High Product Specificity Research Articles

Membraneless organelles assembled by AuNPs-enzyme integration in non-photosynthetic bacteria: Achieving high specificity and selectivity for solar hydrogen production

The emergence of semiartificial photosynthesis holds great promise for improving light energy capture and conversion efficiency. However, achieving high product specificity and selectivity in semiartificial photosynthetic systems remains a challenge. In this study, we employed genetic engineering techniques to construct a membraneless organelle in E. coli, serving as the photoreaction carrier. The self-assembly of photosensitizers and hydrogenase within the carrier has successfully established a semiartificial photosynthetic model characterized by high product specificity and selectivity. In this model system, electrons were directly transferred to hydrogenase upon AuNPs light excitation in membraneless organelle, eliminating the need for additional electron mediators and bypassing the use of intracellular NADH/NAD+ as a reducing agent to avoid byproduct formation. The semiartificial photosynthetic system demonstrated an 11782-fold and 40-fold increase in hydrogen production compared to bare AuNPs and the empty vector E. coli hybrid system, respectively. This membraneless organelle holds the potential to robustly support the specific synthesis of high-energy-density, value-added compounds through solar energy conversion.

Two-step physico-biological production of xylooligosaccharides from sugarcane leaves

The production of xylooligosaccharides (XOS) from sugarcane leaves (SCL) presents high potential to the nutraceutical industries as SCL are cheap and readily available. In this study, a two-step process using hydrothermal pretreatments and enzymatic hydrolysis are developed and optimized for XOS production to obtain high conversion yield and product specificity. Two operation methods for hydrothermal pretreatments, parr reactor, and microwave reactor were compared to extract xylan from SCL. Two hemicellulose enzymes, hemicellulase Amano and xylanase (X2753) were then tested for enzymatic hydrolysis of extracted xylan to XOS. Conditions with high conversion yield after pretreatments were found at 180 °C for 30 min (parr reactor) and 800 W for 8 min (microwave reactor): producing 4.95 mg/mL (22.11 %w/v) and 3.49 mg/mL (15.61 %w/v) XOS respectively. Subjection of derived liquor from hydrothermal pretreatment to enzymatic hydrolysis produced a maximum of 9.42 mg/mL (41.87 %w/v) and 7.81 mg/mL (34.71 %w/v) of XOS for hemicellulase Amano and xylanase (X2753) respectively. The hemicellulase resulted in XOS with degree of polymerization (DP) of 2–5 for the various pretreatment conditions whereas xylanase yielded XOS of DP 2 to 4. However, a negative effect on enzymatic hydrolysis was observed for microwave-assisted pretreated samples. This study revealed that combination of physico-biological methods on SCL could result in high XOS yield with distinct degree of polymerization.

Genome Sequencing-Based Mining and Characterization of a Novel Alginate Lyase from Vibrio alginolyticus S10 for Specific Production of Disaccharides.

Alginate oligosaccharides prepared by alginate lyases attracted great attention because of their desirable biological activities. However, the hydrolysis products are always a mixture of oligosaccharides with different degrees of polymerization, which increases the production cost because of the following purification procedures. In this study, an alginate lyase, Alg4755, with high product specificity was identified, heterologously expressed, and characterized from Vibrio alginolyticus S10, which was isolated from the intestine of sea cucumber. Alg4755 belonged to the PL7 family with two catalytic domains, which was composed of 583 amino acids. Enzymatic characterization results show that the optimal reaction temperature and pH of Alg4755 were 35 °C and 8.0, respectively. Furthermore, Alg4755 was identified to have high thermal and pH stability. Moreover, the final hydrolysis products of sodium alginate catalyzed by Alg4755 were mainly alginate disaccharides with a small amount of alginate trisaccharides. The results demonstrate that alginate lyase Alg4755 could have a broad application prospect because of its high product specificity and desirable catalytic properties.

Toward Unifying the Mechanistic Concepts in Electrochemical CO2 Reduction from an Integrated Material Design and Catalytic Perspective

AbstractElectrocatalytic CO2 reduction (eCO2RR) is one of the avenues with most potential toward achieving sustainable energy economy and global climate change targets by harvesting renewable energy into value‐added fuels and chemicals. From an industrial standpoint, eCO2RR provides specific advantages over thermochemical and photochemical pathways in terms of much broader product scope, high product specificity, and easy adaptability to the renewable electricity infrastructure. However, unlike water electrolyzers, the lack of suitable cathode materials for eCO2RR impedes its commercialization due to material design challenges. The current state‐of‐the‐art catalysts in eCO2RR suffer largely from low reaction rates, insufficient C2+ product selectivity, high overpotentials, and industrial‐scale stability. Overcoming the scientific and applied technical hurdles for commercial realization demands a holistic integration of catalytic designs, deep mechanistic understanding, and efficient process engineering. Special emphasis on mechanistic understanding and performance outcome is sought to guide the future design of eCO2RR catalysts that can play a significant role in closing the anthropogenic carbon loop. This article provides an integrative approach to understand principles of robust eCO2RR catalyst design superimposed with underlying mechanistic projections which strongly depend on experimental conditions viz. choice of electrolyte, reactor and membrane design, pH of the solvent, and partial pressure of the CO2.

Characterisation of ascocorynin biosynthesis in the purple jellydisc fungus Ascocoryne sarcoides

BackgroundNon-ribosomal peptide synthetase-like (NRPS-like) enzymes are highly enriched in fungal genomes and can be discriminated into reducing and non-reducing enzymes. Non-reducing NRPS-like enzymes possess a C-terminal thioesterase domain that catalyses the condensation of two identical aromatic α-keto acids under the formation of enzyme-specific substrate-interconnecting core structures such as terphenylquinones, furanones, butyrolactones or dioxolanones. Ascocoryne sarcoides produces large quantities of ascocorynin, which structurally resembles a terphenylquinone produced from the condensation of p-hydroxyphenylpyruvate and phenylpyruvate. Since the parallel use of two different substrates by a non-reducing NRPS-like enzyme appeared as highly unusual, we investigated the biosynthesis of ascocorynin in A. sarcoides.ResultsHere, we searched the genome of A. sarcoides for genes coding for non-reducing NRPS-like enzymes. A single candidate gene was identified that was termed acyN. Heterologous gene expression confirmed that AcyN is involved in ascocorynin production but only produces the non-hydroxylated precursor polyporic acid. Although acyN is embedded in an ascocorynin biosynthesis gene cluster, a gene encoding a monooxygenase required for the hydroxylation of polyporic acid was not present. Expression analyses of all monooxygenase-encoding genes from A. sarcoides identified a single candidate that showed the same expression pattern as acyN. Accordingly, heterologous co-expression of acyN and the monooxygenase gene resulted in the production of ascocorynin. Structural modelling of the monooxygenase suggests that the hydrophobic substrate polyporic acid enters the monooxygenase from a membrane facing entry site and is converted into the more hydrophilic product ascocorynin, which prevents its re-entry for a second round of hydroxylation.ConclusionThis study characterises the first naturally occurring polyporic acid synthetase from an ascomycete. It confirms the high substrate and product specificity of this non-reducing NRPS-like enzyme and highlights the requirement of a monooxygenase to produce the terphenylquinone ascocorynin.

Statistical correlation between waste macromolecular composition and anaerobic fermentation temperature for specific short-chain fatty acid production

To properly exploit short-chain fatty acids (SCFAs) in the chemical industry, it is of foremost importance to ensure stable SCFA profile production via anaerobic fermentation (AF). The different macromolecular distribution of food wastes (FWs) used as feedstock might be crucial for process outcome. Targeting at a specific SCFAs profile and yield, this study explored the statistical correlation between the macromolecular composition of FWs and the produced SCFAs in batch-AFs at 25 °C and 55 °C. Principal Component Analysis (PCA) revealed that the carbohydrates fraction was directly related with butyric acid accumulation, regardless of process temperature. Nevertheless, operational temperature resulted in a pH change, which ultimately affected the process fate. PCA of 25 °C-batch-AF showed a positive correlation between high carbohydrate content and longer-chain acids accumulation. By contrast, 55 °C-AF resulted in higher product specificity than at 25 °C, mainly due to butyrate-type fermentation of carbohydrates. Batch results were further validated in a semicontinuous reactor. Prevailing SCFAs and high bioconversion efficiencies relied on 3 main FWs characteristics: high carbohydrate content (>77% w/w), high carbohydrate/protein ratio (≥10) and high soluble organic matter content. Results obtained herein allowed predicting a specific SCFAs profile based on FWs composition, which is relevant for setting proper downstream technologies.

Reaction coupling separation for isosteviol production from stevioside catalyzed by acidic ion-exchange resin.

Isosteviol, a prodrug used to be obtained via Wagner-Meerwein rearrangement from steviol with low yield and long reactiontime. Herein, an in-situ separation-coupling-reaction is presented to prepare isosteviol from the natural sweetener stevioside. Simply with in-situ water-washing, the product containing 92.98% purity of isosteviol was obtained with a stevioside conversion of 97.23% from a packet bed reactor without further separation. Within the assayed inorganic acid, organic acids and acidic ionic liquids, the acidic ion-exchange resins provided higher product specificity towards isosteviol. Furthermore, comparing to 5-Fluorouracil, the product presented similar and even stronger inhibition on proliferation of the assayed human cancer cells in a time and dose-dependence by causing cell phase arrest. Isosteviol treatment caused G1 arrest on SGC-7901, HCT-8 and HCT-116 cells, S arrest on HepG2, Huh-7 and HepG3B cells, and G2 arrest on MGC-803 cells, respectively. Reaction coupling separation for isosteviol production catalyzed by acidic ion-exchange resin.

Cyclodextrinase from Thermococcus sp expressed in Bacillus subtilis and its application in the preparation of maltoheptaose

BackgroundMaltoheptaose as malto-oligosaccharides with specific degree of polymerization, has wide applications in food, medicine and cosmetics industries. Currently, cyclodextrinase have been applied as prepared enzyme to prepare maltoheptaose. However, the yield and proportion of maltoheptaose was lower, which is due to limited substrate and product specificity of cyclodextrinase (CDase). To achieve higher maltoheptaose yield, cyclodextrinase with high substrate and product specificity should be obtained.ResultsIn this study, cyclodextrinase derived from Thermococcus sp B1001 (TsCDase) was successfully expressed and characterized in Bacillus subtilis for the first time. The specific activity of TsCDase was 637.95 U/mg under optimal conditions of 90 °C and pH 5.5, which exhibited high substrate specificity for cyclodextrins (CDs). When the concentration of β-CD was 8%, the yield of maltoheptaose achieved by TsCDase was 82.33% across all reaction products, which exceeded the yields of maltoheptaose in other recent reports. Among malto-oligosaccharides generated as reaction products, maltoheptaose was present in the highest proportion, about 94.55%.ConclusionsThis study provides high substrate and product specificity of TsCDase. TsCDase is able to prepare higher yield of maltoheptaose through conversion of β-CD in the food industry.

Enzymatic Modification of Native Chitin and Conversion to Specialty Chemical Products.

Chitin is one of the most abundant biomolecules on earth, occurring in crustacean shells and cell walls of fungi. While the polysaccharide is threatening to pollute coastal ecosystems in the form of accumulating shell-waste, it has the potential to be converted into highly profitable derivatives with applications in medicine, biotechnology, and wastewater treatment, among others. Traditionally this is still mostly done by the employment of aggressive chemicals, yielding low quality while producing toxic by-products. In the last decades, the enzymatic conversion of chitin has been on the rise, albeit still not on the same level of cost-effectiveness compared to the traditional methods due to its multi-step character. Another severe drawback of the biotechnological approach is the highly ordered structure of chitin, which renders it nigh impossible for most glycosidic hydrolases to act upon. So far, only the Auxiliary Activity 10 family (AA10), including lytic polysaccharide monooxygenases (LPMOs), is known to hydrolyse native recalcitrant chitin, which spares the expensive first step of chemical or mechanical pre-treatment to enlarge the substrate surface. The main advantages of enzymatic conversion of chitin over conventional chemical methods are the biocompability and, more strikingly, the higher product specificity, product quality, and yield of the process. Products with a higher Mw due to no unspecific depolymerisation besides an exactly defined degree and pattern of acetylation can be yielded. This provides a new toolset of thousands of new chitin and chitosan derivatives, as the physio-chemical properties can be modified according to the desired application. This review aims to provide an overview of the biotechnological tools currently at hand, as well as challenges and crucial steps to achieve the long-term goal of enzymatic conversion of native chitin into specialty chemical products.

Cross‐linked enzyme aggregates of recombinant cyclodextrin glycosyltransferase for high‐purity β‐cyclodextrin production

AbstractBACKGROUNDHigh‐purity α‐, β‐, or γ‐cyclodextrin (CD) production using cyclodextrin glycosyltransferase (CGTase) as biocatalyst to cyclize starch is advantageous owing to its low cost of purification and product specificity. Several approaches have been investigated to enhance product specificity of CGTase, such as new CGTase isolation, CGTase gene (cgt) mutation, and chemical addition.RESULTSRecombinant CGTase was cross‐linked by glutaraldehyde to obtain cross‐linked enzyme aggregates of recombinant CGTase (CLEA‐CGTase) at 85 °C because free CGTase showed high product specificity at 85 °C. The CLEA‐CGTase was prepared under the conditions 75 U mL−1 CGTase with 0.1% (v/v) glutaraldehyde for 10 min at 85 °C after optimization first, and produced a high proportion of β‐CD from soluble starch at 50 °C in the conversion system. The conversion process was carried out with 10 to 200 U mL−1 CLEA‐CGTase, resulting in 100% β‐CD from soluble starch after 420 min conversion of the potato starch solution at 50 °C. And the proportion of β‐CD was still higher than 90% when 8000 U mL−1 CLEA‐CGTase was added.CONCLUSIONSCLEA technology maintained CGTase conformation of 85 °C, and produced a high proportion of β‐CD at 50 °C. This research showed the CLEA technology kept the conformation of enzyme for specific product production. © 2018 Society of Chemical Industry

Modular pathway engineering for the microbial production of branched-chain fatty alcohols

The intrinsic structural properties of branched long-chain fatty alcohols (BLFLs) in the range of C12 to C18 make them more suitable as diesel fuel replacements and for other industrial applications than their straight-chain counterparts. While microbial production of straight long-chain fatty alcohols has been achieved, biosynthesis of BLFLs has never been reported. In this work, we engineered four different biosynthetic pathways in Escherichia coli to produce BLFLs. We then employed a modular engineering approach to optimize the supply of α-keto acid precursors and produced either odd-chain or even-chain BLFLs with high selectivity, reaching 70 and 75% of total fatty alcohols, respectively. The acyl-ACP and alcohol-producing modules were also extensively optimized to balance enzyme expression level and ratio, resulting in a 6.5-fold improvement in BLFL titers. The best performing strain overexpressed 14 genes from 6 engineered operons and produced 350 mg/L of BLFLs in fed-batch fermenter. The modular engineering strategy successfully facilitated microbial production of BLFLs and allowed us to quickly optimize new BLFL pathway with high titers and product specificity. More generally, this work provides pathways and knowledge for the production of BLFLs and BLFL-related, industry-relevant chemicals in high titers and yields.

Cell foundry with high product specificity and catalytic activity for 21-deoxycortisol biotransformation

Background21-deoxycortisol (21-DF) is the key intermediate to manufacture pharmaceutical glucocorticoids. Recently, a Japan patent has realized 21-DF production via biotransformation of 17-hydroxyprogesterone (17-OHP) by purified steroid 11β-hydroxylase CYP11B1. Due to the less costs on enzyme isolation, purification and stabilization as well as cofactors supply, whole-cell should be preferentially employed as the biocatalyst over purified enzymes. No reports as so far have demonstrated a whole-cell system to produce 21-DF. Therefore, this study aimed to establish a whole-cell biocatalyst to achieve 21-DF transformation with high catalytic activity and product specificity.ResultsIn this study, Escherichia coli MG1655(DE3), which exhibited the highest substrate transportation rate among other tested chassises, was employed as the host cell to construct our biocatalyst by co-expressing heterologous CYP11B1 together with bovine adrenodoxin and adrenodoxin reductase. Through screening CYP11B1s (with mutagenesis at N-terminus) from nine sources, Homo sapiens CYP11B1 mutant (G25R/G46R/L52 M) achieved the highest 21-DF transformation rate at 10.6 mg/L/h. Furthermore, an optimal substrate concentration of 2.4 g/L and a corresponding transformation rate of 16.2 mg/L/h were obtained by screening substrate concentrations. To be noted, based on structural analysis of the enzyme-substrate complex, two types of site-directed mutations were designed to adjust the relative position between the catalytic active site heme and the substrate. Accordingly, 1.96-fold enhancement on 21-DF transformation rate (to 47.9 mg/L/h) and 2.78-fold improvement on product/by-product ratio (from 0.36 to 1.36) were achieved by the combined mutagenesis of F381A/L382S/I488L. Eventually, after 38-h biotransformation in shake-flask, the production of 21-DF reached to 1.42 g/L with a yield of 52.7%, which is the highest 21-DF production as known.ConclusionsHeterologous CYP11B1 was manipulated to construct E. coli biocatalyst converting 17-OHP to 21-DF. Through the strategies in terms of (1) screening enzymes (with N-terminal mutagenesis) sources, (2) optimizing substrate concentration, and most importantly (3) rational design novel mutants aided by structural analysis, the 21-DF transformation rate was stepwise improved by 19.5-fold along with 4.67-fold increase on the product/byproduct ratio. Eventually, the highest 21-DF reported production was achieved in shake-flask after 38-h biotransformation. This study highlighted above described methods to obtain a high efficient and specific biocatalyst for the desired biotransformation.

Expression and Characterization of Levansucrase from Clostridium acetobutylicum.

The Clostridium acetobutylicum gene Ca-SacB encoding levansucrase was cloned and expressed in Escherichia coli. Ca-SacB is composed of 1287 bp and encodes 428 amino acid residues, which could convert 150 mmol/L sucrose to levan with the liberation of glucose. The optimum pH and temperature of this enzyme for levan formation were pH 6 and 60 °C, respectively. Levansucrase activity of Ca-SacB was completely abolished by 5 mmol/L Ag+ and Hg2+. The Km and Vmax values for levansucrase were calculated to be 64 mmol/L and 190 μmol/min/mg, respectively. Interestingly, Ca-SacB was found to have high product specificity, and no fructooligosaccharide was identified in the product, indicating that Ca-SacB may be valuable for industrial production of levan. In addition, Ca-SacB is the first characterized levansucrase isolated from an anaerobic bacterium, which should be valuable for exploring new enzyme resources and deepening the understanding of the catalytic mechanisms of levansucrases.

Three important amino acids control the regioselectivity of flavonoid glucosidation in glycosyltransferase-1 from Bacillus cereus.

Glycosyltransferase-1 from Bacillus cereus (BcGT1) catalyzes a reaction that transfers a glucosyl moiety to flavonoids, such as quercetin, kaempferol, and myricetin. The enzymatic glucosidation shows a broad substrate specificity when the reaction is catalyzed by wild-type BcGT1. Preliminary assays demonstrated that the F240A mutant significantly improves the regioselectivity of enzymatic glucosidation toward quercetin. To unveil and further to control the catalytic function of BcGT1, mutation of F240 to other amino acids, such as C, E, G, R, Y, W, and K, was performed. Among these mutants, F240A, F240G, F240R, and F240K greatly altered the regioselectivity. The quercetin-3-O-glucoside, instead of quercetin-7-O-glucoside as for the wild-type enzyme, was obtained as the major product. Among these mutants, F240R showed nearly 100% product specificity but only retained 25% catalytic efficiency of wild-type enzyme. From an inspection of the protein structure, we found two other amino acids, F132 and F138, together with F240, are likely to form a hydrophobic binding region, which is sufficiently spacious to accommodate substrates with varied aromatic moieties. Through the replacement of a phenylalanine by a tyrosine residue in the substrate-binding region, the mutants may be able to fix the orientation of flavonoids, presumably through the formation of a hydrogen bond between substrates and mutants. Multiple mutants-F240R_F132Y, F240R_F138Y, and F240R_F132Y_F138Y-were thus constructed for further investigation. The multiple points of mutants not only maintained the high product specificity but also significantly improved the catalytic efficiency, relative to F240R. The same product specificity was obtained when kaempferol and myricetin were used as a substrate.

High Acetic Acid Production Rate Obtained by Microbial Electrosynthesis from Carbon Dioxide.

High product specificity and production rate are regarded as key success parameters for large-scale applicability of a (bio)chemical reaction technology. Here, we report a significant performance enhancement in acetate formation from CO2, reaching comparable productivity levels as in industrial fermentation processes (volumetric production rate and product yield). A biocathode current density of -102 ± 1 A m(-2) and an acetic acid production rate of 685 ± 30 (g m(-2) day(-1)) have been achieved in this study. High recoveries of 94 ± 2% of the CO2 supplied as the sole carbon source and 100 ± 4% of electrons into the final product (acetic acid) were achieved after development of a mature biofilm, reaching an elevated product titer of up to 11 g L(-1). This high product specificity is remarkable for mixed microbial cultures, which would make the product downstream processing easier and the technology more attractive. This performance enhancement was enabled through the combination of a well-acclimatized and enriched microbial culture (very fast start-up after culture transfer), coupled with the use of a newly synthesized electrode material, EPD-3D. The throwing power of the electrophoretic deposition technique, a method suitable for large-scale production, was harnessed to form multiwalled carbon nanotube coatings onto reticulated vitreous carbon to generate a hierarchical porous structure.

Mechanism-Based Post-Translational Modification and Inactivation in Terpene Synthases.

Terpenes are ubiquitous natural chemicals with diverse biological functions spanning all three domains of life. In specialized metabolism, the active sites of terpene synthases (TPSs) evolve in shape and reactivity to direct the biosynthesis of a myriad of chemotypes for organismal fitness. As most terpene biosynthesis mechanistically involves highly reactive carbocationic intermediates, the protein surfaces catalyzing these cascade reactions possess reactive regions possibly prone to premature carbocation capture and potentially enzyme inactivation. Here, we show using proteomic and X-ray crystallographic analyses that cationic intermediates undergo capture by conserved active site residues leading to inhibitory self-alkylation. Moreover, the level of cation-mediated inactivation increases with mutation of the active site, upon changes in the size and structure of isoprenoid diphosphate substrates, and alongside increases in reaction temperatures. TPSs that individually synthesize multiple products are less prone to self-alkylation then TPSs possessing relatively high product specificity. In total, the results presented suggest that mechanism-based alkylation represents an overlooked mechanistic pressure during the evolution of cation-derived terpene biosynthesis.

Why Don’t We See A Greater Uptake Of Computational Chemistry Approaches By The Medicinal Chemistry Community?

Future Medicinal ChemistryVol. 4, No. 15 OpinionWhy don’t we see a greater uptake of computational chemistry approaches by the medicinal chemistry community?Michael James BodkinMichael James BodkinEli Lilly Research Laboratories, Surrey, UK, GU20 6PH. Search for more papers by this authorEmail the corresponding author at m.bodkin@lilly.comPublished Online:22 Oct 2012https://doi.org/10.4155/fmc.12.154AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail View articleKeywords: computational chemisthypothesismedicinal chemistserendipityworkflowReferences1 Ritchie TJ, McLay IM. Should medicinal chemists do molecular modelling? Drug Discov. Today17(11–12),534–537 (2012).Crossref, Medline, CAS, Google Scholar2 Warr WA. Scientific workflow systems: Pipeline Pilot and KNIME. J. Comput. Aided Mol. Des.26(7),801–804 (2012).Crossref, Medline, CAS, Google Scholar101 KNIME. Homepage. www.knime.comGoogle Scholar102 Accelrys. Pipeline Pilot. http://accelrys.com/products/pipeline-pilotGoogle Scholar103 KNIME. ErlWood Cheminformatics. http://tech.knime.org/community/erlwoodGoogle Scholar104 Richards S. How KNIME can empower the medicinal chemist at the bench. www.ukqsar.org/2011/07/14/spring-2012Google ScholarFiguresReferencesRelatedDetailsCited ByChemists: AI Is Here; Unite To Get the Benefits27 May 2020 | Journal of Medicinal Chemistry, Vol. 63, No. 16Cell foundry with high product specificity and catalytic activity for 21-deoxycortisol biotransformation13 June 2017 | Microbial Cell Factories, Vol. 16, No. 1 Vol. 4, No. 15 Follow us on social media for the latest updates Metrics Downloaded 356 times History Published online 22 October 2012 Published in print October 2012 Information© Future Science LtdKeywordscomputational chemisthypothesismedicinal chemistserendipityworkflowDisclaimerThe opinions expressed in this editorial are entirely those of the author and do not reflect those of the Eli Lilly and Co.Financial & competing interests disclosureM Bodkin is a Research Advisor and Group Leader at Eli Lilly and Co. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.PDF download

Microbial sucrose isomerases: Producing organisms, genes and enzymes

Sucrose isomerase (SI) activity is used industrially for the conversion of sucrose into isomers, particularly isomaltulose or trehalulose, which have properties advantageous over sucrose for some food uses. All of the known microbial SIs are TIM barrel proteins that convert sucrose without need for any cofactors, with varying kinetics and product specificities. The current analysis was undertaken to bridge key gaps between the information in patents and scientific publications about the microbes and enzymes useful for sucrose isomer production. This analysis shows that microbial SIs can be considered in 5 structural classes with corresponding functional distinctions that broadly align with the taxonomic differences between producing organisms. The most widely used bacterial strain for industrial production of isomaltulose, widely referred to as “ Protaminobacter rubrum” CBS 574.77, is identified as Serratia plymuthica. The strain producing the most structurally divergent SI, with a high product specificity for trehalulose, widely referred to as “ Pseudomonas mesoacidophila” MX-45, is identified as Rhizobium sp. Each tested SI-producer is shown to have a single SI gene and enzyme, so the properties reported previously for the isolated proteins can reasonably be associated with the products of the genes subsequently cloned from the same isolates and SI classes. Some natural isolates with potent SI activity do not catabolize the isomer under usual production conditions. The results indicate that their industrial potential may be further enhanced by selection for variants that do not catabolize the sucrose substrate.

Transition States in a Protein Environment - ONIOM QM:MM Modeling of Isopenicillin N Synthesis.

To highlight the role of the protein in metal enzyme catalysis, we optimize ONIOM QM:MM transition states and intermediates for the full reaction of the nonheme iron enzyme isopenicillin N synthase (IPNS). Optimizations of transition states in large protein systems are possible using our new geometry optimizer with quadratic coupling between the QM and MM regions [Vreven, T. et al. Mol. Phys. 2006, 104, 701-704]. To highlight the effect of the metal center, results from the protein model are compared to results from an active site model containing only the metal center and coordinating residues [Lundberg, M. et al. Biochemistry 2008, 47, 1031-1042]. The analysis suggests that the main catalytic effect comes from the metal center, while the protein controls the reactivity to achieve high product specificity. As an example, hydrophobic residues align the valine substrate radical in a favorable conformation for thiazolidine ring closure and contribute to product selectivity and high stereospecificity. A low-barrier pathway for β-lactam formation is found where the proton required for heterolytic O-O bond cleavage comes directly from the valine N-H group of the substrate. The alternative mechanism, where the proton in O-O bond cleavage initially comes from an iron water ligand, can be disfavored by the electrostatic interactions with the surrounding protein. Explicit protein effects on transition states are typically 1-6 kcal/mol in the present enzyme and can be understood by considering whether the transition state involves large movements of the substrate as well as whether it involves electron transfer.

Enhanced benzaldehyde tolerance in Zymomonas mobilis biofilms and the potential of biofilm applications in fine-chemical production.

Biotransformation plays an increasingly important role in the industrial production of fine chemicals due to its high product specificity and low energy requirement. One challenge in biotransformation is the toxicity of substrates and/or products to biocatalytic microorganisms and enzymes. Biofilms are known for their enhanced tolerance of hostile environments compared to planktonic free-living cells. Zymomonas mobilis was used in this study as a model organism to examine the potential of surface-associated biofilms for biotransformation of chemicals into value-added products. Z. mobilis formed a biofilm with a complex three-dimensional architecture comprised of microcolonies with an average thickness of 20 microm, interspersed with water channels. Microscopic analysis and metabolic activity studies revealed that Z. mobilis biofilm cells were more tolerant to the toxic substrate benzaldehyde than planktonic cells were. When exposed to 50 mM benzaldehyde for 1 h, biofilm cells exhibited an average of 45% residual metabolic activity, while planktonic cells were completely inactivated. Three hours of exposure to 30 mM benzaldehyde resulted in sixfold-higher residual metabolic activity in biofilm cells than in planktonic cells. Cells inactivated by benzaldehyde were evenly distributed throughout the biofilm, indicating that the resistance mechanism was different from mass transfer limitation. We also found that enhanced tolerance to benzaldehyde was not due to the conversion of benzaldehyde into less toxic compounds. In the presence of glucose, Z. mobilis biofilms in continuous cultures transformed 10 mM benzaldehyde into benzyl alcohol at a steady rate of 8.11 g (g dry weight)(-1) day(-1) with a 90% molar yield over a 45-h production period.