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The disproportionation activity of cyclodextrin glucosyltransferase (CGTase; EC 2.4.1.19) can be used to convert small molecules into glycosides, thereby enhancing their solubility and stability. However, CGTases also exhibit a competing hydrolysis activity. The +2 subsite of the substrate binding cleft plays an important role in both the disproportionation and hydrolysis activities, but almost all known mutations at this site decrease disproportionation activity. In this study, Leu(277) of the CGTase from Bacillus stearothermophilus NO2, located near both the +2 subsite and the catalytic acid/base Glu(253), was modified to assess the effect of side chain size at this position on disproportionation and hydrolysis activities. The best mutant, L277M, exhibited a reduced K(m) for the acceptor substrate maltose (0.48 mM versus 0.945 mM) and an increased k(cat)/K(m) (1,175 s(−1) mM(−1) versus 686.1 s(−1) mM(−1)), compared with those of the wild-type enzyme. The disproportionation-to-hydrolysis ratio of L277M was 2.4-fold greater than that of the wild type. Existing structural data were combined with a multiple-sequence alignment and Gly(282) mutations to examine the mechanism behind the effects of the Leu(277)mutations. The Gly(282) mutations were included to aid a molecular dynamics (MD) analysis and the comparison of crystal structures. They reveal that changes to a hydrophobic cluster near Glu(253) and the hydrophobicity of the +2 subsite combine to produce the observed effects. IMPORTANCE In this study, mutations that enhance the disproportionation to hydrolysis ratio of a CGTase have been discovered. For example, the disproportionation-to-hydrolysis ratio of the L277M mutant of Bacillus stearothermophilus NO2 CGTase was 2.4-fold greater than that of the wild type. The mechanism behind the effects of these mutations is explained. This paper opens up other avenues for future research into the disproportionation and hydrolysis activities of CGTases. Productive mutations are no longer limited to the acceptor subsite, since mutations that indirectly affect the acceptor subsite also enhance enzymatic activity.

Previous studies on the hydrolysis of polyacrylates by cutinase have found that cutinase from Humicola insolens can fulfill the requirement for a thermostable cutinase in the treatment of stickies from papermaking, but it has poor hydrolysis ability. To further improve its ability to hydrolyze the polymers in papermaking, we analyzed the structure of cutinase from H. insolens, and constructed three mutants L66A, I169A, and L66A/I169A to reduce the steric hindrance of the substrate binding region. The hydrolysis results for poly(methyl acrylate), poly(ethyl acrylate), and poly(vinyl acetate) showed the catalytic ability of the mutant L66A/I169A most significantly improved. Using polymer macroporous resin composites as substrate, the released products of L66A/I169A were 1.3-4.4 times higher than that of the wild-type enzyme. When polymer suspensions were no longer being deposited, that is, when the turbidity decrease was less than 1%, the amount of L66A/I169A added was reduced by 19%-51% compared with that of the wild-type enzyme. These results indicated that the removal of the gatekeeper structure above the substrate binding region of H. insolens cutinase enhances its ability to hydrolyze polymers, and provided a basis for the application of cutinase in the practical treatment of stickies.

Plastic products made of polyethylene (PE), polypropylene (PP), and polystyrene (PS) are widely used in daily life and industrial production. Polyolefins—which have a very stable structure and do not contain any active molecular groups—are difficult to degrade and pose a serious global environment threat. This study selected latex clearing protein (LcpK30) derived from Streptomyces sp. Strain K30. The natural substrate of the enzyme is rubber (cis-1, 4-polyisoprene), and the site of action is the carbon‑carbon double bond. LcpK30 was incubated with UV-irradiated polyolefin PE, PP and PS (UV-PE, UV-PP, and UV-PS containing carbon‑carbon double bonds) for 5 d at 37 °C. The results showed that UV-PE-LcpK30 was more fragmented than UV-PE-blank; the Fourier transform infrared spectroscopy results showed that UV-PE-LcpK30 and UV-PP-LcpK30 produced new active groups (e.g., –OH and –C=O); however, the effect on UV-PS was not significant. Scanning electron microscopy results showed that the treated group had more obvious roughness, cracks, and pits than the control group. The results of high-temperature gel permeation chromatography showed that the average molecular weight (Mw) of UV-PE-LcpK30 and UV-PP-LcpK30 decreased; the Mw of UV-PE5-LcpK30 was reduced by 42.02%. The results of gas chromatography–mass spectrometry showed the production of ketones. Therefore, the LcpK30 latex clearing protein degrade UV-oxidized polyolefin plastics and has great potential for PE and PP degradation but may not be suitable for PS. Furthermore, other Lcps (such as LcpNRRL, LcpNVL3) can also degrade UV-PE.

The aim of this study was to explore the anti-tumor potential of a polysaccharide isolated from Boschniakia rossica (BRP) in Hep2 human larynx squamous carcinoma cells. High performance size-exclusion chromatography analysis showed that BRP was a homogeneous polysaccharide and had a molecular weight of 22 kDa. Total carbohydrate content in BRP was determined to be 96.9%, without the presence of protein and nucleic acid. BRP suppressed the proliferation of Hep2 cells in a time- and dose-dependent manner. Cell cycle analysis revealed that exposure to BRP (200 μg/ml) caused a G0/G1 cell cycle arrest in Hep2 cells. Moreover, treatment with BRP at 100-400 μg/ml for 24h induced a significant apoptosis Hep2 cells compared to untreated control cells, as determined by flow cytometry with annexin-V/propidium iodide double staining. Additionally, BRP treatment promoted the cleavage of pro-caspase-3, pro-caspase-8, and pro-caspase-9, coupled with increased expression of death receptor DR5 and Bax and reduced expression of Bcl-2. Taken together, our data demonstrate that BRP shows potent anti-tumor activity in human larynx squamous carcinoma, largely through induction of G0/G1 cell cycle arrest and apoptosis. Activation of both mitochondria-mediated and death receptor-mediated apoptosis pathways is involved in the cytotoxicity of BRP.

Nasopharyngeal carcinoma (NPC) is a highly metastatic cancer, frequently occurring in Southeast Asia and Southern China. Several microRNAs (miRNAs) have been shown to have an inhibitive effect on NPC, while the effect of miR-15a on NPC remains unclear. Thus, our study aimed to investigate the potential effect of miR-15a on NPC cell proliferation, apoptosis, and possible functional mechanism. Human NPC CNE1 cells were transfected with miR-15a mimics, miR-15a inhibitors, or a control. Afterward, cell viability and apoptosis were assayed by using CCK-8, BrdU assay, and flow cytometry. Moreover, Western blot was used to detect the expression changes of proliferation and apoptosis of related proteins. As a result, miR-15a overexpression significantly reduced cell proliferation (p p p

This study was designed to evaluate bone matrix gelatin (BMG)/fibrin glue and chitosan/gelatin composite scaffolds for cartilage tissue engineering. Chondrocytes were isolated from costal cartilage of Sprague-Dawley rats and seeded on BMG/fibrin glue or chitosan/gelatin composite scaffolds. After different in vitro culture durations, the scaffolds were subjected to hematoxylin and eosin, Masson's trichrome, and toluidine blue staining, anti-collagen II and anti-aggrecan immunohistochemistry, and scanning electronic microscopy (SEM) analysis. After 2 weeks of culture, chondrocytes were distributed evenly on the surfaces of both scaffolds. Cell numbers and the presence of extracellular matrix components were markedly increased after 8 weeks of culture, and to a greater extent on the chitosan/gelatin scaffold. The BMG/fibrin glue scaffold showed signs of degradation after 8 weeks. Immunofluorescence analysis confirmed higher levels of collagen II and aggrecan using the chitosan/gelatin scaffold. SEM revealed that the majority of cells on the surface of the BMG/fibrin glue scaffold demonstrated a round morphology, while those in the chitosan/gelatin group had a spindle-like shape, with pseudopodia. Chitosan/gelatin scaffolds appear to be superior to BMG/ fibrin glue constructs in supporting chondrocyte attachment, proliferation, and biosynthesis of cartilaginous matrix components.