Search

Godkänd;2023;Nivå 0;2023-04-21 (hanlid); Green and sustainable approach to valorise high saline and oily fish processing effluents for the production of nu-traceuticals (INVENTION; 2020-01028)

Thermophilic enzyme systems are of major importance nowadays in all industrial processes due to their great performance at elevated temperatures. In the present review, an overview of the current knowledge on the properties of thermophilic and thermotolerant carbohydrate esterases and oxidative enzymes with great thermostability is provided, with respect to their potential use in biotechnological applications. A special focus is given to the lytic polysaccharide monooxygenases that are able to oxidatively cleave lignocellulose through the use of oxygen or hydrogen peroxide as co-substrate and a reducing agent as electron donor. Structural characteristics of the enzymes, including active site conformation and surface properties are discussed and correlated with their substrate specificity and thermostability properties.

Lignin, one of the most abundant natural polymers, has been successfully used as an effective lubricant additive with high value. The chemical structure of lignin is very diverse and strongly affected by both the source of lignin (i.e. plant species) and the lignin extraction process. In this work, a series of lignin from different biomass sources (hard or soft wood) and extraction process (organosolv with or without acid catalyst) has been successfully incorporated into poly(ethylene glycol) (PEG) and fortified lubricating properties were achieved. The effects of different lignin on the rheological, thermal and tribological properties of the lignin/EG lubricants were systematically investigated by different characterization techniques. Lignin in PEG significantly improves the lubricating property, where a wear reduction of 93.8% was observed. The thermal and lubrication properties of the PEG lubricants filled with different kinds of lignin are tightly related to the synergistic state of hydrogen bonding and molecular weight distribution. Lignin with broader molecular weight distribution and higher hydroxyl content shows better adhesion on metal surfaces and strengthened lubricating film, which could be used as the efficient lubricating additives. This work provides a criterion for selecting appropriate lignin as the efficient lubricant additive and accelerates the application of lignin.

Nanocellulose isolation fromlignocellulose is a tedious and expensive processwith high energy and harsh chemical requirements, primarily due to the recalcitrance of the substrate, which otherwise would have been costeffective due to its abundance. Replacing the chemical steps with biocatalytic processes offers opportunities to solve this bottleneck to a certain extent due to the enzymes substrate specificity and mild reaction chemistry. In this work, we demonstrate the isolation of sulphate-free nanocellulose from organosolv pretreated birch biomass using different glycosyl-hydrolases, along with accessory oxidative enzymes including a lytic polysaccharide monooxygenase (LPMO). The suggested process produced colloidal nanocellulose suspensions (zeta-potential-19.4 mV) with particles of 7-20 nm diameter, high carboxylate content and improved thermostability (T-o= 301 degrees C, T-max= 337 degrees C). Nanocelluloseswere subjected to post-modification using LPMOs of different regioselectivity. The sample from chemical route was the least favorable for LPMO to enhance the carboxylate content, while that from the C1-specific LPMO treatment showed the highest increase in carboxylate content. (c) 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).

Thraustochytrids are considered natural producers of omega-3 fatty acids as they can synthesize up to 70% docosahexaenoic acids (DHA) of total lipids. However, commercial and sustainable production of microbial DHA is limited by elevated cost of carbon substrates for thraustochytrids cultivation. This problem can be addressed by utilizing low-cost renewable substrates. In the present study, growth, lipid accumulation and fatty acid profiles of the marine thraustochytrid Aurantiochytrium sp. T66 (ATCC-PRA-276) cultivated on volatile fatty acids (C1, formic acid, C2, acetic acid, C3, propionic acid, C4, butyric acid, C5, valeric acid and C6, caproic acid) and glucose as control were evaluated for the first time. This strain showed an inability to utilize C3, C5 and C6 as a substrate when provided at &gt, 2 g/L, while efficiently utilizing C2 and C4 up to 40 g/L. The highest cell dry weight (12.35 g/L) and total lipid concentration (6.59 g/L) were attained when this strain was cultivated on 40 g/L of butyric acid, followed by cultivation on glucose (11.87 g/L and 5.34 g/L, respectively) and acetic acid (8.70 g/L and 3.43 g/L, respectively). With 40 g/L butyric acid, the maximum docosahexaenoic acid content was 2.81 g/L, corresponding to 42.63% w/w of total lipids and a yield of 0.23 g/gcell dry weight (CDW). This marine oleaginous microorganism showed an elevated potential for polyunsaturated fatty acids production at higher acetic and butyric acid concentrations than previously reported. Moreover, fluorescence microscopy revealed that growth on butyric acid caused cell size to increase to 45 &micro, m, one of the largest values reported for oleaginous microorganisms, as well as the presence of numerous tiny lipid droplets.

The feruloyl esterases FaeA1, FaeA2, FaeB1, FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464 were used as biocatalysts for the transesterification of vinyl ferulate (VFA) with Larabinose in detergentless microemulsions. The effect of parameters such as the microemulsion composition, the substrate concentration, the enzyme load, the pH, the temperature and the agitation was investigated. FaeA1 offered the highest transesterification yield (52.2 ± 4.3%) after 8 h of incubation at 50 °C using 80 mM VFA, 55 mM L-arabinose and 0.02 mg FAE mL−1 in a mixture comprising of 19.8: 74.7: 5.5 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0. The ability of L-arabinose ferulate (AFA) to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals was significant (IC50 386.5 μM). AFA was not cytotoxic even at high concentrations (1 mM) however was found to be pro-oxidant at concentrations higher than 20 μM when the antioxidant activity was determined with the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay in human skin fibroblasts.

International audience; The need to develop competitive and eco-friendly processes in the cosmetic industry leads to the search for new enzymes with improved properties for industrial bioconversions in this sector. In the present study, a complete methodology to generate, express and screen diversity for the type C feruloyl esterase from Fusarium oxysporium FoFaeC was set up in a high-throughput fashion. A library of around 30,000 random mutants of FoFaeC was generated by error prone PCR of fofaec cDNA and expressed in Yarrowia lipolytica. Screening for enzymatic activity towards the substrates 5-bromo-4-chloroindol-3-yl and 4-nitrocatechol-1-yl ferulates allowed the selection of 96 enzyme variants endowed with improved enzymatic activity that were then characterized for thermo- and solvent- tolerance. The five best mutants in terms of higher activity, thermo- and solvent- tolerance were selected for analysis of substrate specificity. Variant L432I was shown to be able to hydrolyze all the tested substrates, except methyl sinapate, with higher activity than wild type FoFaeC towards methyl p-coumarate, methyl ferulate and methyl caffeate. Moreover, the E455D variant was found to maintain completely its hydrolytic activity after two hour incubation at 55 °C, whereas the L284Q/V405I variant showed both higher thermo- and solvent- tolerance than wild type FoFaeC. Small molecule docking simulations were applied to the five novel selected variants in order to examine the binding pattern of substrates used for enzyme characterization of wild type FoFaeC and the evolved variants.

The feruloyl esterases Fae125, Fae7262 and Fae68 from Talaromyces wortmannii were screened in 10 different solvent: buffer systems in terms of residual hydrolytic activity and of the ability for the transesterification of vinyl ferulate with prenol or l -arabinose. Among the tested enzymes, the acetyl xylan-related Fae125 belonging to the phylogenetic subfamily 5 showed highest yield and selectivity for both products in alkane: buffer systems (n-hexane or n-octane). Response surface methodology, based on a 5-level and 6-factor central composite design, revealed that the substrate molar ratio and the water content were the most significant variables for the bioconversion yield and selectivity. The effect of agitation, the possibility of DMSO addition and the increase of donor concentration were investigated. After optimization, competitive transesterification yields were obtained for prenyl ferulate (87.5–92.6%) and l -arabinose ferulate (56.2–61.7%) at reduced reaction times (≤24 h) resulting in good productivities (>1 g/L/h, >300 kg product/kg FAE). The enzyme could be recycled for six consecutive cycles retaining 66.6% of the synthetic activity and 100% of the selectivity.

The type C feruloyl esterase FoFaeC from Fusarium oxysporum is a newly discovered enzyme with high potential for use in the hydrolysis of lignocellulosic biomass but it shows low activity towards sinapates. In this work, small molecule docking simulations were employed in order to identify important residues for the binding of the four model methyl esters of hydroxycinnamic acids, methyl ferulate/caffeate/sinapate/p-coumarate, to the predicted structure of FoFaeC. Subsequently rational redesign was applied to the enzyme' active site in order to improve its specificity towards methyl sinapate. A double mutation (F230H/T202V) was considered to provide hydrophobic environment for stabilization of the methoxy substitution on sinapate and a larger binding pocket. Five mutant clones and the wild type were produced in Pichia pastoris and biochemically characterized. All clones showed improved activity, substrate affinity, catalytic efficiency and turnover rate compared to the wild type against methyl sinapate, with clone P13 showing a 5-fold improvement in catalytic efficiency. Although the affinity of all mutant clones was improved against the four model substrates, the catalytic efficiency and turnover rate decreased for the substrates containing a hydroxyl substitution.

International audience; The chemical syntheses currently employed for industrial purposes, including in the manufacture of cosmetics, present limitations such as unwanted side reactions and the need for harsh chemical reaction conditions. In order to overcome these drawbacks, novel enzymes are developed to catalyze the targeted bioconversions. In the present study, a methodology for the construction and the automated screening of evolved variants library of a Type B feruloyl esterase from Myceliophthora thermophila (MtFae1a) was developed and applied to generation of 30,000 mutants and their screening for selecting the variants with higher activity than the wild-type enzyme. The library was generated by error-prone PCR of mtfae1a cDNA and expressed in Saccharomyces cerevisiae. Screening for extracellular enzymatic activity towards 4-nitrocatechol-1-yl ferulate, a new substrate developed ad hoc for high-throughput assays of feruloyl esterases, led to the selection of 30 improved enzyme variants. The best four variants and the wild-type MtFae1a were investigated in docking experiments with hydroxycinnamic acid esters using a model of 3D structure of MtFae1a. These variants were also used as biocatalysts in transesterification reactions leading to different target products in detergentless microemulsions and showed enhanced synthetic activities, although the screening strategy had been based on improved hydrolytic activity.

Non-digestible oligosaccharides (NDOs) are likely prebiotic candidates that have been related to the prevention of intestinal infections and other disorders for both humans and animals. Lignocellulosic biomass is the largest carbon source in the biosphere, therefore cello-oligosacharides (COS), especially cellobiose, are potentially the most widely available choice of NDOs. Production of COS and cellobiose with enzymes offers numerous benefits over acid-catalyzed processes, as it is milder, environmentally friendly and produces fewer by-products. Cellobiohydrolases (CBHs) and a class of endoglucanases (EGs), namely processive EGs, are key enzymes for the production of COS, as they have higher preference toward glycosidic bonds near the end of cellulose chains and are able to release soluble products. In this work, we describe the heterologous expression and characterization of two CBHs from the filamentous fungus Thermothelomyces thermophila, as well as their synergism with proccessive EGs for cellobiose release from organosolv pretreated spruce and birch. The properties, inhibition kinetics and substrate specific activities for each enzyme are described in detail. The results show that a combination of EGs belonging to Glycosyl hydrolase families 5, 6, and 9, with a CBHI and CBHII in appropriate proportions, can enhance the production of COS from forest materials, underpinning the potential of these biocatalysts in the production of NDOs.

Enzymatically liquefied sweet sorghum stalks and beet molasses were evaluated for butyrate production using Clostridium tyrobutyricum in 1 L scale fed-batch fermentations. The hydrolysates used for the fermentations were prepared separately by liquefying the sorghum stalks at 50 °C, pH 5.0 for 18 h, with 30% (w/v) DM content using the enzyme preparation Cellic® CTec2 at an activity of 26.5 FPU/g DM. To enhance sucrose consumption, the fermentations were supplemented with invertase at an activity equivalent to 8.3 U/g DM. With the hydrolysate as the feedstock, a butyrate concentration of 37.2 ± 0.8 g/L, a productivity of 0.86 ± 0.02 g/L h and a yield of 0.39 ± 0.02 g/g (p = 0.05) consumed sugars were obtained. Finally, a butyrate concentration of 58.8 g/L, a productivity of 1.9 g/L h, a butyrate yield of 0.52 g/g consumed sugars and a dry cell mass concentration of 15.7 g/L were obtained with fed-batch cultivation and a constant feed consisting of 64% sorghum hydrolysate juice and 36% molasses. Evidence for inducible saccharolytic activity was also proven, as the cellulase activity in the culture supernatant was found more than double during feed with limiting sugar concentrations. The present study clearly demonstrates that combinations of low cost raw materials can be used for efficient butyrate production, also without cell immobilization.

Single cell oils (SCOs) are considered potential raw material for the production of biodiesel. Rhodosporidium sp. and Lipomyces sp. are good candidates for SCO production. Lipid extractability differs according to yeast species and literature on the most suitable method for each oleaginous yeast species is scarce. This work aimed to investigate the efficiency of the most cited strategies for extracting lipids from intact and pretreated cells of Rhodosporidium toruloides and Lipomyces starkeyi . Lipid extractions were conducted using hexane or combinations of chloroform and methanol. The Folch method resulted in the highest lipid yields for both yeasts (42% for R. toruloides and 48% for L. starkeyi ). Also, this method eliminates the cell pretreatment step. The Bligh and Dyer method underestimated the lipid content in the tested strains (25% for R. toruloides and 34% for L. starkeyi ). Lipid extractability increased after acid pretreatment for the Pedersen, hexane, and Bligh and Dyer methods. For R. toruloides unexpected fatty acid methyl esters (FAME) composition were found for some lipid extraction strategies tested. Therefore, this work provides useful information for analytical and process development aiming at biodiesel production from the SCO of these two yeast species.

L-arginine, an amino acid with a growing range of applications within the pharmaceutical, cosmetic, food, and agricultural industries, can be produced by microbial fermentation. Although it is the most nitrogen-rich amino acid, reports on the nitrogen supply for its fermentation are scarce. In this study, the nitrogen supply for the production of l-arginine by a genetically modified Escherichia coli strain was optimised in bioreactors. Different nitrogen sources were screened and ammonia solution, ammonium sulphate, ammonium phosphate dibasic, and ammonium chloride were the most favourable nitrogen sources for l-arginine synthesis. The key role of the C/N ratio for l-arginine production was demonstrated for the first time. The optimal C/N molar ratio to maximise l-arginine production while minimising nitrogen waste was found to be 6, yielding approximately 2.25 g/L of l-arginine from 15 g/L glucose with a productivity of around 0.11 g/L/h. Glucose and ammonium ion were simultaneously utilized, showing that this ratio provided a well-balanced equilibrium between carbon and nitrogen metabolisms.

Lytic Polysaccharide Monooxygenases have now been evolved as one of the most promising enzymes, attracting huge research attention due to their potential use in saccharification of lignocellulosic biomass for the production of fuels and value added chemicals. In the presence of molecular oxygen, these copper depended enzymes break the recalcitrant cellulose chain by a combined oxidative and hydrolytic action, and increase the substrate accessibility for other cellulases to work. This ‘boosting effect’ and ability to act in synergy makes them important subject to research, towards the future goal of sustainable bioeconomy. Diversity of this enzyme group ranges from early discovered chitin and cellulose active ones, to the recently identified hemicellulose and starch active ones. In this chapter we present a brief summary about LPMOs and the findings related to them from their discovery to the recent developments.

The methanol inducible alcohol oxidase 1 promoter and the Saccharomyces cerevisiae alpha-factor prepro secretion signal were used to drive expression and secretion of a mucin-type fusion protein by Pichia pastoris in 1 L scale bioreactors. The aim of the study was to understand how varying expression rates influenced the secretion dynamics of the fusion protein in terms of intracellular- and extracellular concentrations. Endoplasmic reticulum (ER) folding stress was assessed by the relative expression of the unfolded protein response controlled KAR2 gene. Three predefined methanol feeding models were applied to control the fusion protein synthesis rate. To track the fusion protein synthesis in a non-invasive manner and to follow its intracellular distribution, its C-terminal was linked to the green fluorescent protein. Under all conditions the fusion protein was found to partially accumulate intracellularly, where the major fraction was an insoluble, fluorescent full-sized protein. The high degree of glycosylation of the insoluble fusion protein indicated a secretory bottle-neck in the Golgi-system. This result was consistent with low ER folding stress as quantified by the relative expression of the KAR2 gene. Reduction of recombinant protein synthesis rate, by using lower feed rates of methanol, enhanced extracellular concentrations from 8 to 18 mg·L–1 and reduced the rate of intracellular accumulation. This clearly demonstrates the importance of tuning the synthesis rate with secretory bottle-necks to maintain secretion.

Mannose-binding proteins like the macrophage mannose receptor (MR), the dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and mannose-binding lectin (MBL) play crucial roles in both innate and adaptive immune responses. Immunoglobulin fusion proteins of the P-selectin glycoprotein ligand-1 (PSGL-1/mIgG(2b)) carrying mostly O-glycans and, as a control, the α1-acid glycoprotein (AGP/mIgG(2b)) carrying mainly N-linked glycans were stably expressed in the yeast Pichia pastoris. Pichia pastoris-produced PSGL-1/mIgG(2b) was shown to carry O-glycans that mediated strong binding to mannose-specific lectins in a lectin array and were susceptible to cleavage by α-mannosidases including an α1,2- but not an α1,6-mannosidase. Electrospray ionization ion-trap mass spectrometry confirmed the presence of O-glycans containing up to nine hexoses with the penta- and hexasaccharides being the predominant ones. α1,2- and α1,3-linked, but not α1,6-linked, mannose residues were detected by (1)H-nuclear magnetic resonance spectroscopy confirming the results of the mannosidase cleavage. The apparent equilibrium dissociation constants for binding of PNGase F-treated mannosylated PSGL-1/mIgG(2b) to MR, DC-SIGN and MBL were shown by surface plasmon resonance to be 126, 56 and 16 nM, respectively. In conclusion, PSGL-1/mIgG(2b) expressed in P. pastoris carried O-glycans mainly comprised of α-linked mannoses and with up to nine residues. It bound mannose-specific receptors with high apparent affinity and may become a potent targeting molecule for these receptors in vivo.

During dual-phase fermentations using Escherichia coli engineered for succinic acid production, the productivity and viable cell concentration decrease as the concentration of succinic acid increases. The effects of succinic acid on the fermentation kinetics, yield, and cell viability were investigated by resuspending cells in fresh media after selected fermentation times. The cellular succinic acid productivity could be restored, but cell viability continuously decreased throughout the fermentations by up to 80% and subsequently the volumetric productivity was reduced. Omitting complex nutrients in the resuspension media had no significant effect on cellular succinate productivity and yield, although the viable cell concentration and thus the volumetric productivity was reduced by approximately 20%. By resuspending the cells, the amount of succinate produced during a 100-h fermentation was increased by more than 60%. The results demonstrate that by product removal succinic acid productivity can be maintained at high levels for extended periods of time.

In this work an Escherichia coli metabolically engineered to ferment lignocellulosic biomass sugars to succinic acid was tested for growth and fermentation of detoxified softwood dilute sulfuric acid hydrolyzates, and the minimum detoxification requirements were investigated with activated carbon and/or overliming treatments. Detoxified hydrolyzates supported fast growth and complete fermentation of all hydrolyzate sugars to succinate at yields comparable to pure sugar, while untreated hydrolyzates were unable to support either growth or fermentation. Activated carbon treatment was able to remove significantly more HMF and phenolics than overliming. However, in some cases, overliming treatment was capable of generating a fermentable hydrolyzate where activated carbon treatment was not. The implications of this are that in addition to the known organic inhibitors, the changes in the inorganic content and/or composition due to overliming are significant to the hydrolyzate toxicity. It was also found that any HMF remaining after detoxification was completely metabolized during aerobic cell growth on the hydrolyzates that were capable of supporting growth.

The economical viability of biochemical succinic acid production is a result of many processing parameters including final succinic acid concentration, recovery of succinate, and the volumetric productivity. Maintaining volumetric productivities2.5 g L(-1) h(-1) is important if production of succinic acid from renewable resources should be competitive. In this work, the effects of organic acids, osmolarity, and neutralizing agent (NH4OH, KOH, NaOH, K2CO3, and Na2CO3), and Na2CO3) on the fermentative succinic acid production by Escherichia coli AFP184 were investigated. The highest concentration of succinic acid, 77 g L(-1), was obtained with Na2CO3. In general, irrespective of the base used, succinic acid productivity per viable cell was significantly reduced as the concentration of the produced acid increased. Increased osmolarity resulting from base addition during succinate production only marginally affected the productivity per viable cell. Addition of the osmoprotectant glycine betaine to cultures resulted in an increased aerobic growth rate and anaerobic glucose consumption rate, but decreased succinic acid yield. When using NH4OH productivity completely ceased at a succinic acid concentration of approximately 40 g L(-1). Volumetric productivities remained at 2.5 g L(-1) h(-1) for up to 10 h longer when K- or Na-bases where used instead of NH4OH. The decrease in cellular succinic acid productivity observed during the anaerobic phase was found to be due to increased organic acid concentrations rather than medium osmolarity.

Background Arginine is a high-value product, especially for the pharmaceutical industry. Growing demand for environmental-friendly and traceable products have stressed the need for microbial production of this amino acid. Therefore, the aim of this study was to improve arginine production in Escherichia coli by metabolic engineering and to establish a fermentation process in 1-L bioreactor scale to evaluate the different mutants. Results Firstly, argR (encoding an arginine responsive repressor protein), speC, speF (encoding ornithine decarboxylases) and adiA (encoding an arginine decarboxylase) were knocked out and the feedback-resistant argA214 or argA215 were introduced into the strain. Three glutamate independent mutants were assessed in bioreactors. Unlike the parent strain, which did not excrete any arginine during glucose fermentation, the constructs produced between 1.94 and 3.03 g/L arginine. Next, wild type argA was deleted and the gene copy number of argA214 was raised, resulting in a slight increase in arginine production (4.11 g/L) but causing most of the carbon flow to be redirected toward acetate. The V216A mutation in argP (transcriptional regulator of argO, which encodes for an arginine exporter) was identified as a potential candidate for improved arginine production. The combination of multicopy of argP216 or argO and argA214 led to nearly 2-fold and 3-fold increase in arginine production, respectively, and a reduction of acetate formation. Conclusions In this study, E. coli was successfully engineered for enhanced arginine production. The ∆adiA, ∆speC, ∆speF, ∆argR, ∆argA mutant with high gene copy number of argA214 and argO produced 11.64 g/L of arginine in batch fermentation, thereby demonstrating the potential of E. coli as an industrial producer of arginine.

Ribonucleotide reductase catalyzes all de novo synthesis of deoxyribonucleotides. The mammalian enzyme consists of two non-identical subunits, the R1 and R2 proteins, each inactive alone. The R1 subunit contains the active site, whereas the R2 protein harbors a binuclear iron center and a tyrosyl free radical essential for catalysis. It has been proposed that the radical properties of the R2 subunit are transferred approximately 35 A to the active site of the R1 protein, through a coupled electron/proton transfer along a conserved hydrogen-bonded chain, i.e. a radical transfer pathway (RTP). To gain a better insight into the properties and requirements of the proposed RTP, we have used site-directed mutagenesis to replace the conserved tyrosine 370 in the mouse R2 protein with tryptophan or phenylalanine. This residue is located close to the flexible C terminus, known to be essential for binding to the R1 protein. Our results strongly indicate that Tyr(370) links the RTP between the R1 and R2 proteins. Interruption of the hydrogen-bonded chain in Y370F inactivates the enzyme complex. Alteration of the same chain in Y370W slows down the RTP, resulting in a 58 times lower specific activity compared with the native R2 protein and a loss of the free radical during catalysis.

Clostridium tyrobutyricum ATCC 25755 is an acidogenic bacterium capable of utilizing xylose for the fermentation production of butyrate. Hot water extraction of hardwood lingocellulose is an efficient method of producing xylose where autohydrolysis of xylan is catalysed by acetate originating from acetyl groups present in hemicellulose. The presence of acetic acid in the hydrolysate might have a severe impact on the subsequent fermentations. In this study the fermentation kinetics of C. tyrobutyricum cultures after being classically adapted for growth at 26.3 g/L acetate equivalents were studied. Analysis of xylose batch fermentations found that even in the presence of high levels of acetate, acetate adapted strains had similar fermentation kinetics as the parental strain cultivated without acetate. The parental strain exposed to acetate at inhibitory conditions demonstrated a pronounced lag phase (over 100 hours) in growth and butyrate production as compared to the adapted strain (25 hour lag) or non-inhibited controls (0 lag). Additional insight into the metabolic pathway of xylose consumption was gained by determining the specific activity of the acetate kinase (AK) enzyme in adapted versus control batches. AK activity was reduced by 63% in the presence of inhibitory levels of acetate, whether or not the culture had been adapted.

Targeting antigens to antigen-presenting cells (APC) improve their immunogenicity and capacity to induce Th1 responses and cytotoxic T lymphocytes (CTL). We have generated a mucin-type immunoglobulin fusion protein (PSGL-1/mIgG2b), which upon expression in the yeast Pichia pastoris became multivalently substituted with O-linked oligomannose structures and bound the macrophage mannose receptor (MMR) and dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) with high affinity in vitro. Here, its effects on the humoral and cellular anti-ovalbumin (OVA) responses in C57BL/6 mice are presented. OVA antibody class and subclass responses were determined by ELISA, the generation of anti-OVA CTLs was assessed in 51Cr release assays using in vitro-stimulated immune spleen cells from the different groups of mice as effector cells and OVA peptide-fed RMA-S cells as targets, and evaluation of the type of Th cell response was done by IFN-γ, IL-2, IL-4 and IL-5 ELISpot assays. Immunizations with the OVA − mannosylated PSGL-1/mIgG2b conjugate, especially when combined with the AbISCO®-100 adjuvant, lead to faster, stronger and broader (with regard to IgG subclass) OVA IgG responses, a stronger OVA-specific CTL response and stronger Th1 and Th2 responses than if OVA was used alone or together with AbISCO®-100. Also non-covalent mixing of mannosylated PSGL-1/mIgG2b, OVA and AbISCO®-100 lead to relatively stronger humoral and cellular responses. The O-glycan oligomannoses were necessary because PSGL-1/mIgG2b with mono- and disialyl core 1 structures did not have this effect. Mannosylated mucin-type fusion proteins can be used as versatile APC-targeting molecules for vaccines and as such enhance both humoral and cellular immune responses.

Succinic acid (SA) is an important platform molecule in the synthesis of a number of commodity and specialty chemicals. In the present work, dual-phase batch fermentations with the E. coli strain AFP184 were performed using a medium suited for large-scale industrial production of SA. The ability of the strain to ferment different sugars was investigated. The sugars studied were sucrose, glucose, fructose, xylose, and equal mixtures of glucose and fructose and glucose and xylose at a total initial sugar concentration of 100 g L-1. AFP184 was able to utilize all sugars and sugar combinations except sucrose for biomass generation and succinate production. For sucrose as a substrate no succinic acid was produced and none of the sucrose was metabolized. The succinic acid yield from glucose (0.83 g succinic acid per gram glucose consumed anaerobically) was higher than the yield from fructose (0.66 g g-1). When using xylose as a carbon source, a yield of 0.50 g g-1 was obtained. In the mixed-sugar fermentations no catabolite repression was detected. Mixtures of glucose and xylose resulted in higher yields (0.60 g g-1) than use of xylose alone. Fermenting glucose mixed with fructose gave a lower yield (0.58 g g-1) than fructose used as the sole carbon source. The reason is an increased pyruvate production. The pyruvate concentration decreased later in the fermentation. Final succinic acid concentrations were in the range of 25-40 g L-1. Acetic and pyruvic acid were the only other products detected and accumulated to concentrations of 2.7-6.7 and 0-2.7 g L-1. Production of succinic acid decreased when organic acid concentrations reached approximately 30 g L-1. This study demonstrates that E. coli strain AFP184 is able to produce succinic acid in a low cost medium from a variety of sugars with only small amounts of byproducts formed.

Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1. families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1 L culture. Further, the N-terminal His-tag could be removed by thrombin cleavage while the tagged protein was bound to metal affinity resin. Recombinant Munc18c produced in this way is functional, in that it forms a stable complex with the SNARE interacting partner, syntaxin4. Thus we have developed a method for producing and purifying large amounts of functional Munc18c-both tagged and detagged-from a baculovirus expression system. We have also developed a method to purify the Munc18c:syntaxin4 complex. These methods will be employed for future functional and structural studies. Crown copyright (C) 2003 Published by Elsevier Inc. All rights reserved.

Next Section Abstract Class I ribonucleotide reductases consist of two subunits, R1 and R2. The active site is located in R1; active R2 contains a diferric center and a tyrosyl free radical (Tyr⋅), both essential for enzymatic activity. The proposed mechanism for the enzymatic reaction includes the transport of a reducing equivalent, i.e. electron or hydrogen radical, across a 35-A distance between Tyr⋅ in R2 and the active site in R1, which are connected by a hydrogen-bonded chain of conserved, catalytically essential amino acid residues. Asp266 and Trp103 in mouse R2 are part of this radical transfer pathway. The diferric/Tyr⋅ site in R2 is reconstituted spontaneously by mixing iron-free apoR2 with Fe(II) and O2. The reconstitution reaction requires the delivery of an external reducing equivalent to form the diferric/Tyr⋅ site. Reconstitution kinetics were investigated in mouse apo-wild type R2 and the three mutants D266A, W103Y, and W103F by rapid freeze-quench electron paramagnetic resonance with ≥4 Fe(II)/R2 at various reaction temperatures. The kinetics of Tyr⋅ formation in D266A and W103Y is on average 20 times slower than in wild type R2. More strikingly, Tyr⋅ formation is completely suppressed in W103F. No change in the reconstitution kinetics was found starting from Fe(II)-preloaded proteins, which shows that the mutations do not affect the rate of iron binding. Our results are consistent with a reaction mechanism using Asp266 and Trp103 for delivery of the external reducing equivalent. Further, the results with W103F suggest that an intact hydrogen-bonded chain is crucial for the reaction, indicating that the external reducing equivalent is a H⋅. Finally, the formation of Tyr⋅ is not the slowest step of the reaction as it is in Escherichia coli R2, consistent with a stronger interaction between Tyr⋅ and the iron center in mouse R2. A new electron paramagnetic resonance visible intermediate named mouseX, strikingly similar to species X found inE. coli R2, was detected only in small amounts under certain conditions. We propose that it may be an intermediate in a side reaction leading to a diferric center without forming the neighboring Tyr⋅.

Mouse and Escherichia coli ribonucleotide reductases (RR) both belong to the same class of RR, where the enzyme consists of two non-identical subunits, proteins R1 and R2. A transient free radical was observed by EPR spectroscopy in the mouse RR reaction with the suicidal inhibitor 2′-azido-2′-deoxycytidine 5′-diphosphate. The detailed hyperfine structure of the EPR spectrum of the transient radical is somewhat different for the mouse and previously studied E. coli enzymes. When the positive allosteric effector ATP was replaced by the negative effector dATP, no transient radical was observed, showing that ‘normal’ binding of the inhibitor to the substrate binding site is required. Using the mouse protein R2 mutants W 103Y and D266A, where the mutations have been shown to specifically block long range electron transfer between the active site of the R1 protein to the iron/radical site in protein R2, no evidence of transient radical was found. Taken together, the data suggest that the radical is located at the active site in protein R1, and is probably of the sulfenylimine type.

Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, each inactive alone. The R1 protein binds the ribonucleotide substrates while the R2 protein contains a binuclear iron center and a tyrosyl free radical, essential for activity. The crystal structures of the corresponding Escherichia coli proteins suggest that the distance from the active site in R1 to the tyrosyl radical buried in R2 is about 35 A. Therefore, an electron pathway was suggested between the active site and the tyrosyl radical. Such a pathway could include a conserved tryptophan on the suggested R1 interaction surface of R2 and a conserved aspartic acid hydrogen bonded both to the tryptophan and to a histidine iron ligand. To find experimental support for such an electron pathway, we have replaced the conserved tryptophan in mouse R2 with phenylalanine or tyrosine and the aspartic acid with alanine. All the mutated R2 proteins were shown to bind metal with the same affinity as native R2 and to form the binuclear iron center. In addition, the W103Y and D266A proteins formed a normal tyrosyl free radical while only low amounts of radical were observed in the W103F protein. Neither the kinetic rate constants nor the equilibrium dissociation constant of the R1/R2 complex was affected by the mutations as shown by BIAcore biosensor technique. However, all mutant R2 proteins were completely inactive in the enzymatic assay, supporting the hypothesis that the tryptophan and aspartic acid residues are important links in an amino acid residue specific long-range electron transfer.

O-glycan variability of glycoproteins expressed by Pichia pastoris and its effects on mannose receptor binding properties