycan strands connected via peptides cross-linked by penicillin-binding proteins (PBPs). We recently reported the emergence of β-lactam resistance resulting from a bypass of PBPs by the YcbB L,D-transpeptidase (LdtD), which form chemically distinct 3→3 cross-links compared to 4→3 formed by PBPs. Here we show that peptidoglycan expansion requires controlled hydrolysis of cross-links and identify among eight endopeptidase paralogues the minimum enzyme complements essential for bacterial growth with 4→3 (MepM) and 3→3 (MepM and MepK) cross-links. Purified Mep endopeptidases unexpectedly displayed a 4→3 and 3→3 dual specificity implying recognition of a common motif in the two cross-link types. Uncoupling of the polymerization of glycan chains from the 4→3 cross-linking reaction was found to facilitate the bypass of PBPs by YcbB. These results illustrate the plasticity of the peptidoglycan polymerization machinery in response to the selective pressure of β-lactams.

Industrial biotransformations catalyzed by microbial lipases: screening platform and commercial aspects.

The successfulness of a lipase-catalyzed industrial process depends on a proper lipase selection. In this work, an alternative screening platform for industrially important biotransformations catalyzed by microbial lipases was proposed. Thus, the reactivity of sixty lipase activities from spore-forming microorganisms towards hydrolytic and transesterification reactions by using p-nitrophenyl palmitate as a chromogenic acyl donor substrate was explored. Only three biocatalysts were capable of catalyzing all reactions tested. Fourteen biocatalysts did not show hydrolytic activity at all; however, they displayed transesterification activities using ethanol, starch, low-methoxyl (LM) pectin, high-methoxyl (HM) pectin, or vitamin C as acyl acceptors. Using heat-treated biocatalysts, hydrolytic activities were not highly correlated with the corresponding transesterification activities using ethanol (r = -0.058, p = 0.660), starch (r = 0.431, p = 0.001), LM pectin (r = -0.010, p = 0.938), HM pectin (r = 0.167, p = 0.202), and vitamin C (r = -0.048, p = 0.716) as acyl acceptor. In addition, to the best of our knowledge, several transesterification activities produced from microorganisms of the genus Bacillus, Brevibacillus, Lysinibacillus, Geobacillus, or Sporosarcina were reported for first time. Finally, the global lipase market was presented and segmented by date, application, geography and player highlighting the commercial contribution of microbial lipases.

Molecular and structural characterization of agmatine coumaroyltransferase in Triticeae, the key regulator of hydroxycinnamic acid amide accumulation

Hydroxycinnamic acid amides (HCAAs) are involved in stress-induced defense in many plant species. Barley accumulates high concentrations of HCAAs irrespective of exogenous stressors, while other major cereals such as wheat and rice accumulate relatively low levels of HCAAs in intact tissues. The primary HCAA species in barley are biosynthesized by agmatine p-coumaroyltransferase (ACT), an N-acyltransferase of the BAHD superfamily. However, the molecular basis underlying barley's uniquely high HCAA accumulation has not been elucidated, and information regarding the structural details of BAHD N-acyltransferases is limited. Hence, we aimed to investigate the ACTs of family Poaceae. We isolated ACT (-like) genes, including those previously undescribed, and investigated their enzymatic and genetic features. All the identified enzymes belonged to clade IVa of the BAHD superfamily. The barley and wheat ACTs were further categorized, based on catalytic properties and primary structures, into ACT1 and ACT2 groups, the encoding loci of which are neighbors on the same chromosome. While all ACTs exhibited similar Km values for CoA-thioesters (acyl-group donors), members of the ACT1 group showed a distinctly higher affinity for agmatine (acyl-acceptor). Among the ACTs tested, an ACT isozyme in barley (HvACT1-1) showed the highest catalytic efficiency and transcript level, indicating that ACT regulates high-level HCAA accumulation in barley. For further enzymatic characterization of the ACTs, we crystalized wheat ACT2 (TaACT2) and determined its structure at 2.3 Å resolution. Structural alignment of TaACT2 and HvACT1-1 showed that the architectures of the substrate binding pockets were well conserved. However, the structure of a loop located at the entrance to acyl-acceptor binding site may be more flexible in TaACT2, which could be responsible for the lower affinity of TaACT2 to agmatine. Mutations of HvACT1-1 at Glu372 and Asp374 within one of the clade-IV specific motifs facing the deduced acyl-acceptor binding pocket caused significant catalytic deterioration toward agmatine both in Km and kcat, suggesting their key roles in acyl acceptor binding by the clade-IV enzymes. This study elucidated the molecular basis of how plants accumulate defensive specialized metabolites and provided insights into developing efficient and eco-friendly agricultural methods.

Biodiesel production with enzymatic technology: progress and perspectives

AbstractRapid changes in the global climate resulting from anthropogenic activities and greenhouse gas emissions from fossil resources are encouraging commercial and academic research to seek out new routes for the production of sustainable fuels. Biodiesel, as a renewable fuel, has long been recognized as one of the solutions to meet energy demand in a climate‐constrained world. Although, several routes have been devised for biodiesel production, the enzymatic route has attracted substantial research interest. Here we discuss enzymatic esterification / transesterification technology and its contribution to the green synthesis of biodiesel. This review provides a comprehensive assessment of the fundamentals of enzymatic reaction, such as the impact of different reaction components (lipase, acyl acceptor, substrate, and reaction media) on the process parameters, along with recent developments in improving enzymatic biodiesel production, such as different lipases, combinations of lipases, and two‐step methods. Kinetics and the mechanism of enzymatic reaction when lipase is used as a catalyst are also explained in greater detail. Finally, the opportunities and challenges for the development of potentially sustainable and eco‐friendly enzymatic biodiesel technology are discussed. © 2021 Society of Industrial Chemistry and John Wiley & Sons Ltd.

One-pot fungal biomass-to-biodiesel process: Influence of the molar ratio and the concentration of acid heterogenous catalyst on reaction yield and costs

This work evaluates a microbial-based biodiesel production process through simultaneous esterification and transesterification of the lipid-rich fungal biomass using ethanol as both extractor and acyl acceptor. Two of the most influential parameters were evaluated, the molar ratio of ethanol to the oil, and the concentration of a heterogeneous acid catalyst (H3PMo/Al2O3), with responses in terms of conversion to ethyl esters and an estimated cost of the process variables. Fungal biomass of Mucor circinelloides was cultivated in sugarcane molasses media and the obtained oleaginous cells were used as the source of acylglycerols and free fatty acids for the trans/esterification reactions in a pressurized stainless-steel reactor at 200 °C for 6 h. Our results demonstrate that the effects of the two factors analyzed were significant, with an indication that increases in the molar ratio and catalyst favor the reaction yield. In the reaction system, a molar ratio of 120:1 (ethanol: oil) and 15 wt% of catalyst yielded 96.6% of ester content, which meets the minimum limit established by the international standards. Production costs were estimated in function of ethanol and catalyst price and indicated the selected parameters reflected the adequate configuration to reach the stablished minimum ester content.

L-tryptophan-histidine synthesis by Pseudomonas serine peptidase, an amino acid ester hydrolase of the peptidase family S9

Pseudomonas sp. KM1 produces an amino acid ester hydrolase (KM1AEH) that catalyzes peptide bond formation by acting on carboxylic ester bonds. The KM1AEH gene was cloned from genomic DNA and expressed in Escherichia coli. The recombinant enzyme (rKM1AEH) was purified, and gel filtration showed that it is a 68 kDa monomeric protein. rKM1AEH can synthesize the vasoactive dipeptide tryptophan-histidine from tryptophan methyl ester and histidine as acyl donor and acceptor, respectively. The enzyme showed maximum activity at pH 9.5 and 45 °C and was specifically inhibited by silver (Ag+). Mutation of the catalytic Ser459 residue in the active site of rKM1AEH with Ala, Cys, or Thr eliminated all catalytic activity. The enzyme is a novel ester hydrolase that belongs to the peptidase family S9 based on the phylogenetic analysis.

In vitro inhibition of shikimate hydroxycinnamoyltransferase by acibenzolar acid, the first metabolite of the plant defence inducer acibenzolar-S-methyl

Acibenzolar acid, the first metabolite formed in planta from the defence inducer acibenzolar-S-methyl (ASM), has been shown to be an inhibitor of the enzyme shikimate hydroxycinnamoyltransferase (HST), extracted from grapevine or tobacco cell suspension cultures. Using a purified recombinant Arabidopsis thaliana HST, the inhibition was found to be competitive, acibenzolar acid binding reversibly to the shikimate binding site of the HST:p-coumaroyl-CoA complex, with a Ki value of 250 μM. The other hydroxycinnamoyltransferases tested in the course of this study, using either hydroxypalmitic acid, putrescine, tyramine, or quinic acid as acyl acceptors were not, or only slightly, inhibited by acibenzolar acid. To understand the specificity of the interaction of acibenzolar acid with HST, we analyzed the structure-activity relationship of a series of benzoic or acibenzolar acid analogues, tested either as AtHST substrates or as inhibitors. This analysis confirmed previously published data on the substrate flexibility of HST and demonstrated that both the carboxyl group and the thiadiazole moiety of acibenzolar acid are playing an important role in the interaction with the shikimate binding site. Acibenzolar acid, which cannot form an ester bond with p-coumaric acid, was however a less potent inhibitor than protocatechuic or 3-hydroxybenzoic acids, which are used as acyl acceptors by HST. Our results show that the interaction of acibenzolar acid with HST, which is probably directly linked to the substrate promiscuity of HST, is unlikely to play a direct role in the defence-inducing properties of ASM in plants.

Versatility in acyltransferase activity completes chicoric acid biosynthesis in purple coneflower

Purple coneflower (Echinacea purpurea (L.) Moench) is a popular native North American herbal plant. Its major bioactive compound, chicoric acid, is reported to have various potential physiological functions, but little is known about its biosynthesis. Here, taking an activity-guided approach, we identify two cytosolic BAHD acyltransferases that form two intermediates, caftaric acid and chlorogenic acid. Surprisingly, a unique serine carboxypeptidase-like acyltransferase uses chlorogenic acid as its acyl donor and caftaric acid as its acyl acceptor to produce chicoric acid in vacuoles, which has evolved its acyl donor specificity from the better-known 1-O-β-D-glucose esters typical for this specific type of acyltransferase to chlorogenic acid. This unusual pathway seems unique to Echinacea species suggesting convergent evolution of chicoric acid biosynthesis. Using these identified acyltransferases, we have reconstituted chicoric acid biosynthesis in tobacco. Our results emphasize the flexibility of acyltransferases and their roles in the evolution of specialized metabolism in plants.

RNAi Modulation of Chlorogenic Acid and Lignin Deposition in Nicotiana tabacum and Insufficient Compensatory Metabolic Cross-Talk.

Chlorogenic acid (CGA) and guaiacyl/syringyl (G/S) lignin formation involves hydroxycinnamoyl ester intermediacy, the latter formed via hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase (HCT) and hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase (HQT) activities. HQT and HCT RNAi silencing of a commercial tobacco (Nicotiana tabacum) K326 line was examined herein. NtHQT gene silencing gave relatively normal plant phenotypes, with CGA levels reduced (down to 1% of wild type) with no effects on lignin. RNAi NtHCT silencing had markedly adverse phenotypes (e.g., stunted, multiple stems, delayed flowering, with senescence delayed by several months). Lignin contents were partially lowered, with a small increase in cleavable p-hydroxyphenyl (H) monomers; those plants had no detectable CGA level differences relative to wild type. In vitro NtHCT kinetic parameters revealed preferential p-coumaroyl CoA and shikimate esterification, as compared to other structurally related potential acyl group donors and acceptors. In the presence of coenzyme A, NtHCT catalyzed the reverse reaction. Site-directed mutagenesis of NtHCT (His153Ala) abolished enzymatic activity. NtHQT, by comparison, catalyzed preferential conversion of p-coumaroyl CoA and quinic acid to form p-coumaroyl quinate, the presumed CGA precursor. In sum, metabolic pathways to CGA and lignins appear to be fully independent, and previous conflicting reports of substrate versatilities and metabolic cross-talk are resolved.

Function of the HYDROXYCINNAMOYL-CoA:SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE is evolutionarily conserved in embryophytes.

The plant phenylpropanoid pathway generates a major class of specialized metabolites and precursors of essential extracellular polymers that initially appeared upon plant terrestrialization. Despite its evolutionary significance, little is known about the complexity and function of this major metabolic pathway in extant bryophytes, which represent the non-vascular stage of embryophyte evolution. Here, we report that the HYDROXYCINNAMOYL-CoA:SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE (HCT) gene, which plays a critical function in the phenylpropanoid pathway during seed plant development, is functionally conserved in Physcomitrium patens (Physcomitrella), in the moss lineage of bryophytes. Phylogenetic analysis indicates that bona fide HCT function emerged in the progenitor of embryophytes. In vitro enzyme assays, moss phenolic pathway reconstitution in yeast and in planta gene inactivation coupled to targeted metabolic profiling, collectively indicate that P. patens HCT (PpHCT), similar to tracheophyte HCT orthologs, uses shikimate as a native acyl acceptor to produce a p-coumaroyl-5-O-shikimate intermediate. Phenotypic and metabolic analyses of loss-of-function mutants show that PpHCT is necessary for the production of caffeate derivatives, including previously reported caffeoyl-threonate esters, and for the formation of an intact cuticle. Deep conservation of HCT function in embryophytes is further suggested by the ability of HCT genes from P. patens and the liverwort Marchantia polymorpha to complement an Arabidopsis thaliana CRISPR/Cas9 hct mutant, and by the presence of phenolic esters of shikimate in representative species of the three bryophyte lineages.

Structural and Biochemical Insights Into Two BAHD Acyltransferases (AtSHT and AtSDT) Involved in Phenolamide Biosynthesis.

Phenolamides represent one of the largest classes of plant-specialized secondary metabolites and function in diverse physiological processes, including defense responses and development. The biosynthesis of phenolamides requires the BAHD-family acyltransferases, which transfer acyl-groups from different acyl-donors specifically to amines, the acyl-group acceptors. However, the mechanisms of substrate specificity and multisite-acylation of the BAHD-family acyltransferases remain poorly understood. In this study, we provide a structural and biochemical analysis of AtSHT and AtSDT, two representative BAHD-family members that catalyze the multisite acylation of spermidine but show different product profiles. By determining the structures of AtSHT and AtSDT and using structure-based mutagenesis, we identified the residues important for substrate recognition in AtSHT and AtSDT and hypothesized that the acyl acceptor spermidine might adopt a free-rotating conformation in AtSHT, which can undergo mono-, di-, or tri-acylation; while the spermidine molecule in AtSDT might adopt a linear conformation, which only allows mono- or di-acylation to take place. In addition, through sequence similarity network (SSN) and structural modeling analysis, we successfully predicted and verified the functions of two uncharacterized Arabidopsis BAHD acyltransferases, OAO95042.1 and NP_190301.2, which use putrescine as the main acyl-acceptor. Our work provides not only an excellent starting point for understanding multisite acylation in BAHD-family enzymes, but also a feasible methodology for predicting possible acyl acceptor specificity of uncharacterized BAHD-family acyltransferases.

From thiol-subtilisin to omniligase: Design and structure of a broadly applicable peptide ligase

Omniligase-1 is a broadly applicable enzyme for peptide bond formation between an activated acyl donor peptide and a non-protected acyl acceptor peptide. The enzyme is derived from an earlier subtilisin variant called peptiligase by several rounds of protein engineering aimed at increasing synthetic yields and substrate range. To examine the contribution of individual mutations on S/H ratio and substrate scope in peptide synthesis, we selected peptiligase variant M222P/L217H as a starting enzyme and introduced successive mutations. Mutation A225N in the S1′ pocket and F189W of the S2′ pocket increased the synthesis to hydrolysis (S/H) ratio and overall coupling efficiency, whereas the I107V mutation was added to S4 pocket to increase the reaction rate. The final omniligase variants appeared to have a very broad substrate range, coupling more than 250 peptides in a 400-member library of acyl acceptors, as indicated by a high-throughput FRET assay. Crystal structures and computational modelling could rationalize the exceptional properties of omniligase-1 in peptide synthesis

Identification and Bioinformatic Analysis of the ASAT Gene Family in <i>Oryza staiva</i>

Rice bran oil is rich in bioactive steryl ferulates that are important not only for seed development, drought stress and lipid metabolism, but also for grain nutrition quality and human healthy. However, its complete compositions and associated metabolic pathways are poorly understood.  Arabidopsis thaliana  Acyl-CoA Sterol Acyltransferase1 (AtASAT1) is the only one in the superfamily of Membrane-Bound O-Acyltransferases that has ASAT activity, which prefers saturated fatty acyl-CoAs as acyl donors and cycloartenol as the acyl acceptor. Overexpression of  AtASAT1  in Arabidopsis results in a strong accumulation of cycloartenol fatty acyl esters accompanied by an increase of the whole steryl esters contents in seeds. Here we present our bioinformatic based analysis data aiming to identify more plant ASAT family members and to in silico study of them in rice. We identified 105 ASAT Family members from public genome database of 10 plant species including Arabidopsis and tomato, and the phylogenetic tree of ASAT family constructed revealed evolutionary relationships of ASAT family members between oil-bearing crop and non-oil-bearing species, and between monocotyledons and dicotyledons. We further investigated into physical and chemical properties of 14 OsASAT proteins, and chromosome distributions, structures and promoter sequences, and expression patterns of corresponding  OsASAT  genes, providing a solid theoretical basis for further study of the biological functions of OsASAT proteins.