Comparative proteomic analysis: SclR is importantly involved in carbohydrate metabolism in Aspergillus oryzae.

The helix-loop-helix (HLH) family of transcriptional factors is a key player in a wide range of developmental processes in organisms from mammals to microbes. We previously identified the bHLH transcription factor SclR in Aspergillus oryzae and found that the loss of SclR function led to significant phenotypic changes, such as rapid protein degradation and cell lysis in dextrin-polypeptone-yeast extract liquid medium. The result implied that SclR is potentially important in both traditional fermentative manufacturing and commercial enzyme production in A. oryzae because of its effect on growth. Therefore, this study presents a comparative assessment at the proteome level of the intracellular differences between an sclR-disrupted strain and a control strain using isobaric tandem mass tag (TMT) labeling for quantification. A total of 5447 proteins were identified, and 568 were differentially expressed proteins (DEPs). Of the DEPs, 251 proteins were increased by 1.5-fold, and 317 proteins were decreased by 1.5-fold in an sclR-disrupted strain compared to the control. The comparison of the quantitative TMT results revealed that SclR was mainly involved in carbon metabolism, especially carbohydrate metabolism. In addition, an enzyme profile by a semi-quantitative method (API-ZYM) indicated that three enzymes (ß-galactosidase, α-glucosidase, and α-mannosidase) were significantly less active in the ∆sclR strain than in the control. Moreover, quantitative RT-PCR showed that the expression of certain genes was changed similarly to their corresponding proteins. These results suggested that a possible function of SclR during growth of A. oryzae is its important involvement in carbohydrate metabolism.

Enzyme characterization of lactic acid bacteria isolated from duck excreta.

Background and Aim: The production of lignocellulosic biomass waste in the agricultural sector of Indonesia is quite high annually. Utilization of lignocellulosic biomass waste through fermentation technology can be used as feed and biofuel. Fermentation technology requires the involvement of micro-organisms such as bacteria (lactic acid bacteria or LAB). LABs can be isolated from various sources, such as duck excreta. However, there have not been many reports of LAB from duck excreta. The present study aimed to characterize LAB enzymes isolated from duck excreta and obtain LAB enzymes with superior fermentation properties. Materials and Methods: A total of 11 LAB cultures obtained from duck excreta in Yogyakarta, Indonesia, were tested. Enzyme characterization of each LAB was performed using the API ZYM kit (BioMérieux, Marcy-I'Etoile, France). The bacterial cell suspension was dropped onto the API ZYM™ cupule using a pipette and incubated for 4 h at 37°C. After incubation, ZYM A and ZYM B were dripped onto the API ZYM cupule, and color changes were observed for approximately 10 s under a strong light source. Results: Esterase activity was moderate for all LABs. The activity of α-chymotrypsin, ß-glucuronidase, α-fucosidase, and α-mannosidase was not observed in a total of 10 LAB. The phosphohydrolase and amino peptidase enzyme activity of seven LABs was strong. Only six LAB samples showed protease activity. The glycosyl hydrolase (GH) activity was observed in a total of 8 LAB, while the activity of 2 LAB was strong (Lactococcus lactis subsp. lactis K5 and Lactobacillus brevis M4A). Conclusion: A total of 2 LABs have superior properties. L. lactis subsp. lactis K5 and L. brevis M4A have a high potential to be used in fermentation. They have the potential for further research, such as their effectiveness in fermentation, lignocellulose hydrolysis, feed additives, molecular characterization to detect specific enzymes, and their specific activities.

Enzymatic Activity of Prototheca zopfii Strains Isolated from Cows with Mastitis.

Bovine mastitis caused by Prototheca spp. can be a disease of high significance because of economic losses and the potential risk to public health. The aim of our study was to evaluate enzymatic activity of Prototheca zopfii. For this study, we used 15 P. zopfii strains previously isolated from cows with clinical and subclinical mastitis in Poland. We determined enzymatic profile of Prototheca species using the API ZYM system. Of the enzymatic activities detected during the study, acid phosphatase, leucine arylamidase, naphthol-as-bi-phosphohydrolase, esterase, lipase esterase, valine arylamidase, alkaline phosphatase, and lipase C14 were found in high percentage of strains.

Mechanism of protective effect of carnosol on pig intestinal epithelial cells.

This study aims to study the protective effect and mechanism of carnosol on intestinal oxidative stress. Porcine intestinal epithelial cells (ZYM-SIEC02) were pretreated with carnosol. Tert-butyl hydroperoxide (t-BHP) was added to stimulate the cells. The cell colonization and viability were detected by Edu staining, MTT, and cell counting kit-8 (CCK8) assays. The expressions of reactive oxygen species (ROS), nitric oxide (NO), superoxide dismutase (SOD), and malondialdehyde (MDA) in intracellular and oxidative stress were detected. The expression of related genes and proteins in cells was detected by real-time PCR and western blot. The regulatory mechanisms were identified by co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (CHIP) assays. The results showed that t-BHP reduced cell proliferation and viability, while cells pretreated with carnosol had resistance to t-BHP, decreased intracellular ROS, MDA and NO levels, and increased SOD content. The mRNA and protein levels of heme oxygenase 1/Nuclear respiratory factor 2 (HO-1/Nrf-2) in ZYM-SIEC02 cells treated with carnosol were significantly increased. Nrf2 was able to bind to cell cycle negative regulatory protein p21 Nrf2 could bind to the promoter regions of cyclin D1 (CCND1) and SOD genes. In conclusion, carnosol has a protective effect on intestinal epithelial cells by up-regulating the expression of Nrf2 and inhibiting p21 protein to promote the expression of CCND1 and SOD.

The effect of fruit on the extracellular enzyme profiles of fungi.

INTRODUCTION: In recent years, the number of diseases caused by fungal pathogens has increased significantly. Many species of fungi are pathogenic for plants, causing a threat to food production and to humans, and are among the causes of chronic diseases. OBJECTIVE: The aim of the study is to determine the enzyme profiles of fungi, depending on the different types of fruit with which they have contact, and to determine the differences in these profiles in relation to the substrate on which they are grown. MATERIAL AND METHODS: Six strains of fungi identified as Cladosporium sphaerospermum, Fusarium poae, Alternaria alternata, Penicillium expansum, Penicillium verucosum and Acremonium strictum, isolated from fruits, were selected and analyzed for enzymatic profiles. The enzymatic activity was assessed using the API ZYM test (bioMerieux, France). RESULTS: In the majority of the 6 fungal strains isolated from fruits, enzymes belonging to glycol-hydrolases were the most active. The exception was Acremonium strictum, where phosphatases dominated. Among most fungal isolates, the enzymes ß- glucosidase and N-acetyl-ß-glucosaminidase showed the highest activity. The highest ß-glucosidase activities were found in Cladosporium sphaerospermum and Penicillium expansum. On the other hand, lipase, α-fucosidase and α-chymotrypsin showed the least activity. The least activity of these enzymes or their complete absence was observed in Fusarium poae, Alternaria alternata, Penicillium expansum and Acremonium strictum. CONCLUSIONS: The activity of hydrolytic enzymes in the isolated fungi depended on the addition of fruit and the type of medium. Individual fruits can increase or decrease the activity of the enzymes. Fungi present in fruit have pathogenic properties and can be possible risk factors for fungal infections.

Applicability of API ZYM to capture seasonal and spatial variabilities in lake and river sediments.

Waters draining into a lake carry with them much of the suspended sediment that is transported by rivers and streams from the local drainage basin. The organic matter processing in the sediments is executed by heterotrophic microbial communities, whose activities may vary spatially and temporally. Thus, to capture and evaluate some of these variabilities in the sediments, we sampled six sites: three from the St. Clair River and three from Lake St. Clair in spring, summer, fall, and winter of 2016. At all sites and dates, we investigated the spatial and temporal variations in 19 extracellular enzyme activities using API ZYM. Our results indicated that a broad range of enzymes were found to be active in the sediments. Phosphatases, lipases, and esterases were synthesized most intensively by the sediment microbial communities. No consistent difference was found between the lake and sediment samples. Differences were more obvious between sites and seasons. Sites with the highest metabolic (enzyme) diversity reflected the capacity of the sediment microbial communities to breakdown a broader range of substrates and may be linked to differences in river and lake water quality. The seasonal variability of the enzymes activities was governed by the variations of environmental factors caused by anthropogenic and terrestrial inputs, and provides information for a better understanding of the dynamics of sediment organic matter of the river and lake ecosystems. The experimental results suggest that API ZYM is a simple and rapid enzyme assay procedure to evaluate natural processes in ecosystems and their changes.

Enzymatic characterization of clinical and environmental Cryptococcus neoformans strains isolated in Italy.

BACKGROUND: Cryptococcus neoformans is an encapsulated yeast causing mainly opportunistic infections. The virulence factors involved in cryptococcosis pathogenesis include the presence and the size of the polysaccharide capsule, the production of melanin by phenoloxidase, the growth at 37°C and the enzyme secretion like proteinase, phospholipase and urease. Many other enzymes are secreted by C. neoformans but their role in the fungus virulence is not yet known. AIMS: In order to investigate this topic, we compared the phospholipase production between strains from patients and from bird droppings, and we examined its relationship to phenoloxidase production. We further characterized the strains by determining the activity of 19 different extracellular enzymes. METHODS: Two hundred and five Italian C. neoformans clinical isolates and 32 environmental isolates were tested. Phenoloxidase production was determined by the development of brown colonies on Staib's agar. Extracellular phospholipase activity was performed using the semiquantitative egg-yolk plate method. API ZYM commercial kit was used to observe the production and the activity of 19 different extracellular enzymes. RESULTS: Statistical analysis of the results showed a significantly higher phospholipase activity in the clinical isolates than in the environmental isolates. No significant difference about the phenoloxidase production between both groups was found. Regarding the 19 extracellular enzymes tested using the API ZYM commercial kit, acid phosphatase showed the highest enzymatic activity in both groups. Concerning the enzyme α-glucosidase, the clinical isolates presented a significantly higher positivity percentage than the environmental isolates. A hundred percent positivity in the enzyme leucine arylamidase production was observed in both groups, but the clinical isolates metabolized a significantly greater amount of substrate. CONCLUSIONS: The higher phospholipase production in the clinical isolates group confirms the possible role of this enzyme in the cryptococcosis pathogenesis. The extracellular activities of the enzymes acid phosphatase, α-glucosidase and leucine arylamidase, tested by means of the API ZYM commercial kit, appear to be very interesting. Many studies indicate that these enzymes are involved in the virulence of bacteria and parasites; our results suggest their possible role as virulence factors in Cryptococcus infections too.

Immunomodulatory Effects of ZYM-201 on LPS-stimulated B Cells.

ZYM-201 is a methyl ester of triterpenoid glycoside from Sanguisorba officinalis which has been used for treatment of inflammatory and metabolic diseases. In this study, immunomodulatory effects of ZYM-201 on B cells were examined in vitro and in vivo. When splenocytes were activated with lipopolysaccharide (LPS), the major population which had shown an increase in cell numbers was B cells. However, when the B cells were treated with ZYM-201 after LPS activation, their cell numbers and the expression of major costimulatory molecules, CD80 and CD86, were decreased. Furthermore, the effect of LPS, which induces activation of NF-κB, was abolished by ZYM-201: LPS-stimulated B cells showed decrease of phosphorylation after treatment of ZYM-201. The same results were shown in vivo experiments. These results suggest that ZYM-201 may play a role in the modulation of inflammatory responses through inhibiting NF-κB activation and downregulating the expression of costimulatory molecules on B cells.

Enzymatic variability among Brazilian Pythium insidiosum isolates.

BACKGROUND: Pythium insidiosum is an oomycete classified in the kingdom Stramenopila. P. insidiosum hyphae are not able to initiate infection without the secretion of hydrolytic enzymes, which are considered an important factor in microbial virulence. AIMS: To evaluate the extracellular enzymatic activity of 14 Brazilian P. insidiosum isolates and a standard strain (ATCC 58637) by the API-ZYM System screening method. METHODS: Zoospores were grown in RPMI 1640 broth, and 65 µL of the liquid phase were inoculated in each cupule of the API-ZYM strips. RESULTS: Differences in the enzymatic activities were observed among the isolates, although phosphohydrolases and ester hydrolases were conspicuous among all isolates. ß-glucosidase was also present in most of the isolates. Enzymatic activities of α-glucosidase and chymotrypsin were not observed, differing from a previous study involving Australian isolates and intracellular enzymes. CONCLUSIONS: The discrepancy in the enzymatic profile observed among Brazilian P. insidiosum isolates reflects the phenotypic variations found in susceptibility tests.

LC-MS/MS method for rapid and concomitant quantification of pro-inflammatory and pro-resolving polyunsaturated fatty acid metabolites.

Lipid autacoids derived from n-3/n-6 polyunsaturated fatty acids (PUFA) are some of the earliest signals triggered by an inflammatory reaction. They are acting also as essential regulators of numerous biological processes in physiological conditions. With regards to their importance, a robust and rapid procedure to quantify a large variety of PUFA metabolites, applicable to diverse biological components needed to be formulated. We have developed a simple methodology using liquid chromatography-tandem mass spectrometry allowing quantification of low-level of PUFA metabolites including bioactive mediators, inactive products and pathway biomarkers. Solid phase extraction was used for samples preparation with an extraction yield of 80% ranging from 65% to 98%. The method was optimized to obtain a rapid (8.5min) and accurate separation of 26 molecules, with a very high sensitivity of detection and analysis (0.6-155pg). When applied to biological samples, the method enabled characterization of eicosanoids and docosanoids production in epithelial cells or foam macrophages stimulated with LPS, in biological fluids and tissues from mouse models of peritonitis or infectious colitis. Our results demonstrate that this new method can be used in cultured cells, in fluids and in colonic tissues to quantify pro-inflammatory and pro-resolving PUFA metabolites mediators.

Isolation and Characterization of Cryptococcus neoformans from Environmental Sources in Busan.

Twenty nine samples of pigeon droppings (n = 12) and soil contaminated with avian excreta (n = 19), collected from different sites in Busan, were examined for isolation and characterization of Cryptococcus neoformans. Of these samples, 5 strains of C. neoformans were recovered from pigeon droppings (5/12 : 41.7%). All isolates were belonged to C. neoformans var. grubii (serotype A). The extracellular enzyme activities of the strains by using the API-ZYM system showed two different enzymatic patterns. The genetic variability among C. neoformans isolates was analyzed by random amplified polymorphic DNA (RAPD) using three 10-mer primers. Two different RAPD patterns, which clearly distinguished the isolates, were identified. Analysis of RAPD patterns provided a good characterization of environmental strains of C. neoformans serotype A as a heterogeneous group and were in good agreement with enzymatic profiles.