1.The Expression,Purification,Characterization And Preparation Of Low Lactose Pasteurized Milk With A Recombinant Thermostable β-glucosidase In Pichia Pastoris2.Dihydro-CDDO-trifluoroethyl Amide Suppresses Inflammatory Responses In Macrophages Via Activa

BackgroundLactose intolerance is increasingly recognized by the people in the world in the past few years. In vivo, lactase is an enzyme secreted by intestinal villi that hydrolyses the disaccharide lactose into glucose and galactose and is essential for the digestion of bovine milk which contains an average of4.8%lactose. Deficiency for this gene leads to malabsorption of lactose and subsequent fermentation of lactose by the gut flora. Lactose intolerance develops when afflicted individuals experience abdominal pain, diarrhea, bloating, flatulence, and other gastrointestinal symptoms following lactose consumption.Avoidance of dairy products by lactose intolerant individuals often leads to insufficient calcium and vitamin D consumption and may cause adverse health outcomes, especially reduced bone mineral density and fractures.Since lactose is a disaccharide composed of glucose and galactose, it can be hydrolyzed into these monosaccharides using either a beta-glucosidase or a beta-galactosidase. Based on this theory, researchers tried to use extrinsic lactase to hydrolyze the lactose in the milk. Consumption of lactose-free milk, which can be produced by adding lactase enzyme directly to milk, provides a means of maintaining good health while avoiding the symptoms of lactose intolerance.Lactase can be synthesized from many sources including animals, plants, bacteria, fungi and yeast. Now, the lactase enzymes most commonly used in the industry are beta-galactosidases from fungi and yeast, which have two major defects. One is the beta-galactosidases commonly used in the industry are mesophilic enzymes, which can be easily denatured by pasteurization conditions and has to be added after the pasteurization. Another is they are generally inhibited as glucose concentration increases. Undoubtedly, this two major defects increase the incidence of pollution and the cost of company.Although pasteurization conditions denature many enzymes, several thermostable lactase have been identified. Hyperthermophilic archeaeon Pyrococcus furiosus harbors several hydrolytic enzyme activities, notably, it has a thermostable beta-glucosidase with a half-life of85h at100℃. This beta-glucosidase serves as a model system to use for lactose hydrolysis since it displays extreme stability and high catalytic activity in the presence of lactose.In the present study, we cloned the Pyrococcus furiosus beta-glucosidase gene into the pGAPZaA vector and electrotransformed it into the pichia pastoris X-33strain for large-scale production of heterologous proteins by high-density cell culture. We then do the purification, characterization and analyzed the lactose hydrolytic action of this beta-glucosidase under pasteurization conditions in order to establish a new method for the production of low lactose pasteurized milk. Our system provides a method for high level production of "food grade" thermostable lactase that is appropriate for use in the milk industry during the process of pasteurization, eliminating the need to add enzyme following pasteurization and eliminating the risk of microbial contamination.Objectives1. Expression, purification and enzyme characterization of a recombinant thermostable beta-glucosidase from Pyrococcus furiosus in Pichia pastoris. 2. Hydrolysis of lactose in milk under pasteurized conditions use the recombinant thermostable beta-glucosidase and analyse parameters.Materials and methods1. Cloning and gene expression.2. Fed-batch fermentation.3. SDS-PAGE analysis.4. Purification of beta-glucosidase.5. Enzyme assay of the recombinant beta-glucosidase.6. Effect of temperature on beta-glucosidase activity and stability.7. Effect of pH on beta-glucosidase activity and stability.8. Effect of metal ions on beta-glucosidase activity.9. Hydrolysis of lactose in milk at the condition of pasteurization.Results1. The expression of recombinant beta-glucosidase.The biomass accumulated at120h was312g/L, enzyme expression reached its peak with a yield of740mg/L, and the corresponding beta-glucosidase activity was271U/mL. A protein corresponding to recombinant beta-glucosidase, with a molecular mass of approximately120kDa, was detected upon performing SDS-PAGE analysis with Coomassie Brilliant Blue staining.2. Purification of recombinant beta-glucosidase.After the one-step weak anion exchange chromatography to isolate the target protein, resulted in1.9fold purification and80.8%recovery from the supernatant.3. Enzyme characterization of recombinant beta-glucosidase.Optimum temperature was100℃, and at30℃the activity decreased to nearly7%of the maximum activity. Beta-glucosidase was stable and retained more than80%of its maximum activity from30℃to120℃. Beta-glucosidase presented optimum activity at pH6.0and nearly inactivated at pH4.0and9.0. Beta-glucosidase was stable and retained more than80%of its maximum activity from pH5.0to pH8.0when pre-incubated at37℃for1h. Although Cu2+can reduce the relative activity to78%, other metal cations have only negligible effects on the activity of the recombinant enzymes.4. Hydrolysis of lactose in milk under pasteurization conditions.Using62U/mL of recombinant beta-glucosidase achieved70%hydrolysis and increasing to498U/mL of recombinant enzyme resulted in greater than90%hydrolysis. We also tested the efficiency of hydrolysis of this enzyme from5-30minutes and found that after20minutes, the amount of hydrolysis was similar to30minutes. The effect of glucose concentration on hydrolysis was also tested, and we found that even high concentrations of glucose had minimal influence on the hydrolysis of lactose, demonstrating that the enzyme had a relatively good tolerance to glucose.Conclusions1. This report is the first on the cloning, expression, purification, and characterization of the beta-glucosidase from Pyrococcus furiosus in Pichia pastoris.2. In this study, we established a new method to produce low lactose milk using this enzyme under pasteurized conditions. This method highlights the potential for low lactose milk production without additional steps required to add enzymes following pasteurization, and thus, decreases the risk of microbial contamination to a great extent. BackgroundInnate immunity is the body’s first line to defense against microbial infection. As an important component of innate immunity, macrophages involved in the inflammatory process indirectly or directly through the secretion of cytokines, mediators and extracellular enzymes of different functions in the inflammatory responses. Although activation of macrophages and secretion of proinflammatory cytokines and mediators are important for the elimination of invading microorganisms, uncontrolled activation may leads to autoimmune and inflammatory diseases. Therefore, strictly control macrophage activation and secretion of proinflammatory cytokines and mediators are very important.Nuclear factor-erythroid2-related factor2(Nrf2), a member of the Cap’n’collar (CNC) family of basic-leucine zipper transcription factors, is the major regulator of cellular defenses against various pathological stresses in a variety of organ systems including lung, liver, gastrointestinal tract, bladder, kidney, brain, skin, ovary, and heart, Nrf2has evolved to be an attractive drug target for the treatment and prevention of human disease. Several natural or synthetic compounds have been studied to target Nrf2-mediated signaling for treatment and prevention, such as curcumin, tea polyphenol, resveratrol and ankaflavin. Of these, the synthetic triterpenoid derivatives of oleanolic acid seem to be the most promising drug candidates.The synthetic oleanolic triterpenoid of dihydro-CDDO-trifluoroethyl amide (Dh404) is a novel and potent Nrf2activitor. Dh404is well tolerated in mice and rats, and has been shown to improve obesity, type II diabetes, cardiac maladaptive remodeling and dysfunction. However, the effect of Dh404-Nrf2signaling axis about inflammation is unclear. Herein, we established a macrophage damage model in vitro to observe the effects of Dh404on LPS induced inflammation. The study can help development of new drugs for inflammation by regulating the Nrf2signaling and provide new evidence for the clinical application of Dh404.Objectives1. To establish LPS induced inflammation damage model in RAW264.7macrophages and mouse bone marrow derived macrophages.2. To observe the effects of Dh404on anti-inflammation, and further explore the possible protective mechanisms.Materials and methods1. Culture RAW264.7macrophages and mouse bone marrow derived macrophages to do the following experiments.2. Cell viability measured by LDH assay.3. Transcriptional activity of NF κB and Neh2detected by Dual-Luciferase reporter assay system.4. Gene expression detected by Q-PCR.5. Protein expression detected by Western blot.6. Protein expression detected by immunofluorescence.7. Bone marrow derived macrophage identified by Flow cytometry.8. Statistical analysis.All data are expressed as X±SD, the differences between groups were compared using ANOVA analysis. SPSS statistical software was used for statistical analysis, and P<0.05was considered statistically significant.Results1. The effects of Dh404on cell viability.lOnM,25nM,50nM,100nM and200nM of Dh404could not influence the viability of RAW264.7macrophages, however, cell viability was decreased in the concentration of500nM,1000nM and2000nM. Therefore,200nM of Dh404was used in subsequent trials for further study.2. Dh404inhibit the expression of iNOS, MCP-1and MIP-1β.Compared with LPS (1ug/mL) treatment group, the mRNA and protein expression of iNOS were significantly decreased by LPS (1ug/mL)+Dh404(200nM) treatment. At the same time, the mRNA expression of MCP-1and MIP-1β were also significantly decreased by LPS (1ug/mL)+Dh404(200nM) treatment, indicating that Dh404inhibited the LPS-induced inflammation in RAW264.7macrophages.3. Dh404treatment minimally regulates MAPKs pathway in RAW264.7macrophages.Compared with the control group, the phosphorylated protein expression of MAPKs (ERK, P38and JNK) were significantly increased by LPS (1ug/mL) treatment, but the phosphorylated protein expression of MAPKs were not decreased by LPS (1ug/mL)+Dh404(200nM) treatment.4. Dh404treatment minimally regulates JAK-STAT pathway in RAW264.7macrophages.Compared with the control group, the phosphorylated protein expression of JAK-STAT (JAK1, JAK2and STAT3) were significantly increased by LPS (1ug/mL) treatment, but the phosphorylated protein expression of JAK-STAT were not decreased by LPS (1ug/mL)+Dh404(200nM) treatment.5. Dh404treatment minimally regulates NFkB pathway in RAW264.7macrophages.Compared with the control group, the protein expression of IκBa was significantly decreased by LPS (lug/mL) treatment, but it was not increased by LPS (1ug/mL)+Dh404(200nM) treatment; the luciferase assay of NFκB was significantly increased by LPS (1ug/mL) treatment, but it was not decreased by LPS (1ug/mL)+Dh404(200nM) treatment; the gene expression of IL-1β, IL-6and TNFa were significantly increased by LPS (1ug/mL) treatment,but they were not decreased by LPS (lug/mL)+Dh404(200nM) treatment.6. Dh404activates Nrf2in RAW264.7macrophages.Compared with the control group, Dh404(200nM) treatment rapidly increased Nrf2protein expression and increased Nrf2downstream genes expression, such as NAD(P)H:quinine oxidoreductase (NQO)1and heme oxygenase1(HO-1), at the mRN A and protein levels, indicating that Dh404can upregulates Nrf2pathway in RAW264.7macrophages.But Dh404(200nM) treatment was not increased the gene expression of Nrf2, indicating that Dh404’s regulation is posttranscriptional level; at the same time, Dh404(200nM) treatment was not increased the luciferase assay of Neh2, indicating that Dh404’s regulation is not through the separation of Nrf2and Keapl.7. Dh404suppresses LPS induced inflammatory responses via Nrf2.(1) Compared to Nrf2WT mice, the expression level of iNOS, MCP-1and MIP-1βin the Nrf2-/-mice bone marrow derived macrophages were much higher both in basal condition and in LPS-inflamed condition.(2) Dh404dramatically inhibited LPS-induced expression of iNOS, MCP-1and MIP-1β in bone marrow derived macrophages from WT mice, however, the anti-inflammatory effect of Dh404was blocked in LPS-inflamed bone marrow-derived macrophages from Nrf2-/-mice. These results clearly demonstrate that Nrf2is an essential mediator for Dh404induced resolution of inflammatory responses in macrophages.Conclusions1. Dh404could inhibit the inflammatory responses induced by LPS in RAW264.7macrophages and mouse bone marrow derived macrophages.2. Dh404treatment minimally regulates MAPKs, JAK-STAT and NFκB pathway in RAW264.7macrophages.3. Dh404could activate Nrf2and the downstream genes expression.4. Dh404suppresses LPS induced inflammatory responses via Nrf2.