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Chen K.,Tianjin University of Science and Technology | Liang N.,Medical Institute | Luo X.,Tianjin University of Science and Technology | Luo X.,Tianjin Key Laboratory of Industrial Microbiology | And 2 more authors.
Journal of Microbiology and Biotechnology | Year: 2013

Previous studies have shown that lactic acid bacteria can inhibit inflammatory responses, but the mechanisms are very little known. In this study, transaction and expression of three proinflammatory factors, iNOS, PTGS-2, and IL8, which are closely related to the inflammatory response, were investigated by luciferase reporter assay and RT-PCR in HT-29 cells treated by Lactobacillus acidophilus. The results showed that the live L. acidophilus sharply down-regulated the transcription of these three genes. Because there was a NF-κB binding site located at-265 bp,-225 bp, and-95 bp upstream of the iNOS, PTGS-2, and IL8 promoters, respectively, we further addressed the effects of NF-κB on transaction of the three promoters by cotransfection. As was expected, NF-κBs remarkably up-regulated the activity of the reporter gene and, no effect of NF-κBs on IL-8 promoter transaction was found after NF-κB binding site mutation of the IL8 promoter in HT-29 cells. In conclusion, the live L. acidophilus decreased the transcriptional activity of NF-κB and, in turn, inhibited the transaction of NF-κB on the three proinflammatory factors mentioned above.© the Korean Society for Microbiology and Biotechnology.


Liu Y.H.,Key Laboratory of Industrial Fermentation Microbiology | Liu Y.H.,National Engineering Laboratory for Industrial Enzymes | Liu Y.H.,Tianjin University of Science and Technology | Hu B.,Key Laboratory of Industrial Fermentation Microbiology | And 11 more authors.
Journal of Applied Microbiology | Year: 2012

Aims: The purpose of this research was to obtain the mutant of Bacillus licheniformis alpha amylase (BLA) with an improved acid stability and elucidate the difference in catalytic mechanism under acidic conditions between wild-type and mutant BLAs. Methods and Results: The stability of BLA under acid condition was enhanced through direct evolution using error-prone polymerase chain reaction. Two mutation sites, T353I and H400R, were obtained in BLA. To identify the mutation of amino acids in Thr353Ile/His400Arg related to its acid stability, single mutants Thr353Ile and His400Arg were obtained via site-directed mutagenesis. Among the resulting mutant enzymes, the kcat/Km values of the mutants Thr353Ile, His400Arg and Thr353Ile/His400Arg under pH 4·5 were 3·5-, 6·0- and 11·3-fold higher, respectively, than that of the wild-type. Thr353Ile/His400Arg exhibited stronger tolerance towards a lower pH without obvious difference in thermostability when compared with wild-type. Conclusions: The results combined with three-dimensional structure analysis of mutant BLAs demonstrated that Thr353Ile/His400Arg showed an improved acid stability under low pH condition as a result of the interactions of hydrogen bonding, hydrophobicity, helix propensity and electrostatic fields. Significance and Impact of Study: It provides theoretical basis and background data for the improvement of acid stability in BLA by protein engineering. © 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.


Wei Q.,Tianjin University of Science and Technology | Wei Q.,Laboratory of Industrial Fermentation Microbiology | Wang H.,Tianjin University of Science and Technology | Wang H.,Laboratory of Industrial Fermentation Microbiology | And 12 more authors.
Applied Microbiology and Biotechnology | Year: 2013

Soy sauce is a traditional condiment manufactured by natural inoculation and mixed culture fermentation. As is well known, it is the microbial community that plays an important role in the formation of its flavors. However, to date, its dynamic changes during the long period of fermentation process are still unclear, intensively constraining the improvement and control of the soy sauce quality. In this work, we revealed the dynamic changes of the microbial community by combining a cultured dependent method and a cultured independent method of polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis. Results indicated that the two methods verified and complemented each other in profiling microbial community, and that significant dynamics of the microbial community existed during the fermentation process, especially the strong inhibition of the growth of most of the microbes when entering into the mash stage from the koji stage. In the analysis of bacterial community, Staphylococcus and Bacillus were found to be the dominant bacteria and detected in the whole fermentation process. Kurthia and Klebsiella began to appear in the koji stage and then fade away in the early stage of the mash fermentation. In the analysis of fungal community, Aspergillus sojae and Zygosaccharomyces rouxii were found to be the dominant fungi in the koji and mash fermentation, respectively. It was clearly shown that when A. sojae decreased and disappeared in the middle stage of the mash fermentation, Z. rouxii appeared and increased at the meantime. Aspergillus parasiticus, Trichosporon ovoides and Trichosporon asahii also appeared in the koji and the early period of the mash fermentation and disappeared thereafter. Similar to Z. rouxii, Millerozyma farinosa and Peronospora farinosa were also found spontaneously which appeared in the mid-late period of the mash fermentation. The principal component analysis suggested that the microbial community underwent significant changes in the early period of the fermentation and, thereafter, tended to the stabilization in the mid-late periods. This study gave us important clues to understand the fermentation process and can serve as a foundation for improving the quality of soy sauce in the future. © 2013 Springer-Verlag Berlin Heidelberg.


Wang Y.,Key Laboratory of Industrial Fermentation Microbiology | Wang Y.,Tianjin University of Science and Technology | Liu Y.,Key Laboratory of Industrial Fermentation Microbiology | Liu Y.,National Engineering Laboratory for Industrial Enzymes | And 9 more authors.
Biotechnology Letters | Year: 2014

To achieve efficient expression and secretion of a biologically-active pullulanase, the effect of promoter and signal peptide on the production of pullulanase was studied. Three types of promoters (PP43, Papr and Pamy) and four types of signal peptides (SPsacB, SPamy, SPaprl and SPaprs) were combined to construct twelve expression cassettes for pullulanase in Bacillus subtilis. The pullulanase activity assay was employed to quantify the level of differential expression, and a real-time PCR assay was applied to comparatively track the transcriptional level. Under the same experimental conditions, the potency ratios among the three promoters were Papr > Pamy > PP43. The secretion efficiency ratios mediated by the signal peptides were SPsacB > SPamy > SPaprs > SPaprl. The highest yield of pullulanase could be achieved under the promotion mediated by Papr and secretion by SPsacB. © 2014 Springer Science+Business Media Dordrecht.


Zheng H.,Key Laboratory of Industrial Fermentation Microbiology | Zheng H.,Tianjin University of Science and Technology | Zheng H.,Chinese Academy of Agricultural Sciences | Liu Y.,Key Laboratory of Industrial Fermentation Microbiology | And 12 more authors.
Journal of Industrial Microbiology and Biotechnology | Year: 2014

The extreme process condition of high temperature and high alkali limits the applications of most of natural xylanases in pulp and paper industry. Recently, various methods of protein engineering have been used to improve the thermal and alkalic tolerance of xylanases. In this work, directed evolution and site-directed mutagenesis were performed to obtain a mutant xylanase improved both on alkali stability and thermostability from the native Paenibacillus campinasensis Family-11 xylanase (XynG1-1). Mutant XynG1-1B43 (V90R/P172H) with two units increased in the optimum pH (pH 7.0-pH 9.0) and significant improvement on alkali stability was selected from the second round of epPCR library. And the further thermoduric mutant XynG1-1B43cc16 (V90R/P172H/T84C- T182C/D16Y) with 10 C increased in the optimum temperature (60-70 C) was then obtained by introducing a disulfide bridge (T84C-T182C) and a single amino acid substitution (D16Y) to XynG1-1B43 using site-directed mutagenesis. XynG1-1B43cc16 also showed higher thermostability and catalytic efficiency (k cat /K m ) than that of wild-type (XynG1-1) and XynG1-1B43. The attractive improved properties make XynG1-1B43cc16 more suitable for bioleaching of cotton stalk pulp under the extreme process condition of high temperature (70 C) and high alkali (pH 9.0). © 2013 Society for Industrial Microbiology and Biotechnology.


Ge Z.,Key Laboratory of Industrial Fermentation Microbiology | Mao S.,Tianjin Key Laboratory of Industrial Microbiology | Li Y.,Tianjin University of Science and Technology | Liu X.,Tianjin Key Laboratory of Industrial Microbiology | Lu F.,Tianjin Key Laboratory of Industrial Microbiology
Shengwu Gongcheng Xuebao/Chinese Journal of Biotechnology | Year: 2014

In order to discover the steroid biotransformation ability of filamentous fungus Aspergillus niger TCCC41650, we studied the fermentation of 4-androstene-3,17-dione with A. niger TCCC41650. The transformation product was purified, crystallized and determined as 16β-hydroxy-androst-4-ene-3,17-dione by X-ray single crystal diffraction method. The best fermentation condition was found to be pH 6.0, ethanol amount 2% with a substrate concentration of 1‰, the transformation rate is 85.81% after 72 h. Based on the best of our knowledge, 16β-hydroxylation rarely occurs in microbial transformations of steroid. This study laid the foundation for the research of 16β-hydroxylation steroids. © 2014 by the Institute of Microbiology, the Chinese Academy of Sciences and the Chinese Society for Microbiology


Liu Y.,Key Laboratory of Industrial Fermentation Microbiology | Liu Y.,National Engineering Laboratory for Industrial Enzymes | Liu Y.,Tianjin Key Laboratory of Industrial Microbiology | Liu Y.,Tianjin University of Science and Technology | And 13 more authors.
Biotechnology Letters | Year: 2012

The gene encoding a novel alkaline pectate lyase (Apel) from Bacillus subtilis was cloned and expressed in B. subtilis WB600. Apel contained an ORF of 1,260 bp, encoding a signal peptide of 21 amino acids and a mature protein of 399 amino acids with a calculated molecular mass of 45497.9 Da. The mature Apel was structurally related to the enzymes in the polysaccharide lyase family 1. After purification, the recombinant Apel had a specific activity of 445 U mg -1. The enzyme was optimally active at 50°C and pH 9. © 2011 Springer Science+Business Media B.V.


Luo X.-G.,Tianjin University of Science and Technology | Luo X.-G.,Tianjin Key Laboratory of Industrial Microbiology | Zhang C.-L.,Tianjin University of Science and Technology | Zhang C.-L.,Tianjin Key Laboratory of Industrial Microbiology | And 17 more authors.
Cancer Letters | Year: 2014

Myocardin-related transcription factor-A (MRTF-A) is a Rho signal-responsive transcriptional coactivator of serum response factor (SRF). Recent studies indicated that MRTF-A might be an important regulator of mammary gland and be involved in cancer metastasis. However, the roles of histone modification in the MRTF-A-dependent signal pathway and tumor migration are still not very clear. Here, we report that histone methylation is required for the MRTF-A-mediated upregulation of myosin regulatory light chain 9 (MYL9), an important cytoskeletal component which is implicated in cell migration. Furthermore, we demonstrate that SET and MYND domain containing protein 3 (SMYD3), a hitone methyltransferase (HMT) associated with carcinogenesis, might be the one which is responsible for the histone methylation occurred in the MRTF-A-mediated- transactivation of MYL9 and migration of breast cancer cells. Overexpression of SMYD3 promotes MRTF-A-mediated upregulation of MYL9 and migration of MCF-7 breast cancer cells, while contrary results were observed when the endogenous MRTF-A and SMYD3 were suppressed with specific siRNAs. In addition, the mutation analysis suggested that this cooperative transactivation is mainly mediated via the proximal binding element of MRTF-A in the promoter of MYL9, and the HMT activity of SMYD3 is required as well. Our findings reveal a new mechanism by which MRTF-A and SMYD3 functions in transcriptional regulation and cell migration, and provide a better understanding for metastasis of breast cancer. © 2013 Elsevier Ireland Ltd.


Zheng H.,Key Laboratory of Industrial Fermentation Microbiology | Zheng H.,Tianjin University of Science and Technology | liu Y.,Key Laboratory of Industrial Fermentation Microbiology | liu Y.,Tianjin Key Laboratory of Industrial Microbiology | And 10 more authors.
Bioresource Technology | Year: 2012

A xylanase gene (xynG1-1) from Paenibacillus campinasensis G1-1 was expressed in Bacillus megaterium MS941 and a high level of extracellular xylansae activity (304.26. IU/mL) was achieved after induction with 0.5% xylose. The purified recombinant xylanase (XynG1-1R) revealed optimal activity at 60. °C and pH 7.0 and retained 79% and 81% activity after incubation without substrate at 60. °C, pH 5.0 and pH 8.0 for 3. h, respectively. Application of XynG1-1R (15. IU/g pulp) to cotton stalk pulp bleaching increased brightness by 3.65% over that of the control without the xylanase and reduced the need for chlorine compounds by 50% without loss of brightness and pulp fiber qualities. When XynG1-1R (80. IU/g paper sludge) was used in combination with mixed cellulolytic enzymes, the saccharification efficiency of recycled paper sludge was increased by 10%. These results indicated that XynG1-1R is a promising candidate for various industrial applications such as biobleaching and bioenergy conversion. © 2012 Elsevier Ltd.


Tian H.,Key Laboratory of Industrial Microbiology | Luo X.-G.,Key Laboratory of Industrial Microbiology | Luo X.-G.,Tianjin University of Science and Technology | Han Z.,Key Laboratory of Industrial Microbiology | And 5 more authors.
Advances in Intelligent and Soft Computing | Year: 2012

Lactobacillus plantarum (L. plantarum) is a flexible and versatile microorganism that inhabits a variety of environmental niches, and some strains are marketed as probiotics. In this study, a strainTH1 which was isolated from silage in our former works was classified asL. plantarum by 16S rDNA sequence analysis and phylogenetic tree. Then its potential abilities of bile tolerance, nitrate reduction and anti-hypertension were investigated. The results showed that L. plantarum TH1 had capacity to resistance to bile salt, directly deoxygenate nitrate to nitrogen or ammonia rather then nitrite, and produce ACE inhibitory components in fermented milk. These works indicated that this novel lactobacillus strain might be considered as candidates for probiotics strains using in the food or drug system to improve the health of patients suffering from hypertension or other chronic disease. © 2012 Springer-Verlag GmbH.

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