• Journal of Microbiology and Biotechnology
• /
• v.27 no.10
• /
• pp.1783-1789
• /
• 2017

Thermostability is an important property of xylanase because high temperature is required for its applications, such as wood pulp bleaching, baking, and animal feedstuff processing. In this study, XynB from Thermobacillus composti, a moderately thermophilic gram-negative bacterium, was modified via site-directed mutagenesis (based on its 3D structure) to obtain thermostable xylanase, and the properties of this enzyme were analyzed. Results revealed that the half-life of xylanase at $65^{\circ}C$ increased from 10 to 50 min after a disulfide bridge was introduced between the ${\alpha}$-helix and its adjacent ${\beta}$-sheet at S98 and N145. Further mutation at the side of A153E named XynB-CE in the C-terminal of this ${\alpha}$-helix enhanced the half-life of xylanase for 60 min at $65^{\circ}C$. Therefore, the mutant may be utilized for industrial applications.

• BMB Reports
• /
• v.40 no.4
• /
• pp.571-576
• /
• 2007

Three anti-apoptosis genes, Ls-iap2, iap3 and p49 were found in Leucania separata multiple nuclear polyhedrovirus. Amino acid sequence homology of Ls-IAP2 and Ls-IAP3 with Op-IAP2 and Op-IAP3 from Orgyia pseddotsugata MNPV were 20% and 42%, while that of Ls-P49 is 28% with Sl-P49 from Spodoptera littorolis MNPV. Ls-IAP2 contains one baculoviral IAP repeat (BIR) domain followed by a RING domain, while Ls-IAP3 contains two BIRs and a RING. Ls-P49 contains a reactive site loop, predicted cleavage site (KKLD$^{74}{\downarrow}$G) that is different from Sl-P49 (TVID$^{94}{\downarrow}$G). Expressed Ls-iap3 or Ls-p49 under presence of actinomycin D in SF9 cells, DNA ladder assayrevealed that Ls- IAP3 or Ls-P49 could block the apoptosis of SF9 cells induced by actinomycin D. Replication of p35 deficient-mutant Autographa californica MNPV in SF9 cells was also rescued when Ls-iap3 or Ls-p49 was expressed transiently. No anti-apoptotic activity was observed for Ls-IAP2. The results showed that both of Ls-IAP3 and Ls-P49 were functional apoptotic suppressors in SF9 cells.

• BMB Reports
• /
• v.43 no.8
• /
• pp.567-572
• /
• 2010

In this study, we cloned the ERF-B3 subfamily transcription factor gene BnaERF-B3-hy15 from Brassica napus L. Huyou15. This 600 bp gene encodes a 199 amino acid classic ethylene responsive factor (ERF), which shown no binding or very weak binding GCC box-binding activity by the yeast one-hybrid assay. We used gene shuffling and the yeast one-hybrid system to obtain three mutated sequences that can bind to the GCC box. Sequence analysis indicated that two residues, Gly156 in the AP2 domain and Phe62 at the N-terminal domain were mutated to arginine and serine, respectively. Changes of Gly156 to arginine and Phe62 to serine increased the GCC-binding activity of BnaERF-B3-hy15 and the alter of Gly156 to arginine changed the AP2-domain structure of BnaERF-B3-hy15.

• BMB Reports
• /
• v.42 no.11
• /
• pp.752-757
• /
• 2009

Copper is essential but toxic in excess for aerobic organisms. Yeast transcription factor ACE1 functions as a sensor for copper and an inducer for the transcription of CUP1. In addition, ACE1 can activate the transcription of superoxide dismutase gene (sod1) in response to copper. In this study, we introduced the yeast ACE1 into Arabidopsis and analyzed its function in plant. Under high copper stress, the transgenic plants over-expressing ACE1 showed higher survival rate than the wild-type. We also found that over-expression of ACE1 in Arabidopsis increased the activities of SOD and POD, which were beneficial to the cell in copper buffering. Excess copper would suppress the expression of chlorophyll biosynthetic genes in Arabidopsis, RT-PCR analysis revealed that over-expression of ACE1 decrease the suppression. Together, our results indicate that ACE1 may play an important role in response to copper stress in Arabidopsis.

• Molecules and Cells
• /
• v.43 no.5
• /
• pp.438-447
• /
• 2020

The aim of this study was to explore the role of IL-6-miR-210 in the regulation of Tregs function and atrial fibrosis in atrial fibrillation (AF). The levels of interleukin (IL)-6 and IL-10 in AF patients were detected by using ELISA. Proportions of Treg cells were detected by fluorescence activated cell sorting analysis in AF patients. The expression of Foxp3, α-SMA, collagen I and collagen III were determined by western blot. The atrial mechanocytes were authenticated by vimentin immunostaining. The expression of miR-210 was performed by quantitative real-time polymerase chain reaction (qRT-PCR). TargetScan was used to predict potential targets of miR-210. The cardiomyocyte transverse sections in AF model group were observed by H&E staining. The myocardial filaments were observed by masson staining. The level of IL-6 was highly increased while the level of IL-10 (Tregs) was significantly decreased in AF patients as compared to normal control subjects, and IL-6 suppressed Tregs function and promoted the expression of α-SMA, collagen I and collagen III. Furthermore, miR-210 regulated Tregs function by targeting Foxp3, and IL-6 promoted expression of miR-210 via regulating hypoxia inducible factor-1α (HIF-1α). IL-6-miR-210 suppresses regulatory T cell function and promotes atrial fibrosis by targeting Foxp3.

• BMB Reports
• /
• v.44 no.2
• /
• pp.87-95
• /
• 2011

Enzymatic catalysis has been pursued extensively in a wide range of important chemical processes for their unparalleled selectivity and mild reaction conditions. However, enzymes are usually costly and easy to inactivate in their free forms. Immobilization is the key to optimizing the in-service performance of an enzyme in industrial processes, particularly in the field of non-aqueous phase catalysis. Since the immobilization process for enzymes will inevitably result in some loss of activity, improving the activity retention of the immobilized enzyme is critical. To some extent, the performance of an immobilized enzyme is mainly governed by the supports used for immobilization, thus it is important to fully understand the properties of supporting materials and immobilization processes. In recent years, there has been growing concern in using polymeric materials as supports for their good mechanical and easily adjustable properties. Furthermore, a great many work has been done in order to improve the activity retention and stabilities of immobilized enzymes. Some introduce a spacer arm onto the support surface to improve the enzyme mobility. The support surface is also modified towards biocompatibility to reduce non-biospecific interactions between the enzyme and support. Besides, natural materials can be used directly as supporting materials owning to their inert and biocompatible properties. This review is focused on recent advances in using polymeric materials as hosts for lipase immobilization by two different methods, surface attachment and encapsulation. Polymeric materials of different forms, such as particles, membranes and nanofibers, are discussed in detail. The prospective applications of immobilized enzymes, especially the enzyme-immobilized membrane bioreactors (EMBR) are also discussed.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.2
• /
• pp.245-253
• /
• 2014

A fibrinolytic enzyme was produced by an edible mushroom of Pleurotus ostreatus using submerged culture fermentation. The enzyme was purified from the culture supernatant by applying a combination of freeze-thaw treatment, ammonium sulfate precipitation, hydrophobic interaction, and gel filtration chromatographies. The enzyme was purified by a 147-fold, with a yield of 7.54%. The molecular masses of the enzyme an determined by gel filtration and SDS-PAGE were 13.6 and 18.2 kDa, respectively. The isoelectric point of the enzyme was 8.52. It hydrolyzed fibrinogen by cleaving the ${\alpha}$ and ${\beta}$ chains of fibrinogen followed by the ${\gamma}$ chains, and also activated plasminogen into plasmin. The enzyme was optimally active at $45^{\circ}C$ and pH 7.4. The enzyme activity was completely inhibited by EDTA, whereas protease inhibitors of TPCK, SBTI, PMSF, aprotinin and pepstatin showed no inhibition on its activity. The partial amino acid sequences of the enzyme as determined by Q-TOF2 were ATFVGCSATR, GGTLIHESSHFTR, and YTTWFGTFVTSR. These sequences showed a high degree of homology with those of metallo-endopeptidases from P. ostreatus and Armillaria mellea. The purified enzyme can also be applied as a natural agent for oral fibrinolytic therapy or prevention of thrombosis.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.6
• /
• pp.771-780
• /
• 2014

A putative lipolytic enzyme gene, named as est9x, was obtained from a marine microbial metagenome of the South China Sea. Sequence analysis showed that Est9X shares lower than 27% sequence identities with the characterized lipolytic enzymes, but possesses a catalytic triad highly conserved in lipolytic enzymes of the ${\alpha}/{\beta}$ hydrolase superfamily. By phylogenetic tree construction, Est9X was grouped into a new lipase/esterase family. To understand Est9X protein in depth, it was recombinantly expressed, purified, and biochemically characterized. Within potential hydrolytic activities, only lipase/esterase activity was detected for Est9X, confirming its identity as a lipolytic enzyme. When using p-nitrophenol esters with varying lengths of fatty acid as substrates, Est9X exhibited the highest activity to the C2 substrate, indicating it is an esterase. The optimal activity of Est9X occurred at a temperature of $65^{\cric}C$, and Est9X was pretty stable below the optimum temperature. Distinguished from other salt-tolerant esterases, Est9X's activity was tolerant to and even promoted by as high as 4 M NaCl. Our results imply that Est9X is a unique esterase and could be a potential candidate for industrial application under extreme conditions.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.22
• /
• pp.9859-9863
• /
• 2014

Genetic epidemiological studies have shown that genetic susceptibility to esophageal cancer (EC) is an important cause of its high incidence within families in some areas of China. The purpose of this study was to obtain evidence of a genetic basis of EC in Xin-an and Xin-xiang counties in China. Familial aggregation and complex segregation analyses were performed of 79 EC families in these counties. The heritability of EC was examined using Falconer's method and complex segregation analysis was conducted with the SEGREG program in Statistical Analysis for Genetic Epidemiology (SAGE version 5.3.1). The results showed that the distribution of EC in families did not fit well into a binomial distribution. The heritability of EC among first-degree and second-degree relatives was $67.0{\pm}7.31%$ and $43.1%{\pm}9.80%$, respectively, and the summing up powered heritability was $53.2{\pm}6.74%$. The segregation ratio was 0.045. Complex segregation analysis showed that the genetic model of EC was additive. The current results provide evidence for an inherited propensity to EC in certain high-risk groups in China, and support efforts to identify the genes that confer susceptibility to this disease.

• Asian Pacific Journal of Cancer Prevention
• /
• v.13 no.6
• /
• pp.2485-2489
• /
• 2012

Purpose: Notch is an important signaling pathway that regulates cell fate, stem cell maintenance and the initiation of differentiation in many tissues. It has been reported that activation of Notch-1 contributes to tumorigenesis. However, whether Notch signaling might have a role in chemoresistance of prostate cancer is unclear. This study aimed to investigate the effects of Notch-1 silencing on the sensitivity of prostate cancer cells to docetaxel treatment. Methods: siRNA against Notch-1 was transfected into PC-3 prostate cancer cells. Proliferation, apoptosis and cell cycle distribution were examined in the presence or absence of docetaxel by MTT and flow cytometry. Expression of $p21^{waf1/cip1}$ and Akt as well as activation of Akt in PC-3 cells were detected by Western blot and Real-time PCR. Results: Silencing of Notch-1 promoted docetaxel induced cell growth inhibition, apoptosis and cell cycle arrest in PC-3 cells. In addition, these effects were associated with increased $p21^{waf1/cip1}$ expression and decreased Akt expression and activation in PC-3 cells. Conclusion: Notch-1 promotes chemoresistance of prostate cancer and could be a potential therapeutic target.