Engineering substrate specificity of quinone-dependent dehydrogenases for efficient oxidation of deoxynivalenol to 3-keto-deoxynivalenol.

The oxidative reaction of Fusarium mycotoxin deoxynivalenol (DON) using the dehydrogenase is a desirable strategy and environmentally friendly to mitigate its toxicity. However, a critical issue for these dehydrogenases shows widespread substrate promiscuity. In this study, we conducted pocket reshaping of Devosia strain A6-243 pyrroloquinoline quinone (PQQ)-dependent dehydrogenase (DADH) on the basis of protein structure and kinetic analysis of substrate libraries to improve preference for particular substrate DON (10a). The variant presented an increased preference for substrate 10a and enhanced catalytic efficiency. A 4.7-fold increase in preference for substrate 10a was observed. Kinetic profiling and molecular dynamics (MD) simulations provided insights into the enhanced substrate specificity and activity. Moreover, the variant exhibited stronger conversion of substrate 10a to 3-keto-DON compared to the wild DADH. Overall, this study provides a feasible protocol for the redesign of PQQ-dependent dehydrogenases with favourable substrate specificity and catalytic activity, which is desperately needed for DON antidote development.

Self-cascade deoxynivalenol detoxification by an artificial enzyme with bifunctions of dehydrogenase and aldo/keto reductase from genome mining.

Due to the severe health risks for human and animal caused by the intake of toxic deoxynivalenol (DON) derived from Fusarium species, elimination DON in food and feed has been initiated as a critical issue. Enzymatic cascade catalysis by dehydrogenase and aldo-keto reductase represents a fascinating strategy for DON detoxification. Here, one quinone-dpendent alcohol dehydrogenase DADH oxidized DON into less-toxic 3-keto-DON and NADPH-dependent aldo-keto reductase AKR13B3 reduced 3-keto-DON into relatively non-toxic 3-epi-DON were identified from Devosia strain A6-243, indicating that degradation of DON on C3 are two-step sequential cascade processes. To establish the bifunctions, fusion enzyme linking DADH and AKR13B3 was successfully assembled to promote one-step DON degradations with accelerated specific activity and efficiency, resulting 93.29 % of DON removal rate in wheat sample. Three-dimensional simulation analysis revealed that the bifunctional enzyme forms an artificial intramolecular channel to minimize the distance of intermediate from DADH to AKR13B3 for two-step enzymatic reactions, and thereby accelerates this enzymatic process. As the first report of directing single step DON detoxification by an interesting bifunctional artificial enzyme, this work revealed a facile and eco-friendly approach to detoxify DON with application potential and gave valuable insights into execute other mycotoxin detoxification for ensuring food safety.

Structure-Guided Steric Hindrance Engineering of Devosia Strain A6-243 Quinone-Dependent Dehydrogenase to Enhance Its Catalytic Efficiency.

Deoxynivalenol (DON), the most widely distributed mycotoxin worldwide, causes severe health risks for humans and animals. Quinone-dependent dehydrogenase derived from Devosia strain A6-243 (DADH) can degrade DON into less toxic 3-keto-DON and then aldo-keto reductase AKR13B3 can reduce 3-keto-DON into relatively nontoxic 3-epi-DON. However, the poor catalytic efficiency of DADH made it unsuitable for practical applications, and it has become the rate-limiting step of the two-step enzymatic cascade catalysis. Here, structure-guided steric hindrance engineering was employed to enhance the catalytic efficiency of DADH. After the steric hindrance engineering, the best mutant, V429G/N431V/T432V/L434V/F537A (M5-1), showed an 18.17-fold increase in specific activity and an 11.04-fold increase in catalytic efficiency (kcat/Km) compared with that of wild-type DADH. Structure-based computational analysis provided information on the increased catalytic efficiency in the directions that attenuated steric hindrance, which was attributed to the reshaped substrate-binding pocket with an expanded catalytic binding cavity and a favorable attack distance. Tunnel analysis suggested that reshaping the active cavity by mutation might alter the shape and size of the enzyme tunnels or form one new enzyme tunnel, which might contribute to the improved catalytic efficiency of M5-1. These findings provide a promising strategy to enhance the catalytic efficiency by steric hindrance engineering.

Allergenicity, assembly and applications of ovalbumin in egg white: a review.

Ovalbumin (OVA), the most abundant protein in egg whites, has been widely used in various industries. Currently, the structure of OVA has been clearly established, and the extraction of high-purified OVA has become feasible. However, the allergenicity of OVA is still a serious problem because it can cause severe allergic reactions and may even be life-threatening. The structure and allergenicity of the OVA can be altered by many processing methods. In this article, a detailed description on the structure and a comprehensive overview on the extraction protocols and the allergenicity of OVA was documented. Additionally, the information on assembly and potential applications of OVA was summarized and discussed in detail. Physical treatment, chemical modification, and microbial processing can be applied to alter the IgE-binding capacity of OVA by changing its structure and linear/sequential epitopes. Furthermore, research indicated that OVA could assemble with itself or other biomolecules into various forms (particles, fibers, gels, and nanosheets), which expanded its application in the food field. OVA also shows excellent application prospects, including food preservation, functional food ingredients and nutrient delivery. Therefore, OVA demonstrates significant investigation value as a food grade ingredient.

Cis-Element Engineering Promotes the Expression of Bacillus subtilis Type I L-Asparaginase and Its Application in Food.

Type I L-asparaginase from Bacillus licheniformis Z-1 (BlAase) was efficiently produced and secreted in Bacillus subtilis RIK 1285, but its low yield made it unsuitable for industrial use. Thus, a combined method was used in this study to boost BlAase synthesis in B. subtilis. First, fifteen single strong promoters were chosen to replace the original promoter P43, with PyvyD achieving the greatest BlAase activity (436.28 U/mL). Second, dual-promoter systems were built using four promoters (PyvyD, P43, PaprE, and PspoVG) with relatively high BlAase expression levels to boost BlAase output, with the engine of promoter PaprE-PyvyD reaching 502.11 U/mL. The activity of BlAase was also increased (568.59 U/mL) by modifying key portions of the PaprE-PyvyD promoter. Third, when the ribosome binding site (RBS) sequence of promoter PyvyD was replaced, BlAase activity reached 790.1 U/mL, which was 2.27 times greater than the original promoter P43 strain. After 36 h of cultivation, the BlAase expression level in a 10 L fermenter reached 2163.09 U/mL, which was 6.2 times greater than the initial strain using promoter P43. Moreover, the application potential of BlAase on acrylamide migration in potato chips was evaluated. Results showed that 89.50% of acrylamide in fried potato chips could be removed when combined with blanching and BlAase treatment. These findings revealed that combining transcription and translation techniques are effective strategies to boost recombinant protein output, and BlAase can be a great candidate for controlling acrylamide in food processing.

Non-classical secretion of a type I L-asparaginase in Bacillus subtilis.

L-asparaginase (EC 3.5.1.1) showed great commercial value owing to its effective treatment of acute lymphoblastic leukemia (ALL), lymphoid system malignancies and Hodgkin disease, and also to its use in the prevention of acrylamide formation in fried and baked foods. In this study, a type I L-asparaginase gene from Bacillus licheniformis Z-1 (BlAase) was cloned and expressed in Bacillus subtilis RIK 1285. Results showed that even without the mediation of any N-terminal signal peptides, BlAase can efficiently secrete into the medium. Further investigation indicated that the secretion of the BlAase was via neither Sec- nor Tat-dependent secretion pathway, and both the N- and C-terminal regions of the BlAase were essential for its expression and secretion, implying that BlAase might be secreted via a non-classical secretion pathway. To explore its secretion ability, BlAase was used as a signal peptide to direct the secretion of various heterologous proteins, where two of five proteins were successfully secreted with the mediation of BlAase. To the best of our knowledge, this is the first time to achieve extracellular expression of L-asparaginase via non-classical protein secretion pathway in B. subtilis, and provide a potential tool for secretion of recombinant proteins expressed in B. subtilis using BlAase as a signal peptide.

Epimerization of Deoxynivalenol by the Devosia Strain A6-243 Assisted by Pyrroloquinoline Quinone.

Deoxynivalenol (DON) is a secondary metabolite produced by several Fusarium species that is hazardous to humans and animals after entering food chains. In this study, by adding cofactors, the Devosia strain A6-243 is identified as the DON-transforming bacteria from a bacterial consortium with the ability to biotransform DON of Pseudomonas sp. B6-24 and Devosia strain A6-243, and its effect on the biotransformation process of DON is studied. The Devosia strain A6-243 completely biotransformed 100 µg/mL of DON with the assistance of the exogenous addition of PQQ (pyrroloquinoline quinone) within 48 h and produced non-toxic 3-epi-DON (3-epi-deoxynivalenol), while Pseudomonas sp. B6-24 was not able to biotransform DON, but it had the ability to generate PQQ. Moreover, the Devosia strain A6-243 not only degraded DON, but also exhibited the ability to degrade 3-keto-DON (3-keto-deoxynivalenol) with the same product 3-epi-DON, indicating that DON epimerization by the Devosia strain A6-243 is a two-step enzymatic reaction. The most suitable conditions for the biodegradation process of the Devosia strain A6-243 were a temperature of 16-37 °C and pH 7.0-10, with 15-30 µM PQQ. In addition, the Devosia strain A6-243 was found to completely remove DON (6.7 µg/g) from DON-contaminated wheat. The results presented a reference for screening microorganisms with the ability of biotransform DON and laid a foundation for the development of enzymes for the detoxification of mycotoxins in grain and its products.

[Value of hsCRP and Alb in Evaluating the Prognosis of Patients with Systemic Lupus Erythematosus].

OBJECTIVE: To study the value of hsCRP and Alb in evaluating the prognosis of patients with systemic lupus erythematosus (SLE). METHODS: 126 SLE patients from January 2011 to January 2016 were enrolled in this study, and their clinical data were collected, including SLEDAI, hsCRP and Alb and complications. The correlation between hsCRP/Alb ratio and SLEDAI after treatment was analyzed. All patients were followed up after discharge, and the prognosis-related factors were analyzed. RESULTS: After treatment, hsCRP/Alb ratio of patients with SLEDAI 10-14 score was significantly higher than that of 5-9 and 0-4 score(P<0.05). hsCRP/Alb ratio was positively correlated with infection (r=0.574), renal damage (r=0.499) and cardiac injury (r=0.516) (P<0.05), while it demonstrated no correlation with blood system damage, CNS damage and lung injury(P>0.05). after treatment SLEDAI ≥10 score, hsCRP/Alb≥0.05 mg/g and complications significantly correlated with prognosis of patients(P<0.05). CONCLUSION: hsCRP/Alb correlates with the prognosis of patients with SLE at a certam level.

Identification of a discordant genotype at the D2S1338 locus between Identifiler Plus®16 kit and Power Plex®21 system kit.

Genotyping errors can perturb estimates of genetic diversity and adversely affects the reliability of forensic identification, which has become increasingly recognized. During our daily laboratory work, we hit upon a discordant short tandem repeat (STR) genotype at the D2S1338 locus between Identifiler Plus®16 system kit and Power Plex®21 system kit. To figure out the reason of the discordance, nested PCR was performed to amplify the D2S1338 locus and then the final amplicons were sequenced. We found a G to G/A transition at the position on which caused an allele dropout of Identifiler Plus®16 kit.

Interleukin-12 improves cytotoxicity of natural killer cells via upregulated expression of NKG2D.

Natural killer (NK) cells are crucial components of the innate immune system, providing the first line of defense against infectious pathogens and tumors. Interleukin (IL)-12 is an interleukin produced primarily by antigen-presenting cells that play an essential role in the interaction between the innate and adaptive arms of immunity acting upon T and NK cells to generate cytotoxic lymphocytes. In the present study, we explored the effect of IL-12 upregulation on the NK receptor NKG2D and on the promotion of NK cell function. IL-12 enhanced the cytotoxicity of NK cells to different solid and hematological tumor cell lines and promoted interferon-gamma secretion by NK cells. The IL-12-induced cytolytic effect was dependent on the interaction of NKG2D with its ligand, MICA, because blockade of either protein attenuated the effect of IL-12 on NK cytolysis. Reverse transcriptase-polymerase chain reaction and fluorescence-activated cell sorting analyses indicated that IL-12 treatment increased NKG2D transcripts and surface expression in NK cells. Also, IL-12 augmented the expression of cytotoxic effector molecules, TRAIL and perforin, and the phosphorylation of STAT1, STAT4, and ERK1/2, which may also contribute to lysis by NK cells. These results are encouraging for the potential use of IL-12 as part of immunotherapy.

Opposing effects of interferon-alpha and interferon-gamma on the expression of major histocompatibility complex class I chain-related A in tumors.

Natural killer cells are an important component of innate resistance to viruses, bacteria, certain parasites, and tumors. The activating receptor natural killer group 2D (NKG2D) plays a critical role in the elimination of tumor cells by cytotoxic effector cells. It has been shown that the strength of an antitumor immune response might be critically dependent on NKG2D ligard surface levels. Thus, it is essential to regulate the expression of NKG2D ligands in order to ensure effective tumor immunosurveillance and the elimination of pathogen-infected cells. In the present study, we found that interferon (IFN)-alpha and IFNgamma exert opposing effects on major histocompatibility complex class I-related chain A (MICA) expression in human tumor cells. IFNalpha promotes expression of the NKG2D ligand MICA in tumor cells and therefore enhances their sensitivity to natural killer lysis. In contrast, IFNgamma exerts the opposite effect. IFNalpha promotes MICA expression at the level of transcription by augmenting MICA promoter activity. IFNgamma modulates MICA expression not only at the transcriptional level, but also at the post-translational level by promoting proteolytic cleavage by matrix metalloproteinases. Further study is needed to clarify the precise regulatory mechanisms. The pathways involved in NKG2D ligand induction might represent a promising target for improving immune responses to cancer or infections.

[Expression and significance of NKG2D ligands in 13 tumor cell lines].

BACKGROUND & OBJECTIVE: The interaction between NKG2D and its ligands plays a major role in immune surveillance against tumor. This study was to observe the expression and analyze the significance of NKG2D ligands in 13 tumor cell lines. METHODS: The mRNA expression of NKG2D ligands in K562, Raji, PG, Hep2, HepG2, HeLa, HT29, M21, MDA231, SGC7901, Caski, HL-60 and Jurkat cells was measured by reverse transcription-polymerase chain reaction (RT-PCR). The cytotoxicity of natural killer (NK) cells to the tumor cells at different effector-to-target cell (E:T) ratios were detected by MTT assay. The expression of MICA protein was measured by SABC immunohistochemistry and Western blot. RESULTS: The 13 tumor cell lines expressed different levels of NKG2D ligands. MICA was highly expressed in Hep2 cells, but not expressed in Caski, PG, HL-60 and Raji cells. The expression of MICA and MICB were positively correlated to the cytotoxicity of NK cells (r=0.851, P<0.001; r=0.652, P<0.05). Except for ULBP3, the expression of ULBP1, 2, 4 had no correlations to the cytotoxicity of NK cells. CONCLUSION: Among the 6 human NKG2D ligands, the expression of MICA is most intimate to the cytotoxicity of NK cells to tumor cells, and its expression level may determine the degree of immune response of NK cells to tumor.

Interleukin-15 improves cytotoxicity of natural killer cells via up-regulating NKG2D and cytotoxic effector molecule expression as well as STAT1 and ERK1/2 phosphorylation.

NK cells are crucial components of the innate immune system, providing a first line of defense against infectious pathogens and tumors. IL-15 is the major physiologic growth factor responsible for NK cell differentiation, survival and cytolytic activity of mature NK cells. However, the exact regulatory mechanism of IL-15 on NK cell function is still unclear. In this study, we try to investigate the mechanism of IL-15 on NK cytolysis. Our results demonstrate that IL-15 treatment increased NKG2D transcripts and surface expression in NK cells. NKG2D or MICA blockade attenuated the up-regulation of IL-15 on NK cytolysis, demonstrating that the up-regulatory effect of IL-15 on NK cytolysis is at least partly dependent of the interaction of NKG2D and MICA. Furthermore, IL-15 augmented the expression of cytotoxic effector molecules (TRAIL and Perforin) and the phosphorylation of STAT1 and ERK1/2, which may also contribute the NK lysis. These results may have therapeutic implications when designing cytokine immunotherapy against cancer.

[Up-regulation of IFN-alpha on expression of MICA in the cervical carcinoma cell lines].

AIM: To investigate the effect of IFN-alpha on expression of MHC class I chain-related protein A (MICA) in the cervical carcinoma cell lines. METHODS: The cervical carcinoma cell lines (HeLa and Caski) were treated with IFN-alpha. The expression of MICA was measured by RT-PCR and by immunohistochemical staining. The cytotoxicity of human NK cells to the IFN-alpha treated cervical carcinoma cells was detected by MTT method. RESULTS: The mRNA and protein expression of MICA was up-regulated by IFN-alpha in HeLa and Caski cells in a time and dose-dependent manner. Compared to Caski cells which weakly expressed MICA, higher cytolytic activity of NK cells was found against HeLa cells, which expressed relatively higher level of MICA. After being treated with IFN-alpha for 3 d, the susceptibility of the two cervical carcinoma cells to NK cytolysis was increased significantly. CONCLUSION: IFN-alpha can up-regulate the MICA expression in the cervical carcinoma cell lines and thereby enhance the susceptibility to cytolysis of NK cells.