Rhizome of Ligusticum chuanxiong Hort. (LC), also known as chuanxiong, is a very common herb widely used to treat cardiovascular and cerebrovascular diseases.
Acetyl xylan esterases are among the key enzymes in the xylan degradation enzyme system. However, acetyl xylan esterases from natural microorganisms have low expression and low enzyme activity and are impure. In this study, a new xylanase gene, est1051, from the metagenomic library, was expressed in the prokaryotic system. Its enzymatic properties were explored, including optimum temperature and pH, thermal and pH stability, and tolerance against organic solvents, metal ions and salt solutions. Then the fermentation conditions of EST1051 were optimized by the response surface method, and the maximum enzyme yield reached 1909.32 U/L. Finally, the synergism with cellulase on straw degradation was evaluated. EST1051 displays high homology with acetylxylan esterases in terms of amino acid sequences and conserved active sites. EST1051 shows high stability across a broad temperature range, and retains more than 60% of its enzymatic activity between 4 and 60°C after 24 h of incubation. Single-factor analysis and orthogonal design were conducted to determine the optimal conditions for the maximizing the saccharification rate of wheat straws. Interestingly, the synergism of EST1051 with cellulase contributes to the efficient transformation of wheat straws. These findings may open the door to significant industrial applications of this novel acetylxylan esterase.
Abstract Cellulose is the cheapest, natural, renewable organic substance that is used as a carbon source in various fields. Water hyacinth, an aquatic plant rich in cellulose, is often used as a raw material in fuel production. However, natural cellulase can be hardly used in industrial production on account of its low thermal stability and activity. In this study, a metagenomic library was constructed. Then a new cellulase gene, cel1029 , was screened by Congo red staining and expressed in the prokaryotic system. Enzymatic properties of Cel1029 were explored, including optimum temperature and pH, thermal and pH stability, and tolerance against organic solvents, metal ions and salt solutions. Finally, its ability of degrading water hyacinth was identified and evaluated. Cel1029 displayed high homology with endoglucanase in the glycoside hydrolase family 5 (GH5) and had high stability across a broad temperature range. More than 86% of its enzymatic activities were retained between 4 and 60°C after 24 h of incubation. Single-factor analysis and orthogonal design were further conducted to determine the optimal conditions for the highest reducing sugar yield of water hyacinth. Interestingly, Cel1029 efficiently transformed water hyacinth with a reducing sugar yield of 430.39 mg/g in 22 h. These findings may open the door for significant industrial applications of a novel GH5 cellulase (NCBI Reference Sequence: MK051001, Cel1029 ) and help identify more efficient methods to degrade cellulose-rich plants.
Background and Purpose— Activation of Notch worsens ischemic brain damage as antisense knockdown or pharmacological inhibition of the Notch pathway reduces the infarct size and improves the functional outcome in a mouse model of stroke. We sought to determine whether Notch activation contributes to postischemic inflammation by directly modulating the microglial innate response. Methods— The microglial response and the attendant inflammatory reaction were evaluated in Notch1 antisense transgenic (Tg) and in nontransgenic (non-Tg) mice subjected to middle cerebral artery occlusion with or without treatment with a γ-secretase inhibitor (GSI). To investigate the impact of Notch on microglial effector functions, primary mouse microglia and murine BV-2 microglial cell line were exposed to oxygen glucose deprivation or lipopolysaccharide in the presence or absence of GSI. Immunofluorescence labeling, Western blotting, and reverse-transcription polymerase chain reaction were performed to measure microglial activation and production of inflammatory cytokines. The nuclear translocation of nuclear factor-κB in microglia was assessed by immunohistochemistry. The neurotoxic potential of microglia was determined in cocultures. Results— Notch1 antisense mice exhibit significantly lower numbers of activated microglia and reduced proinflammatory cytokine expression in the ipsilateral ischemic cortices compared to non-Tg mice. Microglial activation also was attenuated in Notch1 antisense cultures and in non-Tg cultures treated with GSI. GSI significantly reduced nuclear factor-κB activation and expression of proinflammatory mediators and markedly attenuated the neurotoxic activity of microglia in cocultures. Conclusions— These findings establish a role for Notch signaling in modulating the microglia innate response and suggest that inhibition of Notch might represent a complementary therapeutic approach to prevent reactive gliosis in stroke and neuroinflammation-related degenerative disorders.
Abstract Background The origin of breast cancer remains poorly understood. Here, we testify a putative mechanism of “breast cancer origin from inducible nitric oxide synthase (iNOS) activation and oestrogen receptor alpha (ERα) inactivation”, which are classified as the essential outcomes of chronic inflammatory responses. Methods To reverse breast cancer status, iNOS was downregulated by short-hairpin RNA (shRNA)-guided NOS2 knockdown from human triple-negative breast cancer (TNBC) cells. To re-enact breast cancer origin, ERα was downregulated by shRNA-directed ESR1 knockdown in human mammary epithelial cells. Results Upon NOS2 knockdown from HCC1937 cells, the specific TNBC transcription factor genes, RUNX1 and BCL11A , were downregulated, hypoxia was compromised, Warburg effects were attenuated, and tumourigenic proliferation was halted, accompanied by an increase in the tumour marker cyclin D1 (CD1) and a decrease in the tumour suppressor cyclin-dependent kinase inhibitor (CKI). In contrast, ESR1 knockdown from MCF-10A cells led to upregulated BCL11A and RUNX1 expression, augmented hypoxic responses, pronounced Warburg effects, and enhanced PI3K/Akt/mTOR signalling, together with low levels of CD1 and high levels of CKI induction. Conclusions Breast cancer should originate from inflammatory signalling, during which iNOS activation and ERα inactivation elicit hypoxia, oxidation, mitochondrial dysfunction, and breast cancer-like hyperplasia, demonstrating that iNOS inhibitors and ERα activators represent promising candidate prodrugs enabling breast cancer prevention in the early stage.
Esterase, as a type of powerful catabolic enzyme for the degradation of pyrethroid pesticides (PYRs), appears promising in improving the quality of crops and the environment contaminated by pesticide residues. The purpose of this research is to provide a detailed introduction to the enzymatic properties, optimal production and immobilization conditions, and the degradation ability of Est804 for PYRs. The study on enzymatic properties indicated that Est804 was an alkaline esterase with an optimal pH of 8.0 and a broad optimal temperature in the range of 35−50°C. The optimal activity of free Est804 was calculated to be 112.812 U, and the specific enzyme activity was 48.97 U/mg. The kinetic parameters of Est804 were K m = 0.613 mM, k cat = 12,371 s –1 , and V m = 0.095 mM/min. The results of the fermentative optimization demonstrated that the optimal conditions included 1.5% of inoculation amount, 30 mL of liquid volume, 28°C of the fermentation temperature, and 18 h of the fermentation time. The optimal medium consists of 15.87 g of yeast powder, 8.00 g of glycerol, and 9.57 g of tryptone in 1 L of liquid. The optimized enzyme activity was 1.68-fold higher than that before optimization. Immobilized Est804 exhibited the highest activity under the optimum preparation conditions, including 0.35 g of chitosan dosage, 0.4 mL of an enzyme, and 4 h at 40°C for adsorption. The degradation rates of Cypermethrin (CYP), fenpropathrin (FE), and lambda-cyhalothrin (LCT) by Est804 within 30 min were 77.35%, 84.73%, and 74.16%, respectively. The present study indicated that Est804 possesses great potential for the treatment of pesticide residues on crops and environmental remediation, conducive to the development of SGNH family esterase against pyrethroid accumulation.
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