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BackgroundBiocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co-terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts.ResultsP. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter MhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive.ConclusionOur results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts.

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Introduction: The actin regulatory protein, cofilin plays a key signaling role in many cells for numerous cellular responses including in proliferation, development, motility, migration, secretion and growth. In the pancreas it is important in islet insulin secretion, growth of pancreatic cancer cells and in pancreatitis. However, there are no studies on its role or activation in pancreatic acinar cells. Methods: To address this question, we studied the ability of CCK to activate cofilin in pancreatic acinar cells, AR42J cells and CCK1-R transfected Panc-1 cells, the signaling cascades involved and its effect on enzyme secretion and MAPK activation, a key mediator of pancreatic growth. Results: CCK (0.3 and 100 nM), TPA, carbachol, Bombesin, secretin and VIP decreased phospho-cofilin (i.e., activate cofilin) and both phospho-kinetic and inhibitor studies of cofilin, LIM kinase (LIMK) and Slingshot Protein Phosphatase (SSH1) demonstrated these conventional activators of cofilin were not involved. Serine phosphatases inhibitors (calyculin A and okadaic acid), however inhibited CCK/TPA-cofilin activation. Studies of various CCK-activated signaling cascades showed activation of PKC/PKD, Src, PAK4, JNK, ROCK mediated cofilin activation, but not PI3K, p38, or MEK. Furthermore, using both siRNA and cofilin inhibitors, cofilin activation was shown to be essential for CCK-mediated enzyme secretion and MAPK activation. Conclusion: These results support the conclusion that cofilin activation plays a pivotal convergent role for various cell signaling cascades in CCK mediated growth/enzyme secretion in pancreatic acini. [ABSTRACT FROM AUTHOR]

Introduction: The actin regulatory protein, cofilin plays a key signaling role in many cells for numerous cellular responses including in proliferation, development, motility, migration, secretion and growth. In the pancreas it is important in islet insulin secretion, growth of pancreatic cancer cells and in pancreatitis. However, there are no studies on its role or activation in pancreatic acinar cells.Methods: To address this question, we studied the ability of CCK to activate cofilin in pancreatic acinar cells, AR42J cells and CCK1-R transfected Panc-1 cells, the signaling cascades involved and its effect on enzyme secretion and MAPK activation, a key mediator of pancreatic growth.Results: CCK (0.3 and 100 nM), TPA, carbachol, Bombesin, secretin and VIP decreased phospho-cofilin (i.e., activate cofilin) and both phospho‐kinetic and inhibitor studies of cofilin, LIM kinase (LIMK) and Slingshot Protein Phosphatase (SSH1) demonstrated these conventional activators of cofilin were not involved. Serine phosphatases inhibitors (calyculin A and okadaic acid), however inhibited CCK/TPA-cofilin activation. Studies of various CCK‐activated signaling cascades showed activation of PKC/PKD, Src, PAK4, JNK, ROCK mediated cofilin activation, but not PI3K, p38, or MEK. Furthermore, using both siRNA and cofilin inhibitors, cofilin activation was shown to be essential for CCK-mediated enzyme secretion and MAPK activation.Conclusion: These results support the conclusion that cofilin activation plays a pivotal convergent role for various cell signaling cascades in CCK mediated growth/enzyme secretion in pancreatic acini.

The role of biocontrol products is expected to increase worldwide consumer demand and facilitate the implementation of sustainable agricultural policies. New biocontrol agents must allow for an effective crop-protection strategy in sustainable agriculture. Yeasts are microorganisms living in various niches of the environment that can be antagonists of many plant pathogens. Yeasts rapidly colonize plant surfaces, use nutrients from many sources, survive in a relatively wide temperature range, produce no harmful metabolites and have no deleterious effects on the final food products. Hence, they can be a good biocontrol agent. In this paper, the biological characteristics and potential of yeast are summarized. Additionally, the mechanisms of yeasts as plant-protection agents are presented. This includes the production of volatile organic compounds, production of killer toxins, competition for space and nutrient compounds, production of lytic enzymes, induction of plant immunity and mycoparasitism. The mechanisms of yeast interaction with plant hosts are also described, and examples of yeasts used for pre- and postharvest biocontrol are provided. Commercially available yeast-based products are listed and challenges for yeast-based products are described. [ABSTRACT FROM AUTHOR]

Lignocellulosic biomass is an inexpensive and abundant renewable carbon feedstock available for the sustainable production of fuels and chemicals. However, the process for obtaining pentose and hexose sugars from the hemicellulosic components of plant biomass requires the use of expensive purified enzymes. In this study, Bacillus subtilis strains were first constructed to enable the extracellular depolymerization of hemicellulose at a higher rate. Three different signal peptides (YwmC sp , SacC sp , and AmyE sp) were explored for the secretion of two endo-1,4-β-xylanases (from Trichoderma reesei and Bacillus pumilis), leading to the identification of an optimal design by which B. subtilis could secrete xylanase and effectively depolymerize xylan (the major hemicellulose component). In situ depolymerization of xylan by the engineered B. subtilis (SSL26) produced a maximum xylose titer of 7.1 g/L, corresponding to 66.7% of the total xylose initially present in 13.3 g/L of xylan. To demonstrate the application of this strain in consolidated bioprocessing, a B. subtilis-Escherichia coli consortium was developed by culturing SSL26 together with an E. coli strain X2S, a xylose assimilating succinate producer. Lastly, to demonstrate the generalizability of this approach for fuels and chemicals production, coculture studies were conducted for the production of ethanol and D-lactate from xylan. Together, this novel coculture consolidated bioprocessing (CCBP) enabled the production of succinate, ethanol, and D-lactate directly from xylan at a maximum titre of 3.9 g/L, 2 g/L, and 2 g/L respectively. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: Fungal saccharification of lignocellulosic biomass occurs concurrently with the secretion of a diverse collection of proteins, together functioning as a catalytic system to liberate soluble sugars from insoluble composite biomaterials. How different fungi respond to different substrates is of fundamental interest to the developing biomass saccharification industry. Among the cornerstones of fungal enzyme systems are the highly expressed cellulases (endo-β-glucanases and cellobiohydrolases). Recently, a cyclophellitol-derived activity-based probe (ABP-Cel) was shown to be a highly sensitive tool for the detection and identification of cellulases. Results: Here we show that ABP-Cel enables endo-β-glucanase profiling in diverse fungal secretomes. In combination with established ABPs for β-xylanases and β-d-glucosidases, we collected multiplexed in-gel fluorescence activity-based protein profiles of 240 secretomes collected over ten days from biological replicates of ten different basidiomycete fungi grown on maltose, wheat straw, or aspen pulp. Our results reveal the remarkable dynamics and unique enzyme fingerprints associated with each species substrate combination. Chemical proteomic analysis identifies significant arsenals of cellulases secreted by each fungal species during growth on lignocellulosic biomass. Recombinant production and characterization of a collection of probe-reactive enzymes from GH5, GH10, and GH12 confirm that ABP-Cel shows broad selectivity towards enzymes with endo-β-glucanase activity. Conclusion: Using small-volume samples with minimal sample preparation, the results presented here demonstrate the ready accessibility of sensitive direct evidence for fungal enzyme secretion during early stages of growth on complex lignocellulosic substrates. [ABSTRACT FROM AUTHOR]

Teratosphaeria destructans is one of the most aggressive foliar pathogens of Eucalyptus. The biological factors underpinning T. destructans infections, which include shoot and leaf blight on young trees, have never been interrogated. Thus, the means by which the pathogen modifies its host environment to overcome host defences remain unknown. By applying transcriptome sequencing, the aim of this study was to compare gene expression in a South African isolate of T. destructans grown on nitrogen-deficient and complete media. This made it possible to identify upregulated genes in a nitrogen-starved environment, often linked to the pathogenicity of the fungus. The results support the hypothesis that nitrogen starvation in T. destructans likely mirrors an in planta genetic response. This is because 45% of genes that were highly upregulated under nitrogen starvation have previously been reported to be associated with infection in other pathogen systems. These included several CAZymes, fungal effector proteins, peptidases, kinases, toxins, lipases and proteins associated with detoxification of toxic compounds. Twenty-five secondary metabolites were identified and expressed in both nitrogen-deficient and complete conditions. Additionally, the most highly expressed genes in both growth conditions had pathogenicity-related functions. This study highlights the large number of expressed genes associated with pathogenicity and overcoming plant defences. As such, the generated baseline knowledge regarding pathogenicity and aggressiveness in T. destructans is a valuable reference for future in planta work. [ABSTRACT FROM AUTHOR]

The role of biocontrol products is expected to increase worldwide consumer demand and facilitate the implementation of sustainable agricultural policies. New biocontrol agents must allow for an effective crop-protection strategy in sustainable agriculture. Yeasts are microorganisms living in various niches of the environment that can be antagonists of many plant pathogens. Yeasts rapidly colonize plant surfaces, use nutrients from many sources, survive in a relatively wide temperature range, produce no harmful metabolites and have no deleterious effects on the final food products. Hence, they can be a good biocontrol agent. In this paper, the biological characteristics and potential of yeast are summarized. Additionally, the mechanisms of yeasts as plant-protection agents are presented. This includes the production of volatile organic compounds, production of killer toxins, competition for space and nutrient compounds, production of lytic enzymes, induction of plant immunity and mycoparasitism. The mechanisms of yeast interaction with plant hosts are also described, and examples of yeasts used for pre- and postharvest biocontrol are provided. Commercially available yeast-based products are listed and challenges for yeast-based products are described.

Approximately a tenth of all described fungal species can cause diseases in plants. A common feature of this process is the necessity to pass through the plant cell wall, an important barrier against pathogen attack. To this end, fungi possess a diverse array of secreted enzymes to depolymerize the main structural polysaccharide components of the plant cell wall, i.e., cellulose, hemicellulose, and pectin. Recent advances in genomic and systems-level studies have begun to unravel this diversity and have pinpointed cell wall-degrading enzyme (CWDE) families that are specifically present or enhanced in plant-pathogenic fungi. In this review, we discuss differences between the CWDE arsenal of plant-pathogenic and non-plant-pathogenic fungi, highlight the importance of individual enzyme families for pathogenesis, illustrate the secretory pathway that transports CWDEs out of the fungal cell, and report the transcriptional regulation of expression of CWDE genes in both saprophytic and phytopathogenic fungi.

Abstract Background β-Xylosidases are glycoside hydrolases (GHs) that cleave xylooligosaccharides and/or xylobiose into shorter oligosaccharides and xylose. Aspergillus nidulans is an established genetic model and good source of carbohydrate-active enzymes (CAZymes). Most fungal enzymes are N-glycosylated, which influences their secretion, stability, activity, signalization, and protease protection. A greater understanding of the N-glycosylation process would contribute to better address the current bottlenecks in obtaining high secretion yields of fungal proteins for industrial applications. Results In this study, BxlB—a highly secreted GH3 β-xylosidase from A. nidulans, presenting high activity and several N-glycosylation sites—was selected for N-glycosylation engineering. Several glycomutants were designed to investigate the influence of N-glycans on BxlB secretion and function. The non-glycosylated mutant (BxlBnon-glyc) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlBwt), while a partially glycosylated mutant (BxlBN1;5;7) exhibited increased activity. Additionally, there was no enzyme secretion in the mutant in which the N-glycosylation context was changed by the introduction of four new N-glycosylation sites (BxlBCC), despite the high transcript levels. BxlBwt, BxlBnon-glyc, and BxlBN1;5;7 formed similar secondary structures, though the mutants had lower melting temperatures compared to the wild type. Six additional glycomutants were designed based on BxlBN1;5;7, to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlBN1;5 and BxlBN5;7) showed improved catalytic efficiency, whereas the other four mutants’ catalytic efficiencies were reduced. The N-glycosylation site N5 is important for improved BxlB catalytic efficiency, but needs to be complemented by N1 and/or N7. Molecular dynamics simulations of BxlBnon-glyc and BxlBN1;5 reveals that the mobility pattern of structural elements in the vicinity of the catalytic pocket changes upon N1 and N5 N-glycosylation sites, enhancing substrate binding properties which may underlie the observed differences in catalytic efficiency between BxlBnon-glyc and BxlBN1;5. Conclusions This study demonstrates the influence of N-glycosylation on A. nidulans BxlB production and function, reinforcing that protein glycoengineering is a promising tool for enhancing thermal stability, secretion, and enzymatic activity. Our report may also support biotechnological applications for N-glycosylation modification of other CAZymes.

In a previous study, we found that plasma can enhance spore germination and α-amylase secretion in A. oryzae, a beneficial fungus used in fermentation. To confirm this, in the current study, we investigated the effects of plasma on development and α-amylase secretion using an enlarged sample size and a different plasma source: a plasma jet. There was a ~10% (p < 0.01) increase in spore germination upon non-thermal atmospheric pressure plasma jet (NTAPPJ) treatment for 5 min and 10 min, as compared with the control (no plasma treatment). The activity of α-amylase detected in potato dextrose broth (PDB) media during incubation was significantly elevated in plasma-treated samples, with a more obvious increase upon 10 min and 15 min treatments and 24–96 h incubation periods. The levels of the oxidation reduction potential (ORP) and NOX (nitrogen oxide species) were higher in the plasma-treated samples than in the control samples, suggesting that these two variables could serve as standard indicators for enhancing α-amylase activity after plasma treatment. Genome sequencing analysis showed approximately 0.0016–0.0017% variations (changes in 596–655 base pairs out of a total of 37,912,014 base pairs) in the genomic DNA sequence of A. oryzae after plasma treatment. Our results suggest that NATPPJ can enhance the spore germination and extracellular activity of α-amylase, probably by increasing the levels of ORP and NOX to an optimum level. [ABSTRACT FROM AUTHOR]

The opportunistic pathogens comprising the Candida haemulonii complex (C. haemulonii , C. duobushaemulonii and C. haemulonii var. vulnera) are notable for their intrinsic resistance to different antifungal classes. Little is known about the virulence attributes in this emerging fungal complex. However, it is well-recognized that enzymes play important roles in virulence/pathogenesis of candidiasis. Herein, we aimed to identify aspartyl-type peptidases in 12 clinical isolates belonging to the C. haemulonii complex. All isolates were able to grow in a chemically defined medium containing albumin as the sole nitrogen source, and a considerable consumption of this protein occurred after 72–96 h. C. haemulonii var. vulnera isolates showed the lowest albumin degradation capability and the poorest growth rate. The measurement of secreted aspartyl peptidase (Sap) activity, using the cathepsin D fluorogenic substrate, varied from 91.6 to 413.3 arbitrary units and the classic aspartyl peptidase inhibitor, pepstatin A, significantly blocked the Sap released by C. haemulonii complex. No differences were observed in the Sap activity among the three fungal species. Flow cytometry, using a polyclonal antibody against Sap1-3 of C. albicans , detected homologous proteins at the surface of C. haemulonii complex (anti-Sap1-3-labeled cells ranged from 24.6 to 79.1%). Additionally, the immunoblotting assay, conducted with the same Sap1-3 antibody, recognized a protein of ∼50 kDa in all fungal isolates. A glimpse in the genome of these fungi revealed several potential proteins containing Sap1-3-like conserved domain. Altogether, our results demonstrated the potential of C. haemulonii species complex to produce Saps, an important virulence factor of Candida spp. • Enzymes play an important role in virulence/pathogenesis of candidiasis. • Candida haemulonii species complex are able to produce aspartyl peptidases in vitro. • The genome of C. haemulonii possess proteins containing Sap-like conserved domain. [ABSTRACT FROM AUTHOR]

A major hurdle in the production of bioethanol with second-generation feedstocks is the high cost of the enzymes for saccharification of the lignocellulosic biomass into fermentable sugars. Simultaneous saccharification and fermentation with Saccharomyces cerevisiae yeast that secretes a range of lignocellulolytic enzymes might address this problem, ideally leading to consolidated bioprocessing. However, it has been unclear how many enzymes can be secreted simultaneously and what the consequences would be on the C6 and C5 sugar fermentation performance and robustness of the second-generation yeast strain. We have successfully expressed seven secreted lignocellulolytic enzymes, namely endoglucanase, β-glucosidase, cellobiohydrolase I and II, xylanase, β-xylosidase and acetylxylan esterase, in a single second-generation industrial S. cerevisiae strain, reaching 94.5 FPU/g CDW and enabling direct conversion of lignocellulosic substrates into ethanol without preceding enzyme treatment. Neither glucose nor the engineered xylose fermentation were significantly affected by the heterologous enzyme secretion. This strain can therefore serve as a promising industrial platform strain for development of yeast cell factories that can significantly reduce the enzyme cost for saccharification of lignocellulosic feedstocks. Image 1 • Successful expression of seven secreted lignocellulolytic enzymes in second-generation industrial yeast strain. • Combined secreted activity of nearly 100 FPU/g CDW. • Direct fermentation of multiple lignocellulosic polymers into bioethanol. • No significant decline in glucose or xylose fermentation capacity. [ABSTRACT FROM AUTHOR]

In a previous study, we found that plasma can enhance spore germination and α-amylase secretion in A. oryzae, a beneficial fungus used in fermentation. To confirm this, in the current study, we investigated the effects of plasma on development and α-amylase secretion using an enlarged sample size and a different plasma source: a plasma jet. There was a ~10% (p < 0.01) increase in spore germination upon non-thermal atmospheric pressure plasma jet (NTAPPJ) treatment for 5 min and 10 min, as compared with the control (no plasma treatment). The activity of α-amylase detected in potato dextrose broth (PDB) media during incubation was significantly elevated in plasma-treated samples, with a more obvious increase upon 10 min and 15 min treatments and 24–96 h incubation periods. The levels of the oxidation reduction potential (ORP) and NOX (nitrogen oxide species) were higher in the plasma-treated samples than in the control samples, suggesting that these two variables could serve as standard indicators for enhancing α-amylase activity after plasma treatment. Genome sequencing analysis showed approximately 0.0016–0.0017% variations (changes in 596–655 base pairs out of a total of 37,912,014 base pairs) in the genomic DNA sequence of A. oryzae after plasma treatment. Our results suggest that NATPPJ can enhance the spore germination and extracellular activity of α-amylase, probably by increasing the levels of ORP and NOX to an optimum level.

[Display omitted] • Pseudomonas putida is a promising strain for lignin valorisation. • A secretion system for recombinant lignin-depolymerising peroxidase was developed. • PpDyP was added to the flagellar type III secretion system. • Efficient oxidation activity was obtained against lignin model compounds. • The engineered strain showed efficient assimilation of various lignin substrates. Pseudomonas putida is a promising strain for lignin valorisation. However, there is a dearth of stable and efficient systems for secreting enzymes to enhance the process. Therefore, a novel secretion system for recombinant lignin–depolymerising peroxidase was developed. By adopting a flagellar type III secretion system, P. putida KT-M2, a secretory host strain, was constructed and an optimal secretion signal fusion partner was identified. Application of the dye-decolourising peroxidase of P. putida to this system resulted in efficient oxidation activity of the cell-free supernatant against various chemicals, including lignin model compounds. This peroxidase-secreting strain was examined to confirm its lignin utilisation capability, resulting in the efficient assimilation of various lignin substrates with 2.6-fold higher growth than that of the wild-type strain after 72 h of cultivation. Finally, this novel system will lead efficient bacterial lignin breakdown and utilization through enzyme secretion, paving the way for sustainable lignin-consolidated bioprocessing. [ABSTRACT FROM AUTHOR]

Abstract: BACKGROUND: Recently, probiotics have increasingly been used as feed additives in poultry diets as an alternative to antibiotic growth promoters fostering resistance development. RESULTS: This study was aimed at assessing the potential of Bacillus amyloliquefaciens US573 as a direct‐fed microbial. The US573 strain was found to be free of harmful enzymatic activities and sensitive to antibiotics. In addition, it showed a good acid and bovine bile tolerance, high adhesion efficacy to chicken enterocytes, and an ability to form biofilms, which may favor its survival and persistence in the animal gastrointestinal tract. Moreover, besides the previously described extremely salt‐tolerant and highly thermostable phytase, the US573 strain secretes xylanase, β‐glucanase and amylase activities useful in neutralizing antinutritional factors and maximizing the absorption of nutrients. The secretion of such enzymes may be responsible for the good performance of the US573 isolate in the digestibility of wheat in vitro. Indeed, using the vegetative cells, a yield of wheat dry matter digestibility of approximately 48% was achieved, which is slightly lower than the commercial feed additive Rovabio used as a reference (56.73% digestibility). CONCLUSION: The obtained results illustrate the potential of US573 strain as a promising direct‐fed microbial candidate for application in the poultry industry. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Melatonin is an indoleamine produced from the amino acid l-tryptophan, whereas metabolites of melatonin are known as kynuramines. One of the best-known kynuramines is N1-acetyl-N1-formyl-5-methoxykynuramine (AFMK). Melatonin has attracted scientific attention as a potent antioxidant and protector of tissue against oxidative stress. l-Tryptophan and kynuramines share common beneficial features with melatonin. Melatonin was originally discovered as a pineal product, has been detected in the gastrointestinal tract, and its receptors have been identified in the pancreas. The role of melatonin in the pancreatic gland is not explained, however several arguments support the opinion that melatonin is probably implicated in the physiology and pathophysiology of the pancreas. (1) Melatonin stimulates pancreatic enzyme secretion through the activation of entero-pancreatic reflex and cholecystokinin (CCK) release. l-Tryptophan and AFMK are less effective than melatonin in the stimulation of pancreatic exocrine function; (2) Melatonin is a successful pancreatic protector, which prevents the pancreas from developing of acute pancreatitis and reduces pancreatic damage. This effect is related to its direct and indirect antioxidant action, to the strengthening of immune defense, and to the modulation of apoptosis. Like melatonin, its precursor and AFMK are able to mimic its protective effect, and it is commonly accepted that all these substances create an antioxidant cascade to intensify the pancreatic protection and acinar cells viability; (3) In pancreatic cancer cells, melatonin and AFMK activated a signal transduction pathway for apoptosis and stimulated heat shock proteins. The role of melatonin and AFMK in pancreatic tumorigenesis remains to be elucidated. [ABSTRACT FROM AUTHOR]

Horseradish peroxidase (HRP) is a high-demand enzyme for applications in diagnostics, bioremediation, biocatalysis and medicine. Current HRP preparations are isolated from horseradish roots as mixtures of biochemically diverse isoenzymes. Thus, there is a strong need for a recombinant production process enabling a steady supply with enzyme preparations of consistent high quality. However, most current recombinant production systems are limited at titers in the low mg/L range. In this study, we used the well-known yeast Pichia pastoris as host for recombinant HRP production. To enhance recombinant enzyme titers we systematically evaluated engineering approaches on the secretion process, coproduction of helper proteins, and compared expression from the strong methanol-inducible P AOX1 promoter, the strong constitutive P GAP promoter, and a novel bidirectional promoter P HTX1 . Ultimately, coproduction of HRP and active Hac1 under P HTX1 control yielded a recombinant HRP titer of 132 mg/L after 56 h of cultivation in a methanol-independent and easy-to-do bioreactor cultivation process. With regard to the many versatile applications for HRP, the establishment of a microbial host system suitable for efficient recombinant HRP production was highly overdue. The novel HRP production platform in P. pastoris presented in this study sets a new benchmark for this medically relevant enzyme. [ABSTRACT FROM AUTHOR]

In fungi a genetic system ensures that enzymes are secreted mainly at ambient pH values corresponding to their optima of activity. Although a great deal of information has been obtained concerning this environmental response, there is a lack of studies involving phytopathogenic, endophytic and entomopathogenic fungi as well as different aspects of fungus-host interactions. This study compares in a plate-clearing assays, the effect of ambient pH in the secretion of amylase, cellulase, lipase, pectinase and protease by endophytic, phytopathogenic, and entomopathogenic isolates belonging to several species of Colletotrichum. All enzymes were secreted in a pH-dependent manner by all isolates. Endophytes and pathogens showed distinct patterns of protease secretion, with optima at alkaline and acid growth conditions, respectively. In liquid medium, a Pi-repressible acid phosphatase of an endophytic isolate responded to ambient pH, having a 14-fold increase in secreted specific activity at acid pH, as compared to alkaline pH. Furthermore, part of a Colletotrichum pacC homologue gene, coding for a transcriptional factor responsible for pH-regulated gene expression, was cloned. Ambient pH seems to be a general factor controlling enzyme secretion in fungus-host interactions through a conserved genetic circuit.

Melatonin is an indoleamine produced from the amino acid l-tryptophan, whereas metabolites of melatonin are known as kynuramines. One of the best-known kynuramines is N1-acetyl-N1-formyl-5-methoxykynuramine (AFMK). Melatonin has attracted scientific attention as a potent antioxidant and protector of tissue against oxidative stress. l-Tryptophan and kynuramines share common beneficial features with melatonin. Melatonin was originally discovered as a pineal product, has been detected in the gastrointestinal tract, and its receptors have been identified in the pancreas. The role of melatonin in the pancreatic gland is not explained, however several arguments support the opinion that melatonin is probably implicated in the physiology and pathophysiology of the pancreas. (1) Melatonin stimulates pancreatic enzyme secretion through the activation of entero-pancreatic reflex and cholecystokinin (CCK) release. l-Tryptophan and AFMK are less effective than melatonin in the stimulation of pancreatic exocrine function; (2) Melatonin is a successful pancreatic protector, which prevents the pancreas from developing of acute pancreatitis and reduces pancreatic damage. This effect is related to its direct and indirect antioxidant action, to the strengthening of immune defense, and to the modulation of apoptosis. Like melatonin, its precursor and AFMK are able to mimic its protective effect, and it is commonly accepted that all these substances create an antioxidant cascade to intensify the pancreatic protection and acinar cells viability; (3) In pancreatic cancer cells, melatonin and AFMK activated a signal transduction pathway for apoptosis and stimulated heat shock proteins. The role of melatonin and AFMK in pancreatic tumorigenesis remains to be elucidated.

The recombinant thermo-alkali-stable endoxylanase (Bh-xyl) of polyextremophilic bacterium Bacillus halodurans TSEV1 has been produced extracellularly using a combination of cloning strategies and physico-chemical treatment of recombinant Escherichia coli cells. Sixty percent higher secretion of recombinant xylanase has been achieved by cloning Bh-xyl in pET28a(+) and expression followed by optimization of the cultural variables (EDTA, lysozyme and temperature). The pure recombinant endoxylanase is of 42 kDa, which is active in the broad pH and temperature ranges between 7.0 and 12.0, and 30 and 100 °C, with optima at 9.0 and 70 °C. The K m , V max and k cat values of the Bh-xyl (birchwood xylan) are 2.6 mg ml −1 , 252.3 μmol mg −1 min −1 and 3.36 × 10 3 min −1 , respectively. The enzyme has higher affinity for soft wood xylan than most of the xylanases from extremophilic microbes. The endoxylanase efficiently liberated xylooligosaccharides from the renewable agro-residues, which find application in functional foods as prebiotics. [ABSTRACT FROM AUTHOR]

Aspergillus oryzae is used to produce traditional fermented foods and beverages. A. oryzae 3.042 produces a neutral protease and an alkaline protease but rarely an acid protease, which is unfavourable to soy-sauce fermentation. A. oryzae 100-8 was obtained by N ion implantation mutagenesis of A. oryzae 3.042, and the protease secretions of these two strains are different. Sequencing the genome of A. oryzae 100-8 and comparing it to the genomes of A. oryzae 100-8 and 3.042 revealed some differences, such as single nucleotide polymorphisms, nucleotide deletion or insertion. Some of these differences may reflect the ability of A. oryzae to secrete proteases. Transcriptional sequencing and analysis of the two strains during the same growth processes provided further insights into the genes and pathways involved in protease secretion. [ABSTRACT FROM AUTHOR]

The effects of L-sorbose on growth, morphology and production of a multifunctional ß-glucosidase by the thermophilic fungus Humicola grisea var. thermoidea were investigated. Sorbose increased the lag phase period 3-fold and drastically altered the morphology of the fungal hyphae. Cellobiose and lactose were good inducers of the enzyme. The addition of 5 % sorbose to cultures containing 1 % cellobiose enhanced the extracellular levels of the ß-glucosidase 3.3-fold with constant cytosolic and cell-wall bound levels, demonstrating stimulation of both enzyme synthesis and secretion. The stimulation of enzyme production by sorbose was dependent on the presence of cellobiose as inducer, since 2- to 3-fold inhibition was observed in lactose and glucose. Production and secretion of phosphatases and endoglucanases was not stimulated by sorbose, which did not affect the subcellular distribution of the ß-glucosidase also. However, it reduced the uptake rates of glucose and cellobiose. Taken together, the results discarded increased non-specific enzyme secretion and/or increased release of the enzyme from the cell-wall as possible molecular mechanisms for the effects of sorbose on the production of the multifunctional ß-glucosidase by H. grisea. An alternative mechanism, based on a prolonged action of cellobiose as inducer associated with a decreased catabolic repression by glucose, was discussed. [ABSTRACT FROM AUTHOR]

Abstract: In fungi, ambient pH sensing involves the activation of the Pal/PacC signalling pathway. In the dermatophyte Trichophyton rubrum, pH-dependent secretion of keratinases, which are major virulence determinants, is affected by disruption of the pacC gene. Here, the transcription profiling of the genes coding for N- and O-linked mannosyltransferases, enzymes involved in protein glycosylation, was evaluated in T. rubrum in response to disruption of the pacC gene and growth in keratin, glucose, and glucose plus glycine. We show that transcription of these mannosyltransferase genes is affected by nutrients at acidic pH and by PacC. [Copyright &y& Elsevier]

We have developed a simple, rapid, quantitative colorimetric assay to measure cellulose degradation based on the absorbance shift of Congo red dye bound to soluble cellulose. We term this assay 'Congo Red Analysis of Cellulose Concentration,' or 'CRACC.' CRACC can be performed directly in culture media, including rich and defined media containing monosaccharides or disaccharides (such as glucose and cellobiose). We show example experiments from our laboratory that demonstrate the utility of CRACC in probing enzyme kinetics, quantifying cellulase secretion, and assessing the physiology of cellulolytic organisms. CRACC complements existing methods to assay cellulose degradation, and we discuss its utility for a variety of applications. [ABSTRACT FROM AUTHOR]

Abstract: We cultivated a filamentous fungus, Aspergillus oryzae IAM 2706 by three different cultivation methods, i.e., shaking-flask culture (SFC), agar-plate culture (APC), and membrane-surface liquid culture (MSLC), to elucidate the differences of its behaviors by different cultivation methods under the same media, by measuring the growth, secretion of proteases and α-amylase, secreted protein level, and gene transcriptional profile by the DNA microarray analysis. The protease activities detected by MSLC and APC were much higher than that by SFC, using both modified Czapek–Dox (mCD) and dextrin–peptone–yeast extract (DPY) media. The α-amylase activity was detected in MSLC and APC in a much larger extent than that in SFC when DPY medium was used. On the basis of SDS–PAGE analyses and N-terminal amino acid sequences, 6 proteins were identified in the supernatants of the culture broths using DPY medium, among which oryzin (alkaline protease) and α-amylase were detected at a much higher extent for APC and MSLC than those for SFC while only oryzin was detected in mCD medium, in accordance with the activity measurements. A microarray analysis for the fungi cultivated by SFC, APC, and MSLC using mCD medium was carried out to elucidate the differences in the gene transcriptional profile by the cultivation methods. The gene transcriptional profile obtained for the MSLC sample showed a similar tendency to the APC sample while it was quite different from that for the SFC sample. Most of the genes specifically transcribed in the MSLC sample versus those in the SFC sample with a 10-fold up-regulation or higher were unknown or predicted proteins. However, transcription of oryzin gene was only slightly up-regulated in the MSLC sample and that of α-amylase gene, slightly down-regulated. [Copyright &y& Elsevier]

Abstract: Young landfill leachates (LFL) collected from Djebel Chekir (Tunisia) discharge area were found to be highly loaded with organic matter, ammonia, salts, heavy metals, phenols and hydrocarbons. Despite the possibility of their biodegradability, they represent a threat to the environment and show some resistance to conventional wastewater treatment processes. For these reasons, this study attempted to develop a biological process for the treatment of LFL using selected strains of Trametes trogii, Phanerochaete chrysosporium, Lentinus tigrinus and Aspergillus niger. Experiments were undertaken at different concentrations of the effluent up to 100%. COD removal efficiencies for P. chrysosporium, T. trogii and L. tigrinus were of 68, 79 and 90%, respectively, when LFL underwent a two-fold dilution. COD abatements were accompanied with an important enzyme secretion and a high reduction in the toxicity, expressed as percent bioluminescence inhibition (%BI<20%). Above 50% of LFL, the effluent was toxic to these strains and caused growth inhibition indicating the sensitivity of these strains to concentrated LFL. Comparatively to the other tested strains, A. niger showed to tolerate raw LFL since it grew at 100% of LFL. However, this strain is inefficient in removing phenols and hydrocarbons. Consequently, toxicity abatement was very low (%BI>70%). [Copyright &y& Elsevier]

Hydroperoxide lyase (HPL) gene from tomato leaves was isolated and comprised of 1431 nucleotides encoding a protein of 476 amino acids, corresponding to a molecular mass of 53,480 Da. The protein was expressed in Pichia pastoris as a secreted enzyme (rLeHPL) after 24 h of culture incubation and induction with methanol. However, no intracellular activity was found in the homogenised yeast cells. 13-Hydroperoxy-(9Z,11E)-octadecadienoic acid (HPOD) was the best substrate followed by 9-HPOD and then 13-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid (13-HPOT) while no activity was observed with the 9-HPOT. The pH optima of rLeHPL were at pH 7 and 5, respectively, for the 13-HPOD and 13-HPOT substrates. GC/MS analyses confirmed that the major product was hexanal with a minor peak for nonanal. [Copyright &y& Elsevier]

Leptin originally described as product of the ob gene has been shown to be expressed in various tissues including the gastrointestinal tract. In this study, we investigated the influence of leptin on the secretion of pancreatic juice in biliary–pancreatic duct cannulated anaesthetised rats and in dispersed rat pancreatic acini in vitro. Exogenous leptin was given in boluses intravenously with or without CCK-8 (12 pmol kg−1 body weight) in the presence or absence pharmacological CCK1 receptor blockade, cervical vagotomy, and capsaicin pre-treatment. Administration of leptin (0.1, 1 and 10 μg kg−1 body weight) did not affect the volume of bile and pancreatic juice while the protein and trypsin outputs were reduced in a dose-dependent manner. In the rats, leptin inhibited CCK-8 stimulated protein and trypsin outputs stronger than the basal pancreatic secretion. The inhibition by leptin was abolished by the pharmacological CCK1 receptor blockade, cervical vagotomy, and capsaicin pre-treatment. In contrast, leptin did not affect basal and CCK-8-stimulated amylase release from the dispersed rat pancreatic acini in vitro. In conclusion, the results of the present study suggest that leptin does not act directly on the rat pancreatic acinar cells but inhibits the secretion of pancreatic enzymes acting indirectly via a neurohormonal CCK-vagal-dependent mechanism. [Copyright &y& Elsevier]

Ca2+-induced enzyme secretion in the exocrine pancreas is not completely understood. We have proposed that Ca2+-induced enzyme secretion in the exocrine pancreas involves activation of ion conductances in the membrane of zymogen granules (ZG). Here we have identified a Ca2+-activated anion conductance in rat pancreatic ZG membranes (ZGM). Ca2+ (2.5–50 μM) increased the conductance for I−, NO3−, Br−, or HCO3−, but not for Cl−, as determined by the rate of valinomycin-induced osmotic lysis of ZG suspended in isotonic K+-salts. 4,4′-Diisothiocyanatodihydrostilbene-2,2′-disulfonate (100 μM) or 25 μM dithiothreitol strongly inhibited Ca2+-dependent lysis. The permeability sequence, Ca2+ dependence, and inhibitor sensitivity of ZG anion conductance are reminiscent of a family of epithelial Ca2+-activated anion channels (CLCA). CLCA expression was confirmed by RT-PCR with rat pancreatic mRNA and mouse CLCA1 primers. A PCR product (580 bp) exhibited 81%, 77%, and 57% amino acid similarity to the three mouse isoforms mCLCA-1, -2, and -3 (mgob-5), respectively. Antibodies against bovine tracheal CLCA1 showed CLCA expression in ZGM by immunoblotting, immunoperoxidase light microscopy, and immunogold labeling. These findings suggest that a CLCA-related protein could account for the Ca2+-activated HCO3− conductance of rat pancreatic ZGM and contribute to hormone-stimulated enzyme secretion. [Copyright &y& Elsevier]

Experimental objectives: Stimulation of low-affinity CCK-1 receptors on pancreatic acini leads to inhibition of enzyme secretion. We studied signal transduction mechanisms to identify potential causes for the reduced secretion. Results: Co-stimulation experiments with CCK, CCK-JMV-180, and bombesin revealed an inhibition of bombesin-stimulated enzyme secretion by low-affinity CCK-1 receptors. Binding of 125I-gastrin-releasing peptide (the mammalian analogue of bombesin) to acini after CCK preincubation was not altered. After a short preincubation of acini with high concentrations of CCK, intracellular calcium remained responsive to bombesin. In contrast to bombesin or CCK at concentrations of 10−10 M or lower, high concentrations of CCK caused a strong activation of p125 focal adhesion kinase (p125FAK) and a marked reorganisation of the actin cytoskeleton. Conclusions: Inhibitory mechanisms triggered by low-affinity CCK-1 receptors interrupt enzyme secretion from pancreatic acini at late stages in the signal transduction cascades since bombesin receptor binding and early signalling events remained intact after CCK preincubation. A reorganisation of the actin cytoskeleton is suggested to be the mechanism by which low-affinity CCK-1 receptors actively interrupt enzyme secretion stimulated by other receptors. [Copyright &y& Elsevier]

The distribution of nitric oxide synthase in both neuronal and non-neuronal pancreatic tissues and the role of nitric oxide in the control of exocrine pancreatic secretion are reviewed in this article. Earlier reports based on in vivo studies suggested that nitric oxide can affect the secretory activity of the exocrine pancreas through changes in pancreatic blood flow. More recently, the employment of either nitric oxide synthase inhibitors or nitric oxide donors in in vitro preparations has provided evidence that nitric oxide can exert a direct action on this gland independently on its vascular effects. Most research in this area seems to indicate that modulation of exocrine pancreatic function by nitric oxide is exerted via activation of guanylate cyclase and generation of cGMP, although other pathways cannot be excluded. Experiments performed over the last year in our laboratory reveal a novel and interesting mechanism based on the ability of nitric oxide to control the release of endogenous neurotransmitter in the pancreas and, subsequently, the nerve-mediated enzyme secretion. [ABSTRACT FROM AUTHOR]