Carbohydrate-binding modules enhance H2O2 tolerance by promoting lytic polysaccharide monooxygenase active site H2O2 consumption.

Lytic polysaccharide monooxygenases (LPMOs) oxidatively depolymerize recalcitrant polysaccharides, which is important for biomass conversion. The catalytic domains of many LPMOs are linked to carbohydrate-binding modules (CBMs) through flexible linkers, but the function of these CBMs in LPMO catalysis is not well understood. In this study, we utilized MtLPMO9L and MtLPMO9G derived from Myceliophthora thermophila to investigate the impact of CBMs on LPMO activity, with particular emphasis on their influence on H2O2 tolerance. Using truncated forms of MtLPMO9G generated by removing the CBM, we found reduced substrate binding affinity and enzymatic activity. Conversely, when the CBM was fused to the C terminus of the single-domain MtLPMO9L to create MtLPMO9L-CBM, we observed a substantial improvement in substrate binding affinity, enzymatic activity, and notably, H2O2 tolerance. Furthermore, molecular dynamics simulations confirmed that the CBM fusion enhances the proximity of the active site to the substrate, thereby promoting multilocal cleavage and impacting the exposure of the copper active site to H2O2. Importantly, the fusion of CBM resulted in more efficient consumption of H2O2 by LPMO, leading to improved enzymatic activity and reduced auto-oxidative damage of the copper active center.

A lytic polysaccharide monooxygenase from Myceliophthora thermophila and its synergism with cellobiohydrolases in cellulose hydrolysis.

Lytic polysaccharide monooxygenases (LPMOs) have attracted vast attention because of their unique mechanism of oxidative degradation of carbohydrate polymers and the potential application in biorefineries. This study characterized a novel LPMO from Myceliophthora thermophila, denoted MtLPMO9L. The structure model of the enzyme indicated that it belongs to the C1-oxidizing LPMO, which has neither an extra helix in the L3 loop nor extra loop region in the L2 loop. This was confirmed subsequently by the enzymatic assays since MtLPMO9L only acts on cellulose and generates C1-oxidized cello-oligosaccharides. Moreover, synergetic experiments showed that MtLPMO9L significantly improves the efficiency of cellobiohydrolase (CBH) II. In contrast, the inhibitory rather than synergetic effect was observed when combining used MtLPMO9L and CBHI. Changing the incubation time and concentration ratio of MtLPMO9L and CBHI could attenuate the inhibitory effects. This discovery suggests a different synergy detail between MtLPMO9L and two CBHs, which implies that the composition of cellulase cocktails may need reconsideration.

Effects of lipopolysaccharides stimulated Kupffer cells on activation of rat hepatic stellate cells.

AIM: To study the effects of Kupffer cell-conditioned medium (KCCM) derived from lipopolysaccharide (LPS) treatment on proliferation of rat hepatic stellate cells (HSC). METHODS: HSC and Kupffer cells were isolated from the liver of Wistar rats by in situ perfusion with pronase and collagenase and density gradient centrifugation with Nycodenz and cultured. KCCM was prepared and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to detect HSC proliferation. The content of type IV collagen and laminin secreted by HSC in the HSC-conditioned medium was determined by radioimmunoassay. TGF-beta(1) production in the KCCM was detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: HSC and Kupffer cells isolated had high purity. One microgram per mililiter LPS-activated KCCM and unstimulated KCCM could significantly promote HSC proliferation [0.132+/-0.005 and 0.123+/-0.008 vs control group (0.100+/-0.003), P<0.01], and there was a difference between them (P<0.05). Ten microgram per mililiter LPS-activated KCCM (0.106+/-0.010) was unable to promote HSC proliferation (P>0.05). Adding anti-TGF-beta(1) antibodies could suppress the proliferation promoted by unstimulated KCCM and LPS (1 microg/ml)-activated KCCM (0.109+/-0.009 vs 0.123+/-0.008, 0.115+/-0.008 vs 0.132+/-0.005, P<0.01). LPS (1 microg/ml or 10 microg/ml) could not promote HSC proliferation immediately (0.096+/-0.003 and 0.101+/-0.004 vs 0.100+/-0.003, P>0.05). There was a parallel behavior between HSC proliferation and increased ECM level. One microgram per mililiter LPS-activated KCCM contained a larger amount of TGF-beta(1) than unstimulated KCCM. CONCLUSION: The technique for isolation of HSC and Kupffer cells described here is simple and reliable. KCCM stimulated by LPS may promote HSC proliferation and collagen accumulation, which are associated with hepatic fibrogenesis.

[Animal model of acute spinal cord injury in rats].

OBJECTIVE: To investigate a animal model of spinal cord injury in different degrees of impact. METHODS: A new weight-drop device was designed with the character of controlled degree of impact and time. After thirty-five rats underwent different degrees of impact, their motor function and pathological changes were observed. RESULTS: In control group, the rats could walk after reviving, and the micro-structure of spinal cord was normal. With 0.5 mm depth of impact, the rats also could walk, and the micro-structure of spinal cord did not change obviously. With 0.8 mm depth of impact, the rats could walk after several days of injury and only slight damage could be found in spinal cord. When the impact depth increased to 1.0 or 1.5 mm, the rats were paralyzed completely and could not walk after four weeks of injury. Severe injury was observed in spinal cord. CONCLUSION: This animal model of spinal cord injury is based on different degrees of impact. It has stable and repetitive characters for the research on spinal cord injury.