Production, purification, and characterization of a novel β-1,3-1,4-glucanase (lichenase) from thermophilic Rhizomucor miehei CAU432 were investigated. High-level extracellular β-1,3-1,4-glucanase production of 6230 U/mL was obtained when oat flour (3%, w/v) was used as a carbon source at 50 °C. The crude enzyme was purified to homogeneity with a specific activity of 28818 U/mg. The molecular weight of purified enzyme was estimated to be 35.4 kDa and 33.7 kDa by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature of the enzyme were pH 5.5 and 60 °C, respectively. The K(m) values of purified β-1,3-1,4-glucanase for barley β-glucan and lichenan were 2.0 mM and 1.4 mM, respectively. Furthermore, the gene (RmLic16A) encoding the β-1,3-1,4-glucanase was cloned and its deduced amino acid sequence showed the highest identity (50%) to characterized β-1,3-1,4-glucanase from Paecilomyces thermophila. The high-level production and biochemical properties of the enzyme enable its potential industrial applications.
BackgroundThe zygomycete fungi like Rhizomucor miehei have been extensively exploited for the production of various enzymes. As a thermophilic fungus, R. miehei is capable of growing at temperatures that approach the upper limits for all eukaryotes. To date, over hundreds of fungal genomes are publicly available. However, Zygomycetes have been rarely investigated both genetically and genomically.ResultsHere, we report the genome of R. miehei CAU432 to explore the thermostable enzymatic repertoire of this fungus. The assembled genome size is 27.6-million-base (Mb) with 10,345 predicted protein-coding genes. Even being thermophilic, the G + C contents of fungal whole genome (43.8%) and coding genes (47.4%) are less than 50%. Phylogenetically, R. miehei is more closerly related to Phycomyces blakesleeanus than to Mucor circinelloides and Rhizopus oryzae. The genome of R. miehei harbors a large number of genes encoding secreted proteases, which is consistent with the characteristics of R. miehei being a rich producer of proteases. The transcriptome profile of R. miehei showed that the genes responsible for degrading starch, glucan, protein and lipid were highly expressed.ConclusionsThe genome information of R. miehei will facilitate future studies to better understand the mechanisms of fungal thermophilic adaptation and the exploring of the potential of R. miehei in industrial-scale production of thermostable enzymes. Based on the existence of a large repertoire of amylolytic, proteolytic and lipolytic genes in the genome, R. miehei has potential in the production of a variety of such enzymes.
Natural polysaccharides, particularly galactomannans, are potential candidates for treatment of alcoholic liver diseases (ALD). However, applications are restricted due to the physicochemical properties associated with the high molecular weight. In this work, guar gum galactomannans were partially hydrolyzed by β-mannanase, and the molecular mechanisms of hepatoprotective effects were elucidated both in vitro and in vivo. Release of lactate dehydrogenase and cytochrome C were attenuated by partially hydrolyzed guar gum (PHGG) in HepG2 cells, due to protected cell and mitochondrial membrane integrity. PHGG co-administration decreased serum amino transaminases and cholinesterase levels of acute alcohol intoxicated mice, while hepatic pathologic morphology was depleted. Activity of superoxide dismutase, catalase, and glutathione peroxidase was recovered to 198.2, 34.5, 236.0 U/mg protein, respectively, while malondialdehyde level was decreased by 76.3% (PHGG, 1000 mg/kg∙day). Co-administration of PHGG induced a 4.4-fold increment of p-AMPK expression, and lipid metabolism was mediated. PHGG alleviated toll-like-receptor-4-mediated inflammation via the signaling cascade of MyD88 and IκBα, decreasing cytokine production. Moreover, mediated expression of Bcl-2 and Bax was responsible for inhibited acute alcohol-induced apoptosis with suppressed cleavage of caspase 3 and PARP. Findings gained suggest that PHGG can be used as functional food supplement for the treatment of acute alcohol-induced liver injury.
Two novel glycoside hydrolase (GH) family 12 xyloglucanase genes (designated RmXEG12A and RmXEG12B) were cloned from the thermophilic fungus Rhizomucor miehei. Both genes contained open reading frames of 729 bp encoding 242 amino acids. Their deduced amino acid sequences shared 68% identity with each other and less than 60% with other xyloglucanases. The two genes, without the sequences for the signal peptides, were cloned and successfully expressed in Escherichia coli as active xyloglucanases, designated RmXEG12A and RmXEG12B, with similar molecular masses--25.6 and 25.9 kDa, respectively. RmXEG12A showed optimal activity at pH 6.5 and 65 °C, RmXEG12B at pH 5.0 and 60 °C. Both recombinant xyloglucanases displayed very high specific activities, 6,681.4 and 3,092.2 U mg(-1), respectively, toward tamarind xyloglucan, but no activity toward carboxymethylcellulose, Avicel, or p-nitrophenyl derivatives. The main products of tamarind xyloglucan hydrolysis by the two xyloglucanases were XXXG, XXLG/XLXG, and XLLG (where G is an unsubstituted β-D-Glc residue, X is a xylosylated β-D-Glc residue, and L is a β-D-Glc residue substituted by xylosyl-galactose).
Background Central post-stroke pain (CPSP) is an intractable and disabling central neuropathic pain that severely affects patients’ lives, well-being, and socialization abilities. However, CPSP has been poorly studied mechanistically and its treatment remains challenging. Here, we used a rat model of CPSP induced by thalamic hemorrhage to investigate its underlying mechanisms and the effect of stellate ganglion block (SGB) on CPSP and emotional comorbidities. Methods Thalamic hemorrhage was produced by injecting collagenase IV into the ventral-posterolateral nucleus (VPL) of the right thalamus. The up-and-down method with von Frey hairs was used to measure the mechanical allodynia. Behavioral tests were carried out to examine depressive and anxiety-like behaviors including the open field test (OFT), elevated plus maze test (EPMT), novelty-suppressed feeding test (NSFT), and forced swim test (FST). The peri-thalamic lesion tissues were collected for immunofluorescence, western blotting, and enzyme-linked immunosorbent assay (ELISA). Genetic knockdown of thalamic hypoxia-inducible factor-1α (HIF-1α) and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) with microinjection of HIF-1α siRNA and NLRP3 siRNA into the VPL of thalamus were performed 3 days before collagenase injection into the same regions. Microinjection of lificiguat (YC-1) and MCC950 into the VPL of thalamus were administrated 30 min before the collagenase injection in order to inhibited HIF-1α and NLRP3 pharmacologically. Repetitive right SGB was performed daily for 5 days and laser speckle contrast imaging (LSCI) was conducted to examine cerebral blood flow. Results Thalamic hemorrhage caused persistent mechanical allodynia and anxiety- and depression-like behaviors. Accompanying the persistent mechanical allodynia, the expression of HIF-1α and NLRP3, as well as the activities of microglia and astrocytes in the peri-thalamic lesion sites, were significantly increased. Genetic knockdown of thalamic HIF-1α and NLRP3 significantly attenuated mechanical allodynia and anxiety- and depression-like behaviors following thalamic hemorrhage. Further studies revealed that intra-thalamic injection of YC-1, or MCC950 significantly suppressed the activation of microglia and astrocytes, the release of pro-inflammatory cytokines, the upregulation of malondialdehyde (MDA), and the downregulation of superoxide dismutase (SOD), as well as mechanical allodynia and anxiety- and depression-like behaviors following thalamic hemorrhage. In addition, repetitive ipsilateral SGB significantly restored the upregulated HIF-1α/NLRP3 signaling and the hyperactivated microglia and astrocytes following thalamic hemorrhage. The enhanced expression of pro-inflammatory cytokines and the oxidative stress in the peri-thalamic lesion sites were also reversed by SGB. Moreover, LSCI showed that repetitive SGB significantly increased cerebral blood flow following thalamic hemorrhage. Most strikingly, SGB not only prevented, but also reversed the development of mechanical allodynia and anxiety- and depression-like behaviors induced by thalamic hemorrhage. However, pharmacological activation of thalamic HIF-1α and NLRP3 with specific agonists significantly eliminated the therapeutic effects of SGB on mechanical allodynia and anxiety- and depression-like behaviors following thalamic hemorrhage. Conclusion This study demonstrated for the first time that SGB could improve CPSP with comorbid anxiety and depression by increasing cerebral blood flow and inhibiting HIF-1α/NLRP3 inflammatory signaling.
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