Optimized expression of the Starmerella bombicola lactone esterase in Pichia pastoris through temperature adaptation, codon-optimization and co-expression with HAC1.

The Starmerella bombicola lactone esterase (SBLE) is a novel enzyme that, in vivo, catalyzes the intramolecular esterification (lactonization) of acidic sophorolipids in an aqueous environment. In fact, this is an unusual reaction given the unfavorable conditions for dehydration. This characteristic strongly contributes to the potential of SBLE to become a 'green' tool in industrial applications. Indeed, lactonization occurs normally in organic solvents, an application for which microbial lipases are increasingly used as biocatalysts. Previously, we described the production of recombinant SBLE (rSBLE) in Pichia pastoris (syn. Komagataella phaffii). However, expression was not optimal to delve deeper into the enzyme's potential for industrial application. In the current study, we explored codon-optimization of the SBLE gene and we optimized the rSBLE expression protocol. Temperature reduction had the biggest impact followed by codon-optimization and co-expression of the HAC1 transcription factor. Combining these approaches, we achieved a 32-fold improvement of the yield during rSBLE production (from 0.75 mg/l to 24 mg/L culture) accompanied with a strong reduction of contaminants after affinity purification.

Vegetative Bacillus amyloliquefaciens cells do not confer protection against necrotic enteritis in broilers despite high antibacterial activity of its supernatant against Clostridium perfringens in vitro.

In this study, the effect of Bacillus amyloliquefaciens on Clostridium perfringens was tested in vitro and in vivo. Using an agar well diffusion assay, the inhibitory activity of B. amyloliquefaciens supernatant was analysed against a large collection of netB-positive and netB-negative C. perfringens strains. Although strong growth inhibiting activity was detected against all C. perfringens isolates, it was significantly higher against virulent netB-positive C. perfringens strains compared with avirulent netB-negative isolates. Subsequently, the efficacy of in-feed administration of lyophilised vegetative cells of B. amyloliquefaciens to prevent necrotic enteritis was tested in vivo using an established experimental infection model in broilers. Ross 308 broilers received either B. amyloliquefaciens supplemented or unsupplemented feed throughout the experiment. No significant differences could be detected between the untreated positive control group and the B. amyloliquefaciens treated group in body weight, the number of chickens that developed necrotic lesions and in pathological lesion scores. These results demonstrate that despite its substantial inhibitory activity in vitro, lyophilised vegetative B. amyloliquefaciens cells had no beneficial effect against necrotic enteritis in the in vivo model used here.

Extending the honey bee venome with the antimicrobial peptide apidaecin and a protein resembling wasp antigen 5.

Honey bee venom is a complex mixture of toxic proteins and peptides. In the present study we tried to extend our knowledge of the venom composition using two different approaches. First, worker venom was analysed by liquid chromatography-mass spectrometry and this revealed the antimicrobial peptide apidaecin for the first time in such samples. Its expression in the venom gland was confirmed by reverse transcription PCR and by a peptidomic analysis of the venom apparatus tissue. Second, genome mining revealed a list of proteins with resemblance to known insect allergens or venom toxins, one of which showed homology to proteins of the antigen 5 (Ag5)/Sol i 3 cluster. It was demonstrated that the honey bee Ag5-like gene is expressed by venom gland tissue of winter bees but not of summer bees. Besides this seasonal variation, it shows an interesting spatial expression pattern with additional production in the hypopharyngeal glands, the brains and the midgut. Finally, our immunoblot study revealed that both synthetic apidaecin and the Ag5-like recombinant from bacteria evoke no humoral activity in beekeepers. Also, no IgG4-based cross-reactivity was detected between the honey bee Ag5-like protein and its yellow jacket paralogue Ves v 5.

The growing family of photoactive yellow proteins and their presumed functional roles.

For several years following the discovery and characterization of the first PYP, from Halorhodospira halophila, it was thought that this photoactive protein was quite unique, notwithstanding the isolation of two additional examples in rapid succession. Mainly because of genomic and metagenomic analyses, we have now learned that more than 140 PYP genes occur in a wide variety of bacteria and metabolic niches although the protein has not been isolated in most cases. The amino acid sequences and physical properties permit their organization into at least seven groups that are also likely to be functionally distinct. Based upon action spectra and the wavelength of maximum absorbance, it was speculated nearly 20 years ago but never proven that Hr. halophila PYP was involved in phototaxis. Nevertheless, in only one instance has the functional role and interaction partner for a PYP been experimentally proven, in Rs. centenum Ppr. Genetic context is one of several types of evidence indicating that PYP is potentially involved in a number of diverse functional roles. The interaction with other sensors to modulate their activity stands out as the single most prominent role for PYP. In this review, we have attempted to summarize the evidence for the functional roles and interaction partners for some 26 of the 35 named species of PYP, which should be considered the basis for further focused molecular and biochemical research.

Age-dependent sensitization to the 7S-vicilin-like protein Cor a 11 from hazelnut (Corylus avellana) in a birch-endemic region.

BACKGROUND: Hazelnut (Corylus avellana) allergy exhibits age and geographically distinct sensitization patterns that have not yet been fully resolved. OBJECTIVE: To study sensitization to Cor a 11 in different age groups of hazelnut-allergic patients and infants with atopic dermatitis (AD) sensitized to hazelnut in a birch-endemic region. METHODS: Sera from 80 hazelnut-allergic patients, 33 infants under 1 year of age with AD (24 sensitized and 9 not sensitized to hazelnut), 32 healthy control individuals, and 29 birch pollen-allergic but hazelnut-tolerant individuals were tested for immunoglobulin (Ig) E reactivity to Cor a 11 by ImmunoCAP. IgE reactivity to Cor a 1.01, Cor a 1.04, Cor a 8, and Cor a 9 was studied by ISAC microarray. RESULTS: Forty patients (22 preschool children, 10 schoolchildren, and 8 adults) with systemic reactions on consumption of hazelnut were sensitized to Cor a 11 (respective rates of 36%, 40%, and 12.5%). Forty patients (6 preschool children, 10 schoolchildren, and 24 adults) reported oral allergy syndrome but only 2 of them (of preschool age) were sensitized to Cor a 11. Two (8%) of the AD infants sensitized to hazelnut showed IgE reactivity to Cor a 11. This reactivity was not observed in any of the AD infants without sensitization to hazelnut, in any of the birch-pollen allergic patients without hazelnut allergy, or in any of the healthy control individuals. CONCLUSION: Sensitization to Cor a 11 in a birch-endemic region is predominantly found in children with severe hazelnut allergy, a finding that is consistent with observations concerning sensitization to Cor a 9.

Stability of milk fat globule membrane proteins toward human enzymatic gastrointestinal digestion.

The milk fat globule membrane (MFGM) fraction refers to the thin film of polar lipids and membrane proteins that surrounds fat globules in milk. It is its unique biochemical composition that renders MFGM with some beneficial biological activities, such as anti-adhesive effects toward pathogens. However, a prerequisite for the putative bioactivity of MFGM is its stability during gastrointestinal digestion. We, therefore, subjected MFGM material, isolated from raw milk, to an in vitro enzymatic gastrointestinal digestion. Sodium dodecyl sulfate PAGE, in combination with 2 staining methods, Coomassie Blue and periodic acid Schiff staining, was used to evaluate polypeptide patterns of the digest, whereas mass spectrometry was used to confirm the presence of specific MFGM proteins. Generally, it was observed that glycoproteins showed higher resistance to endogenous proteases compared with non-glycosylated proteins. Mucin 1 displayed the highest resistance to digestion and a considerable part of this protein was still detected at its original molecular weight after gastric and small intestine digestion. Cluster of differentiation 36 was also quite resistant to pepsin. A significant part of periodic acid Schiff 6/7 survived the gastric digestion, provided that the lipid moiety was not removed from the MFGM material. Overall, MFGM glycoproteins are generally more resistant to gastrointestinal digestion than serum milk proteins and the presence of lipids, besides glycosylation, may protect MFGM glycoproteins from gastrointestinal digestion. This gastrointestinal stability makes MFGM glycoproteins amenable to further studies in which their putative health-promoting effects can be explored.

Insights into the venom composition of the ectoparasitoid wasp Nasonia vitripennis from bioinformatic and proteomic studies.

With the Nasonia vitripennis genome sequences available, we attempted to determine the proteins present in venom by two different approaches. First, we searched for the transcripts of venom proteins by a bioinformatic approach using amino acid sequences of known hymenopteran venom proteins. Second, we performed proteomic analyses of crude N. vitripennis venom removed from the venom reservoir, implementing both an off-line two-dimensional liquid chromatography matrix-assisted laser desorption/ ionization time-of-flight (2D-LC-MALDI-TOF) mass spectrometry (MS) and a two-dimensional liquid chromatography electrospray ionization Founer transform ion cyclotron resonance (2D-LC-ESI-FT-ICR) MS setup. This combination of bioinformatic and proteomic studies resulted in an extraordinary richness of identified venom constituents. Moreover, half of the 79 identified proteins were not yet associated with insect venoms: 16 proteins showed similarity only to known proteins from other tissues or secretions, and an additional 23 did not show similarity to any known protein. Serine proteases and their inhibitors were the most represented. Fifteen nonsecretory proteins were also identified by proteomic means and probably represent so-called 'venom trace elements'. The present study contributes greatly to the understanding of the biological diversity of the venom of parasitoid wasps at the molecular level.

Two novel proteins expressed by the venom glands of Apis mellifera and Nasonia vitripennis share an ancient C1q-like domain.

An in-depth proteomic study of previously unidentified two-dimensional polyacrylamide gel electrophoresis spots of honey bee (Apis mellifera, Hymenoptera) venom revealed a new protein with a C1q conserved domain (C1q-VP). BlastP searching revealed a strong identity with only two proteins from other insect species: the jewel wasp, Nasonia vitripennis (Hymenoptera), and the green pea aphid, Acyrthosiphon pisum (Hemiptera). In higher organisms, C1q is the first subcomponent of the classical complement pathway and constitutes a major link between innate and acquired immunity. Expression of C1q-VP in a variety of tissues of honey bee workers and drones was demonstrated. In addition, a wide spatial and temporal pattern of expression was observed in N. vitripennis. We suggest that C1q-VP represents a new member of the emerging group of venom trace elements. Using degenerate primers the corresponding gene was found to be highly conserved in eight hymenopteran species, including species of the Aculeata and the Parasitica groups (suborder Apocrita) and even the suborder Symphyta. A preliminary test using recombinant proteins failed to demonstrate Am_C1q-VP-specific immunoglobulin E recognition by serum from patients with a documented severe bee venom allergy.

The Salmonella Enteritidis TolC outer membrane channel is essential for egg white survival.

Salmonella Enteritidis has developed the potential to contaminate eggs by surviving in the antimicrobial environment of the hen's egg white. This has led to a worldwide pandemic of foodborne salmonellosis infections in humans due to the consumption of contaminated eggs and egg-derived products. The molecular mechanisms of Salmonella Enteritidis egg white survival are not fully clear. Using in vivo expression technology and promoter-reporter fusions we showed that the promoter of the tolC gene, encoding the TolC outer membrane channel that is used by multidrug efflux pumps to export harmful molecules and to secrete bacterial products, is activated by egg white at the chicken body temperature. Using a Salmonella Enteritidis tolC deletion mutant we showed that TolC has an important role in egg white survival. Chromatographic separation techniques and subsequent testing of antimicrobial activities of separated egg white fractions led to the identification of ovotransferrin as the egg white antimicrobial factor which is capable of inhibiting growth of a tolC deletion strain but not the wild type strain. We provide evidence that TolC protects Salmonella Enteritidis against ovotransferrin-mediated growth inhibition in egg white.

Actin proteolysis during ripening of dry fermented sausages at different pH values.

In dry fermented sausages, myofibrillar proteins undergo intense proteolysis generating small peptides and free amino acids that play a role in flavour generation. This study aimed to identify small peptides arising from actin proteolysis, as influenced by the type of processing. Two acidification profiles were imposed, in order to mimic the pH normally obtained in southern-type and northern-type dry fermented sausages. The identification of peptides was done by liquid chromatography coupled to mass spectrometry in a data-independent positive mode of acquisition (LC-MSE). During manufacturing of the dry fermented sausages, actin was highly proteolysed, especially in nine regions of the sequence. After fermentation, 52 and 42 actin-derived peptides were identified at high and low pH, respectively, which further increased to 66 and 144 peptides, respectively, at the end of ripening. Most peptides were released at the cleavage sites of cathepsins B and D, which thus play an important role.

A prediction of DPP IV/CD26 domain structure from a physico-chemical investigation of dipeptidyl peptidase IV (CD26) from human seminal plasma.

Human DPP IV, isolated from seminal plasma by means of immobilised adenosine deaminase, occurs in different forms which are distinguishable by net charge and native molecular weight. Charge differences arise primarily from different degrees of glycosylation containing various amounts of sialic acid. The majority of DPP IV isolated from total seminal plasma consists of the extracellular part of the protein starting at Gly-31. It is a very stable protein resisting high concentrations of denaturant. Unfolding experiments under reducing conditions are indicative of the existence of at least two domains which function independently. One of these domains is highly stabilised by disulfide bonds. Disruption of the disulfide bonds does not affect the activity, the dimeric state nor the adenosine deaminase binding properties of the protein but renders it more susceptible to proteolysis. The low-angle X-ray scattering spectrum is consistent with a model for a protein containing two subunits, each composed of three domains linked by flexible regions with low average mass. The secondary structure composition, determined by FTIR spectrometry, indicates that 45% of the protein consists of beta-sheets, which is higher than expected from computed secondary structure predictions. Our results provide compelling experimental evidence for the three-domain structure of the extracellular part of DPP IV.

A cytochrome c peroxidase from Pseudomonas nautica 617 active at high ionic strength: expression, purification and characterization.

Cytochrome c peroxidase was expressed in cells of Pseudomonas nautica strain 617 grown under microaerophilic conditions. The 36.5 kDa dihaemic enzyme was purified to electrophoretic homogeneity in three chromatographic steps. N-terminal sequence comparison showed that the Ps. nautica enzyme exhibits a high similarity with the corresponding proteins from Paracoccus denitrificans and Pseudomonas aeruginosa. UV-visible spectra confirm calcium activation of the enzyme through spin state transition of the peroxidatic haem. Monohaemic cytochrome c(552) from Ps. nautica was identified as the physiological electron donor, with a half-saturating concentration of 122 microM and allowing a maximal catalytic centre activity of 116,000 min(-1). Using this cytochrome the enzyme retained the same activity even at high ionic strength. There are indications that the interactions between the two redox partners are mainly hydrophobic in nature.

Cross-linking of ubiquitin, HSP27, parkin, and alpha-synuclein by gamma-glutamyl-epsilon-lysine bonds in Alzheimer's neurofibrillary tangles.

The accumulation of misfolded proteins in intracellular inclusions is a generic feature of neurodegenerative disorders. Although heavily ubiquitylated, the aggregated proteins are not degraded by the proteasomes. A possible reason for this phenomenon may be a modification of deposited proteins by transglutaminases forming gamma-glutamyl-epsilon-lysine (GGEL) cross-links between distinct proteins. Here, we show that the frequency of GGEL cross-links is an order of magnitude higher in Alzheimer's brain cortex than in age-matched or younger controls. This difference is due to the accumulation of GGEL cross-links in ubiquitin-immunopositive protein particles present in both Alzheimer's brains and those from aged individuals. The highly cross-linked protein aggregates show immunoreactivity to antibodies against tau and neurofilament proteins, and partially also to alpha-synuclein, indicating that these structures are inherent in Alzheimer's neurofibrillary tangles and Lewy bodies. Using mass sequence analysis, we identified the same six pairs of peptide sequences cross-linked in both senile and Alzheimer's specimens: Gln31 and Gln190 of HSP27 protein are cross-linked with Lys29 and Lys48 of ubiquitin and HSP27 therefore may cross-link two (poly)ubiquitin chains. One lysine residue of parkin and one of alpha-synuclein were also found to be cross-linked. The data suggest that cross-linking of (poly)ubiquitin moieties via HSP27 may have a role in the stabilization of the intraneuronal protein aggregates by interference with the proteasomal elimination of unfolded proteins.

Primary structure of a photoactive yellow protein from the phototrophic bacterium Ectothiorhodospira halophila, with evidence for the mass and the binding site of the chromophore.

The complete amino acid sequence of the 125-residue photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been determined to be MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKEVIGKNFFKDVAP+ ++ CTDSPEFYGKFKEGVASGNLNTMFEYTFDYQMTPTKVKVHMKKALSGDSYWVFVKRV. This is the first sequence to be reported for this class of proteins. There is no obvious sequence homology to any other protein, although the crystal structure, known at 2.4 A resolution (McRee, D.E., et al., 1989, Proc. Natl. Acad. Sci. USA 86, 6533-6537), indicates a relationship to the similarly sized fatty acid binding protein (FABP), a representative of a family of eukaryotic proteins that bind hydrophobic molecules. The amino acid sequence exhibits no greater similarity between PYP and FABP than for proteins chosen at random (8%). The photoactive yellow protein contains an unidentified chromophore that is bleached by light but recovers within a second. Here we demonstrate that the chromophore is bound covalently to Cys 69 instead of Lys 111 as deduced from the crystal structure analysis. The partially exposed side chains of Tyr 76, 94, and 118, plus Trp 119 appear to be arranged in a cluster and probably become more exposed due to a conformational change of the protein resulting from light-induced chromophore bleaching. The charged residues are not uniformly distributed on the protein surface but are arranged in positive and negative clusters on opposite sides of the protein. The exact chemical nature of the chromophore remains undetermined, but we here propose a possible structure based on precise mass analysis of a chromophore-binding peptide by electrospray ionization mass spectrometry and on the fact that the chromophore can be cleaved off the apoprotein upon reduction with a thiol reagent. The molecular mass of the chromophore, including an SH group, is 147.6 Da (+/- 0.5 Da); the cysteine residue to which it is bound is at sequence position 69.

An additional aromatic interaction improves the thermostability and thermophilicity of a mesophilic family 11 xylanase: structural basis and molecular study.

In a general approach to the understanding of protein adaptation to high temperature, molecular models of the closely related mesophilic Streptomyces sp. S38 Xyl1 and thermophilic Thermomonospora fusca TfxA family 11 xylanases were built and compared with the three-dimensional (3D) structures of homologous enzymes. Some of the structural features identified as potential contributors to the higher thermostability of TfxA were introduced in Xyl1 by site-directed mutagenesis in an attempt to improve its thermostability and thermophilicity. A new Y11-Y16 aromatic interaction, similar to that present in TfxA and created in Xyl1 by the T11Y mutation, improved both the thermophilicity and thermostability. Indeed, the optimum activity temperature (70 vs. 60 degrees C) and the apparent Tm were increased by about 9 degrees C, and the mutant was sixfold more stable at 57 degrees C. The combined mutations A82R/F168H/N169D/delta170 potentially creating a R82-D169 salt bridge homologous to that present in TfxA improved the thermostability but not the thermophilicity. Mutations R82/D170 and S33P seemed to be slightly destabilizing and devoid of influence on the optimal activity temperature of Xyl1. Structural analysis revealed that residues Y11 and Y16 were located on beta-strands B1 and B2, respectively. This interaction should increase the stability of the N-terminal part of Xyl1. Moreover, Y11 and Y16 seem to form an aromatic continuum with five other residues forming putative subsites involved in the binding of xylan (+3, +2, +1, -1, -2). Y11 and Y16 might represent two additional binding subsites (-3, -4) and the T11Y mutation could thus improve substrate binding to the enzyme at higher temperature and thus the thermophilicity of Xyl1.

In vitro evidence that an 11-kilodalton N-terminal fragment of proopiomelanocortin is a growth factor specifically stimulating the development of lactotrophs in rat pituitary during postnatal life.

We have previously shown that in reaggregate cell cultures of 14-day-old female rat pituitary, LHRH is capable of stimulating lactotrophs to enter the S-phase of the cell cycle, and that this effect is mediated by paracrine growth factors secreted from gonadotrophs. In the present report we describe the isolation, purification, and chemical characterization of one of these growth factors. Gonadotroph-rich aggregates from 14-day-old female rats were cultured for 6-7 weeks in serum-free and serum albumin-free defined medium in the presence of 0.01 nM LHRH. A total volume of 3.2 liters containing material secreted from 2.5 x 10(8) cells was batchwise concentrated on Sep-Pak C18/125 A cartridges (10 g) and retained molecules ultrafiltrated on Centricon 3 membrane filters [mol wt (M(r)) cut-off, 3 kilodaltons (kDa)]. Material capable of increasing the number of [3H]thymidine ([3H]T)-incorporating lactotrophs in pituitary cell aggregates of 2-week-old rats was isolated by chromatography on two reverse phase HPLC columns, one HPLC gel filtration column, and a final reverse phase HPLC column. A substance was obtained which, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, ran as a single band with an apparent M(r) of 16 kDa. On gel filtration, the apparent M(r) was 11 kDa. N-Terminal amino acid sequence analysis revealed that the substance was a peptide; a sequence of 10 residues was obtained, which was identical to that in the N-terminal part of rat POMC. By electrospray ionization mass spectrometry, six different compounds with slightly different M(r), ranging from 10,796-11,108 daltons, were detected. The latter data suggest that the peptide extends C-terminally at least to residue 74, which in the POMC sequence is flanked by an Arg-Arg dibasic residue, a posttranslational cleavage site. The substance increased the number of [3H]T-incorporating lactotrophs in pituitary cell aggregates without affecting [3H]T labeling of other pituitary cell types. Authentic human POMC-(1-76), at a concentration of 10 nM provoked a similar stimulation of the [3H]T-labeled lactotroph number without affecting other cell types. It is concluded that POMC-(1-74) is a growth factor that specifically targets lactotrophs during postnatal development of the rat anterior pituitary.

Isolation and characterization of an adipokinetic hormone release-inducing factor in locusts: the crustacean cardioactive peptide.

A methanolic extract of 7000 desert locust (Schistocerca gregaria) brains contains several factors that stimulate the in vitro release of adipokinetic hormone (AKH) by glandular cells of locust (Locusta migratoria and Schistocerca gregaria) corpora cardiaca. The most potent one has now been fully identified. Matrix-assisted laser desorption ionization mass spectrometry-time of flight analysis revealed a mass of 954.6 Da. The primary structure of the peptide, Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2, appeared identical to that of a previously identified crustacean cardioactive peptide. This myotropin was first isolated from the shore crab, Carcinus maenas, and later from several insect species, but was never reported in the context of AKH release. The present study shows that synthetic crustacean cardioactive peptide induces the release of AKH from corpora cardiaca in a dose-dependent manner when tested in concentrations ranging from 10(-5)-10(-9) M. This is the first demonstration in invertebrates of a peptide neurohormone controlling the release of a second peptide hormone.