Enzyme Production Research Articles

Molecular Docking Studies of Tribulus Terrestris Phytochemicals Against Bovine Mastitis Caused by Methicillin-resistant Staphylococcus Aureus

Bovine mastitis caused by methicillin-resistant Staphylococcus aureus is among the diseases capable of transferring from animal to human. It often leads to the development of antibiotic-resistant genes in both humans and animals. It is marked by the failure of the active-serine site of the blaZ gene in association with mecA to hydrolyze antibiotics which will inhibit the production of the beta-lactamase enzyme. This research aims to assess the interactions and binding affinities of Tribulus terrestris phytochemicals and important bacterial proteins involved in bovine mastitis. The research process begins with the identification of important bacterial proteins responsible for bovine mastitis and then the preparation of protein and ligand structures. Consequently, docking was performed, and interactions between protein-ligand were analyzed and visualized. Four phytoconstituents of Tribulus Terrestris (2,7-Diphenyl-1,6-dioxopyridazino [4,5:2',3'] pyrrolo [4',5'] pyridazine, gitogenin, tigogenin, and hecogenin) were chosen, and molecular docking simulations revealed all the four compounds exhibit good binding affinities. The docking scores of the ligands with blaZ are as follows; 2,7-Diphenyl-1,6-dioxopyridazino [4,5:2',3'] pyrrolo [4',5'] pyridazine (-7.9), gitogenin (-8.9), tigogenin (-8.9) and hecogenin (-9.0). Also, the docking scores of the ligands with mecA are as follows; 2,7-Diphenyl-1,6-dioxopyridazino [4,5:2',3'] pyrrolo [4',5'] pyridazine (-9.4), gitogenin (-6.4), tigogenin (-6.5) and hecogenin (-6.8). Based on the predicted ADMET values using the Lipinski and Veber rule, compounds with potentially good activities were identified. The results suggest that only tigogenin is likely to exhibit antibacterial activity by binding with the active-serine site of blaZ

Mucin-driven ecological interactions in an in vitro synthetic community of human gut microbes.

Specific human gut microbes inhabit the outer mucus layer of the gastrointestinal tract. Certain residents of this niche can degrade the large and complex mucin glycoproteins that constitute this layer and utilise the degradation products for their metabolism. In turn, this microbial mucin degradation drives specific microbiological ecological interactions in the human gut mucus layer. However, the exact nature of these interactions remains unknown. In this study, we designed and studied an in vitro mucin-degrading synthetic community that included mucin O-glycan degraders and cross-feeding microorganisms by monitoring community composition and dynamics through a combination of 16S rRNA gene amplicon sequencing and qPCR, mucin glycan degradation with PGC-LC-MS/MS, production of mucin-degrading enzymes and other proteins through metaproteomics, and metabolite production with HPLC. We demonstrated that specialist and generalist mucin O-glycan degraders stably co-exist and found evidence for cross-feeding relationships. Cross-feeding on the products of mucin degradation by other gut microbes resulted in butyrate production, hydrogenotrophic acetogenesis, sulfate reduction and methanogenesis. Metaproteomics analysis revealed that mucin glycan degraders Akkermansia muciniphila, Bacteroides spp. and Ruminococcus torques together contributed 92% of the total mucin O-glycan degrading enzyme pool of this community. Furthermore, comparative proteomics showed that in response to cultivation in a community compared to monoculture, mucin glycan degraders increased carbohydrate-active enzymes whereas we also found indications for niche differentiation. These results confirm the complexity of mucin-driven microbiological ecological interactions and the intricate role of carbohydrate-active enzymes in the human gut mucus layer.

Comparative evaluation of the extracellular production of a polyethylene terephthalate degrading cutinase by Corynebacterium glutamicum and leaky Escherichia coli in batch and fed-batch processes

BackgroundWith a growing global population, the generation of plastic waste and the depletion of fossil resources are major concerns that need to be addressed by developing sustainable and efficient plastic recycling methods. Biocatalytic recycling is emerging as a promising ecological alternative to conventional processes, particularly in the recycling of polyethylene terephthalate (PET). However, cost-effective production of the involved biocatalyst is essential for the transition of enzymatic PET recycling to a widely used industrial technology. Extracellular enzyme production using established organisms such as Escherichia coli or Corynebacterium glutamicum offers a promising way to reduce downstream processing costs.ResultsIn this study, we compared extracellular recombinant protein production by classical secretion in C. glutamicum and by membrane leakage in E. coli. A superior extracellular release of the cutinase ICCGDAQI was observed with E. coli in batch and fed-batch processes on a litre-scale. This phenomenon in E. coli, in the absence of a signal peptide, might be associated with membrane-destabilizing catalytic properties of the expressed cutinase. Optimisations regarding induction, expression temperature and duration as well as carbon source significantly enhanced extracellular cutinase activity. In particular, in fed-batch cultivation of E. coli at 30 °C with lactose as carbon source and inducer, a remarkable extracellular activity (137 U mL−1) and cutinase titre (660 mg L−1) were achieved after 48 h. Literature values obtained with other secretory organisms, such as Bacillus subtilis or Komagataella phaffii were clearly outperformed. The extracellular ICCGDAQI produced showed high efficacy in the hydrolysis of PET textile fibres, either chromatographically purified or unpurified as culture supernatant. In less than 18 h, 10 g L−1 substrate was hydrolysed using supernatant containing 3 mg cutinase ICCGDAQI at 70 °C, pH 9 with terephthalic acid yields of up to 97.8%.ConclusionExtracellular production can reduce the cost of recombinant proteins by simplifying downstream processing. In the case of the PET-hydrolysing cutinase ICCGDAQI, it was even possible to avoid chromatographic purification and still achieve efficient PET hydrolysis. With such production approaches and their further optimisation, enzymatic recycling of PET can contribute to a more efficient and environmentally friendly solution to the industrial recycling of plastics in the future.

Optimization of fermentation conditions for Bacillus velezensis TCS001 and evaluation of its growth promotion and disease prevention effects on strawberries

Bacillus velezensis TCS001 is a novel biocontrol bacterium with broad-spectrum antifungal activity and plant growth-promoting effects, holding great potential for development in agricultural production. This study optimized the fermentation conditions for B. velezensis TCS001 through single-factor experiments combined with response surface methodology, and developed a formulation for TCS001 suspension concentrate (TCS001-SC). The efficacy of TCS001-SC to promote the growth of strawberries and to prevent and control strawberry anthracnose was evaluated. The optimal liquid fermentation condition for TCS001 was determined to be 2.89 % soluble peanut cake powder, 3.0 % glucose, 3.0 % soluble starch, 0.002 % FePO4, 0.006 % KCl, 0.6 % NaCl, 0.05 % MgCl2·6H2O, 0.3 % K2HPO4, 0.15 % KH2PO4, 0.05 % CaCO3, 0.005 % MnSO4, a working volume of 31 % (77.5 mL/250 mL), a rotation speed of 173 r/min, a cultivation temperature of 28°C, an inoculum volume of 1.0 %, and a pH of 7.0. The 15 L fermenter upscale culture achieved a spore count of 9.46 × 109 CFU/mL, which was 2.01 times the spore count of 4.7 × 109 CFU/mL before optimization, and a preliminary TCS001-SC was developed with the fermented broth as the main component. Agar plate confrontation tests showed that TCS001 had antifungal activity against five types of anthracnose fungi, with inhibition rates ranging from 70.3 % to 87.2 %. TCS001-SC could promote the growth of strawberries and induce a rapid defense enzyme response in their leaves, enhancing the plant's resistance to pathogens. After treatment, individual strawberry plants showed significant increases in the number of leaves, fresh weight of stems and leaves, root fresh weight, leaf area, plant height, and the content of POD, SOD, CAT enzymes, GA, IAA, and ABA compared to the control.Additionally, it shows good prevention and control effects against strawberry anthracnose, with a control efficacy of 60.45% after five spray treatments at a concentration of 2 × 107 CFU/mL, which is not significantly different from the efficacy of commercial microbial agents such as Bacillus subtilis wettable powder, subsequent field trials will be conducted to determine its potential as a microbial pesticide. This study provides important support for the future industrial production and application of the strain TCS001.

Pathogenicity of psychrotolerant strains of Antarctic Pseudogmynoascus fungi reveals potential opportunistic profiles

Recent studies have demonstrated the presence of fungal taxa in the extreme ecosystems of Antarctica that are known to opportunistically infect humans and animals. Among these are members of the genus Pseudogymnoascus, including some that are genetically similar to P. destructans, known to be pathogenic to bats. We evaluated the in vitro and in vivo pathogenic potential of 11 Pseudogymnoascus spp. strains recovered from Antarctica. All strains were able to grow at temperatures up to 28 °C and displayed in vitro pathogenicity through hemolytic activity, growth at different pH levels, production of hydrolytic enzymes, spore diameters, tolerance to oxidative stress, hypoxia, and halotolerance. Among them, Pseudogymnoascus sp. UFMG 8532 exhibited strong in vitro pathogenicity and in preliminary in vivo assay killed 100 % of Tenebrio molitor larvae within one day. The pathogenicity of the same strain was also tested using immunosuppressed BALB/c mouse models. Survival of BALB/c mice was affected, with oscillations between weight gain and loss, and impacts on sensory function, reflexes and autonomic function. Histopathological data from the organs of infected mice showed evidence of inflammatory processes, with numerous neutrophils, a small number of macrophages, fluid accumulation inside the lungs and intense hyperemia. Our results indicate that Antarctic Pseudogymnoascus spp. strains obtained from various substrates/habitats in maritime Antarctica may possess intrinsic virulence factors and pathogenic potential for immunosuppressed animals and humans in the region. Given that the Antarctic environment is an important reservoir for Pseudogymnoascus species, which display growth performance across a range of temperatures, it is possible that increasing temperatures in the maritime Antarctic could activate dormant genes or biochemical pathways, select virulent species and/or strains, and facilitate their spread within and beyond the region. The ability of Pseudogymnoascus species to grow slowly even at 28°C, coupled with their potential in vitro and in vivo virulence factors, suggests that these fungi might be undergoing an opportunistic transition due to the effects of climate change on the Antarctic Peninsula.

Isolation and Screening of Indigenous Filamentous Fungi Producing Ligninolytic, Cellulolytic and Hemicellulolytic Enzymes from Decomposed Oil Palm Frond

Oil palm frond (OPF) is a palm oil plantation by-product commonly used in animal feeding in Malaysia. The large production, availability, and nutrient content make OPF the best candidate for utilization as animal feed. However, OPF contains high lignin bonds to cellulose and hemicellulose that further limit the digestibility of rumen microbes to produce volatile fatty acids as an energy source for ruminants. This study aims to identify and determine the enzyme activity (ligninolytic, cellulolytic, and hemicellulolytic) of enzymes extracted from filamentous fungi in the pre-treatment of OPF using the solid-state fermentation (SSF) technique. The enzyme extracted from SSF was determined by its enzyme activity (laccase, lignin peroxidase, manganese peroxidase, carboxymethylcellulose, avicelase, and xylanase). Eight fungi were successfully identified to produce enzymes determined in this experiment. Phanerina mellea showed the highest average ligninolytic enzyme activity with a value of 0.37 U/mL and an average cellulolytic + hemicellulolytic of 0.18 U/mL. In this experiment, P. mellea was the most desired fungi for the pre-treatment of OPF. The optimum ligninolytic enzyme production time of OPF pre-treatment is 10 days of SSF.

Characterization of a Polyphenol Oxidase and Lipase Produced by Microbes Derived from Palm Oil Sludge

Introduction: The waste-to-wealth concept was applied in this study. Our main objective was to identify and evaluate several microorganisms responsible for the production of important industrial enzymes using sludge from palm oil processing. We were able to isolate several bacteria that are tyrosinase producers: Bacillus cereus, Acinobacter seifertii, Klebsiella variicola and Pseudomonas stutzeri. Laccase producers Trametes polyzona, Bacillus subtilis, Bacillus pumilus and Staphylococcus condimenti, as well as lipase producers. Methods: In addition, we focused specifically on B. cereus because we knew that it produces tyrosinase. Lipase is another target enzyme, and S. condimenti was found to be a hyperproducer. Production conditions included a 24-hour incubation period at 40°C and a pH of 6.0, while typical substrates such as starch and coconut oil were used. Results: Preliminary protein purification studies identified a 43 kDa lipase that is active at pH 7.0 and 40°C. Salts such as NaCl, various detergents such as Triton X-100 and Tween-80 as well as many metal ions enhanced the activity and made the enzyme unique in its biological function. Conclusion: Even with EDTA (2.5 mM), which does not completely block its function, only 40% inhibition was observed. Only a few organic solvents such as butanol, acetone and DMF are involved in inhibiting its activity.

Stress Responses to Bark Beetle Infestations among Pine (Pinus sylvestris), Fir (Abies alba), and Beech (Fagus sylvatica) Trees

Insect infestation triggers multiple defense responses in plants, both locally at the infection site and systemically throughout the plant, including the production of feeding deterrents, toxins, defensive proteins, enzymes, and secondary metabolites. Our study aimed to compare the endogenous levels of antioxidative enzymes, photosynthetic pigments, phytohormones, total phenols, and flavonoids in bark-beetle-infested and uninfested trees. We evaluated the surviving trees in bark-beetle-infested stands, assessing both the condition and defense of uninfested and infested beech (Fagus sylvatica), pine (Pinus sylvestris), and fir (Abies alba) trees. Sampling was performed at six affected sites in the Czech Republic, targeting trees that were resilient to significant health deterioration caused by abiotic and biotic factors. The results showed different levels of most of the measured compounds in the three species. Among all the tested species, photosynthetic pigment levels showed the strongest association with infestation status, which was generally lower in the infested plants. For chlorophyll a, extremely significant reductions were observed from 123 ± 20.6 to 101 ± 17.9 μg/g dry weight (DW) in pine, from 231 ± 33.1 to 199 ± 22.2 μg/g DW in beech, and from 60 ± 5.66 to 51.3 ± 6.27 μg/g DW in fir. In contrast, enzymatic activities indicated only isolated instances of significant association, whereas antioxidative properties (total phenolic content, flavonoids, and 2,2-diphenyl-1-picrylhydrazyl radical scavenging capacity) were not significantly associated with infestation status. There was a statistically significant increase in glutathione reductase activity in infested fir and pine trees. However, this difference was not statistically significant in beech. In contrast, a significant increase in superoxide dismutase activity was detected in infected beech trees. Phytohormones have emerged as the most diverse group of analyzed compounds. Cytokinins were the most distinct, with many of them being significantly increased in infested pines, whereas both beech and fir showed only one significant association. Additionally, derivatives of jasmonic acid also showed a distinct pattern of change associated with bark beetle infestation, with the levels of three out of the four analyzed jasmonates being significantly decreased in infested pines, whereas no effects were observed in beeches and firs. Notably, many phytohormones were significantly elevated in the infested pine, whereas both beech and fir exhibited only one significant association. Overall, the data showed that pines responded differently to bark beetles than to beeches or firs. The greatest changes in phytohormones were observed in pine, whereas the most significant changes in photosynthetic pigments were observed in beech and fir trees.

Genetic Diversity and Functional Potential of Streptomyces spp. Isolated from Pachmarhi Biosphere Reserve, India.

Streptomyces is a diverse genus, well known for producing a wide array of metabolites that have significant industrial utilization. The present study investigates the genetic and functional diversity of Streptomyces spp. isolated from the Pachmarhi Biosphere Reserve (PBR), India, an unexplored site. The 16S rRNA gene sequencing and analysis revealed 96 isolates belonging to 40 different species indicating a substantial phylogenetic diversity. The strains were clustered into two groups: a major cluster with 94 strains and a small cluster with two strains. BOX- PCR analyses revealed an incredible genetic diversity existing among the strains of Streptomyces spp. in PBR. The analyses revealed the intra-species diversity and inter-species closeness within the genus Streptomyces in the study area. Qualitative screening for enzyme production has shown that 53, 42, 41, 11, and 54 strains tested positive for CMCase, xylanase, amylase, pectinase, and β-glucosidase, respectively. Additionally, 54 strains tested positive for PHB production. The strains were assayed quantitatively for the production of CMCase, xylanase, amylase, and pectinase. Streptomyces sp. MP9-2, Streptomyces sp. MP10-11, Streptomyces sp. MP10-18, and Streptomyces sp. MP10-6 recorded maximum CMCase (0.604 U/mL), xylanase (0.553 U/mL), amylase (1.714 U/mL), and pectinase (13.15 U/mL) activities, respectively. Furthermore, several strains demonstrated plant growth-promoting traits, viz. zinc and phosphate solubilization and production of ammonia, HCN (hydrogen cyanide), and IAA (Indole acetic acid), and nitrogen fixation. Fifty strains showed antifungal activity against Fusarium oxysporum f. sp. lycopersici with inhibitions ranging from 7.5 to 47.5%. Current findings underscore the ecological and biotechnological significance of Streptomyces spp. in the unexplored habitat of PBR.

Screening and Optimization of Pathogenic Strains of Pseudomonas sp. for Cholinesterase Enzyme Activity

The identification and optimization of pathogenic strains of Pseudomonas sp. with significant Cholinesterase enzyme activity has promising implications for environmental and medical applications. This study aimed to screen various Pseudomonas strains to identify those exhibiting high cholinesterase activity and to optimize the conditions for maximal enzyme production. Multiple Pseudomonas strains were isolated and characterized from diverse clinical samples, employing selective media and biochemical assays to determine cholinesterase activity. The strains with maximum enzyme activity were further subjected to optimization experiments, including variations in growth conditions such as Temperature, pH and Nutrient availability. The findings revealed sample no.39 among the collected 50 samples had significant enzyme activity of 0.19151 IU is obtained when choline chloride was 0.3999%, Ammonium Chloride was 0.01M, pH was 7.0, Temperature was 27°C and Time of incubation was 24hrs. These results not only advance our understanding of Pseudomonas sp. enzyme capabilities but also offer potential applications in bioremediation, biosensor development and pharmaceutical industries. Future research will focus on the molecular characterization of the cholinesterase enzyme and its applications in various industrial processes.

Producción de esterasa por Fusarium culmorum crecido en presencia de diferentes concentraciones di(2-etilhexil) ftalato y de ion calcio en fermentación sumergida

Phthalates are plastic additives used as plasticizers in the manufacture of flexible plastic materials. These compounds, such as di(2-ethylhexyl) phthalate (DEHP) are toxic substances to the environment and human health, and have been detected as environmental pollutants. In this study, the activity of esterase produced by Fusarium culmorum grown in media supplemented with different concentrations of DEHP (1.5, 3 and 4 g/L) and calcium ion (0.1 and 0.5 g/L) in submerged fermentation was characterized by biochemical tests and polyacrylamide gel electrophoresis. The greatest esterase activity was observed in cultures grown in media added with 3 g of DEHP/L and 0.5 g of calcium ion/L, followed by those cultures grown in media containing 1.5 g of DEHP/L. Whereas cultures grown on 4 g of DEHP/L had the lowest esterase activity. In general, cultures added with 0.5 g of calcium ion/L had the maximum enzymatic activity and the highest enzymatic productivity in the media tested. High concentration of DEHP were used as the sole source of carbon and energy by F. culmorum. It is suggested that the calcium ion favored the esterase production during the DEHP biodegradation. However, further studies are needed to understand the relationship between calcium ion, fungal growth, and enzyme production to provide the appropriate concentration of this ion in the culture medium.

Enzymatic production, physicochemical characterization, and prebiotic potential of pectin oligosaccharides from pisco grape pomace

The prebiotic capacity of Pectin Oligosaccharides (POS) is influenced by structural factors such as molecular size, composition, and degree of esterification, which affect their interaction with the gut microbiota. While existing literature has predominantly examined POS derived from apple and citrus pectins, the extrapolation of these findings to other pectin sources remains complex due to variations in their composition. This study focused on obtaining POS with prebiotic potential from pisco grape pomace through controlled enzymatic hydrolysis, resulting in three molecular size fractions: <3 kDa, 3–10 kDa, and > 10 kDa. The POS fractions were analyzed using FTIR, HPSEC, HPLC, and MALDI-TOF-MS techniques to characterize their physical-chemical properties. Each fraction presented distinct compositions, with the <3 kDa fraction showing a higher concentration of galacturonic acid and glucose, while the >10 kDa fraction was also composed of rhamnose and arabinose. Notably, the <3 kDa fraction supported greater biomass growth of the probiotic strain Lactobacillus casei ATCC 393 compared to the other fractions. In contrast, the non-probiotic strain Escherichia coli ATCC 25922 achieved the lowest biomass with this fraction. Consequently, the <3 kDa POS fraction exhibited the highest prebiotic index. This fraction, composed of oligomers from the rhamnogalacturonan region and arabino-oligosaccharides with a degree of polymerization between two and five, highlights its potential for further research and applications. Therefore, investigating other sources and optimizing extraction conditions could lead to developing novel prebiotic formulations that supply specific probiotic strains for a symbiotic product.

Elucidating Thermothielavioides terrestris secretome changes for improved saccharification of mild steam-pretreated spruce

BackgroundThe efficient use of softwood in biorefineries is hampered by its recalcitrance to enzymatic saccharification. In the present study, the fungus Thermothielavioides terrestris LPH172 was cultivated on three steam-pretreated spruce materials (STEX180°C/auto, STEX210°C/auto, and STEX210°C/H2SO4), characterized by different hemicellulose content and structure, as well as on untreated biomass. The aim of the study was to map substrate-induced changes in the secretome of T. terrestris grown on differently treated spruce materials and to evaluate the hydrolytic efficiency of the secretome as supplement for a commercial enzyme mixture.ResultsThe cultivation of T. terrestris was monitored by endo-cellulase, endo-xylanase, endo-mannanase, laccase, and peroxidase activity measurements. Proteomic analysis was performed on the secretomes induced by the spruce materials to map the differences in enzyme production. Growth of T. terrestris on STEX180°C/auto and STEX210°C/auto induced higher expression level of mannanases and mannosidases of the GH5_7 CAZy family compared to cultivation on the other materials. Cultivation on untreated biomass led to overexpression of GH47, GH76, and several hemicellulose debranching enzymes compared to the cultivation on the pretreated materials. T. terrestris grown on untreated, STEX180°C/auto and STEX210°C/auto induced three arabinofuranosidases of the GH43 and GH62 families; while growth on STEX210°C/H2SO4 induced a GH51 arabinofuranosidase and a GH115 glucuronidase. All secretomes contained five lytic polysaccharide monooxygenases of the AA9 family. Supplementation of Celluclast® + Novozym188 with the secretome obtained by growing the fungus grown on STEX180°C/auto achieved a twofold higher release of mannose from spruce steam-pretreated with acetic acid as catalyst, compared to the commercial enzyme cocktail alone.ConclusionsMinor changes in the structure and composition of spruce affect the composition of fungal secretomes, with differences in some classes explaining an increased hydrolytic efficiency. As demonstrated here, saccharification of spruce biomass with commercial enzyme cocktails can be further enhanced by supplementation with tailor-made secretomes.

Recombinant production of Paenibacillus wynnii β-galactosidase with Komagataella phaffii

BackgroundThe β-galactosidase from Paenibacillus wynnii (β-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used β-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, β-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce β-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the β-gal-Pw in K. phaffii either extracellularly or intracellularly.ResultsFirstly, 11 different signal peptides were tested for extracellular production of β-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of β-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular β-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric β-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for β-gal-Pw production compared to the AOX1 promoter. After partial purification, a β-gal-Pw enzyme preparation with a total β-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter).ConclusionThis study showed that the β-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular β-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.

Evaluation of lignocellulatic activity of enzymes from microwave-irradiated Pleurotus sajor-caju cultivated with wheat straw

The production of lignocellulytic enzymes by microwave-radiated Pleurotus sajor-caju was assayed. Wheat straw was employed as substrate to P. sajor-caju for production of laccase, manganese peroxidase (MnPase), filter-paperase (FPase), carboxmethyl cellulase (CMCase), and cellulase (as evaluated using microcrystalline cellulose). P. sajor-caju exposed to 10 s of microwave radiation (MR) showed maximum growth with colony radius of 7.17 ± 0.45 cm, while with increasing the exposure time up to 50 s the growth decreased up to 2.67 ± 0.22 cm. Moreover, it failed to grow at 80 s of exposure time. Cellulase, MnPase, FPase, CMCase, and laccase activities were induced to 37 ± .0.54, 49 ± 2.36, 189 ± 2.12, 0.37 ± 0.06, and 1.58 ± 0.03 U/mL compared to that at control 31 ± 0.25, 46 ± 1.25, 177 ± 1.65, 0.28 ± 0.03, and 1.37 ± 0.12 U/mL, respectively as a result of P. sajor-caju exposure to 10 s of MR. As the exposure time increased, these enzymes activity decreased. Different levels of moisture with surfactant (polysorbate 80) were applied to optimize the enzymes activities at 10 s of exposure time. The optimum activities 3.15 ± 0.23, 0.62 ± 0.06, 269 ± 5.36, 65 ± 1.63, and 48 ± 0.98 U/mL were recorded for cellulase, MnPase, FPase, CMCase, and laccase, respectively at 70% of moisture and 0.15 mL/L of polysorbate 80.

Enzymatic Production of Trehalose and Trehalulose by Immobilized Thermostable Trehalose Synthase.

Trehalose, a versatile disaccharide renowned for its unique physical and chemical properties, finds extensive application in the food, pharmaceutical, and cosmetic industries. While conventional extraction methods face challenges, enzymatic conversion offers a promising avenue for the industrial production of trehalose. This study delves into a novel synthetic approach utilizing a recombinant enzyme, merging the thermostable trehalose synthase domain from Thermus thermophiles with a cellulose binding domain. Immobilization of this enzyme on cellulose matrices enhances stability and facilitates product purification, opening avenues for efficient enzymatic synthesis. Notably, the engineered enzyme demonstrates additional activity, converting sucrose into trehalulose. This dual functionality, combined with immobilization strategies, holds immense potential for scalable and cost-effective production of trehalose and trehalulose, offering promising prospects in various industrial and biomedical applications.

Microbial lipases: propitious biocatalysts for various biotechnological applications

The use of microbial lipases is very important because they have a broad spectrum of catalytic reactions. Lipases catalyze reactions in aqueous and non-aqueous media and have advantages when compared to chemical catalysts, due to their specificity, enantioselectivity and stability at pH and temperature. The interest in research for microbial lipases occurs because of their applicability in various sectors and industries, such as food and beverage production, cosmetics, pharmaceuticals, surfactants, dairy products, treatment of effluents containing oils and fats, biofuels, among others. Biocatalysts can be obtained by submerged fermentation (SF) or solid-state fermentation (SSF), which has advantages over SF, because it is possible to use agroindustrial waste as substrate or growth support for microorganisms, becoming an alternative to reduce production costs. SSF is a promising technology for enzyme production, because besides using low-cost substrates, the biocatalyst can be produced in a more concentrated form, facilitating its recovery from the culture medium when necessary. Thus, this paper intends to discuss and review studies on microbial lipases, with direct application of the fermented solid (SSF), focusing on the main microorganisms, substrates and supports used in SSF, applicability of lipases in several industrial sectors, besides presenting conversion results using different microorganisms or/and substrates.

Assessing variability among culturable phylloplane basidiomycetous yeasts from Italian agroecosystems

This study analysed basidiomycetous yeasts isolated from the phylloplane of crops and spontaneous plants in Italian agroecosystems. A total of 25 species belonging to 17 genera were recognized by analysing 83 isolates from vineyards and orchards, that are not treated with synthetic fungicides, and adjacent natural areas. Rhodotorula graminis and Filobasidium magnum were the most frequent species but 13 others were represented by a single isolate (e.g., Buckleyzyma salicina, Pseudozyma prolifica, and Moniliella megachiliensis). Preliminary analysis of (GTG)5-PCR fingerprinting revealed high genetic intraspecific heterogeneity. All isolates were characterized by their production of extracellular hydrolytic enzymes and their sensitivity to six commercial fungicides used in Italy. The isolates displayed great variability in these phenotypic traits, which play an important role in the survival of yeast populations in agroecosystems. Most of them exhibited lipolytic, proteolytic, β-glucosidase and pectinolytic activities, but only three (F. magnum, Kwoniella mangroviensis and Ps. prolifica) also had cellulolytic and amylolytic activity. Most isolates were sensitive to four fungicides, and one R. graminis isolate was resistant to all six. This heterogeneity was not related to the geographical origin of the isolates. The lack of selective factors (i.e. pesticide treatments) in the sampling fields and the presence of adjacent natural areas may have favored the maintenance of an elevated level of strain diversity. This study provides new information on phylloplane basidiomycetous yeasts in agroecosystems and opens the way to further investigations into the impact of agricultural practices on the microbial diversity of these natural habitats.

Sardine Processing Waste: Biological Treatment Strategies and Their Implications

This study explores sustainable methods for Sardine Processing Waste (SPW) valorization. Two approaches were investigated: (a) SPW microbial pretreatment adding Saccharomyces cerevisiae or Bacillus sp. in a two-stage anaerobic digestion (AD) for enzyme and biomethane production and (b) a single-stage AD without SPW pretreatment. Both S. cerevisiae and Bacillus sp. secreted proteases (0.66 and 0.58 U mL−1, respectively) and lipases (3.8 and 4.3 U mL−1, respectively) during hydrolysis, thus reducing viscosity (2.8 and 2.9 cP, respectively) compared with the untreated SPW (4.1 cP). Biomethane production was higher in the single-stage AD (1174 mL CH4 g−1 VS−1) when compared with the two-stage AD (821.5 and 260 mL CH4 g−1 VS−1 with S. cerevisiae and Bacillus sp., respectively). S. cerevisiae addition enhanced SPW degradation as implied by VS and sCOD values (70 and 84%, respectively), but this also resulted in a higher toxicity due to a three-fold increment in NH4-N content, reducing methanogen activity. This research demonstrates the innovative application of S. cerevisiae, a common bread-making yeast, in the biotechnological enhancement of SPW hydrolysis. Non-genetically engineered S. cerevisiae not only co-produced proteases and lipases but also significantly improved solubilization, degradation, and viscosity reduction, thereby rendering the yeast a key player in solid fish waste valorization, beyond its traditional applications.

Multiple Regulatory Mechanisms Synergistically Control the Soluble Expression of CsCE for Enhanced Enzymatic Productivity of Lactulose in E. coli.

The limited expression of cellobiose 2-epimerase poses a significant constraint on the industrial enzymatic production of lactulose. Extensive modifications to the expression cassette offer a means to enhance the yield of recombinant proteins. In this study, an integrated strategy, combining individual and collaborative approaches, is proposed to fine-tune each stage of the CsCE overexpression program. This strategy involves the multidimensional integration of standardized genetic elements at various levels, including transcription, translation, folding, and three-dimensional structure. The volumetric activity of the final recombinant strain was markedly increased by 12-fold compared to the wild-type strain, reaching 2260.62 U/L. The protein expression in the newly developed high-yield recombinant strain exhibited a significant enhancement, with a higher proportion of soluble protein compared to that of inclusion bodies. Our findings offer insights into the multifaceted synergistic regulation of protein expression processes, holding promising implications for the production of heterologous recombinant proteins.