Biochemical and molecular characterization of a novel glycerol dehydratase from Klebsiella pneumoniae 2e with high tolerance against crude glycerol impurities.

BACKGROUND: The direct bioconversion of crude glycerol, a byproduct of biodiesel production, into 1,3-propanediol by microbial fermentation constitutes a remarkably promising value-added applications. However, the low activity of glycerol dehydratase, which is the key and rate-limiting enzyme in the 1,3-propanediol synthetic pathway, caused by crude glycerol impurities is one of the main factors affecting the 1,3-propanediol yield. Hence, the exploration of glycerol dehydratase resources suitable for crude glycerol bioconversion is required for the development of 1,3-propanediol-producing engineered strains. RESULTS: In this study, the novel glycerol dehydratase 2eGDHt, which has a tolerance against crude glycerol impurities from Klebsiella pneumoniae 2e, was characterized. The 2eGDHt exhibited the highest activity toward glycerol, with Km and Vm values of 3.42 mM and 58.15 nkat mg-1, respectively. The optimum pH and temperature for 2eGDHt were 7.0 and 37 °C, respectively. 2eGDHt displayed broader pH stability than other reported glycerol dehydratases. Its enzymatic activity was increased by Fe2+ and Tween-20, with 294% and 290% relative activities, respectively. The presence of various concentrations of the crude glycerol impurities, including NaCl, methanol, oleic acid, and linoleic acid, showed limited impact on the 2eGDHt activity. In addition, the enzyme activity was almost unaffected by the presence of an impurity mixture that mimicked the crude glycerol environment. Structural analyses revealed that 2eGDHt possesses more coil structures than reported glycerol dehydratases. Moreover, molecular dynamics simulations and site-directed mutagenesis analyses implied that the existence of unique Val744 from one of the increased coil regions played a key role in the tolerance characteristic by increasing the protein flexibility. CONCLUSIONS: This study provides insight into the mechanism for enzymatic action and the tolerance against crude glycerol impurities, of a novel glycerol dehydratase 2eGDHt, which is a promising glycerol dehydratase candidate for biotechnological conversion of crude glycerol into 1,3-PDO.

Integrin αvß6 sustains and promotes tumor invasive growth in colon cancer progression.

AIM: To detect the mechanism by which colon tumor escapes the growth constraints imposed on normal cells by cell crowding and dense pericellular matrices. METHODS: An immunohistochemical study of integrin αvß6 and matrix metalloproteinase-9 (MMP-9) was performed on tissue microarrays of 200 spots, including 100 cases of colon tumors. RESULTS: High immunoreactivity for αvß6 (73.7%; 28/38) and MMP-9 (76.5%; 52/68) was observed in invasive tumor portions. Furthermore, the effects of integrin αvß6 on tumor invasive growth in nude mice were detected. Tumor invasive growth and high expression of both αvß6 and MMP-9 were only seen in tumors resulting from WiDr cells expressing αvß6 in the tumorigenicity assay. Flow cytometry was applied to analyze αvß6 expression in colon cancer WiDr and SW480 cells. The effects of cell density on αvß6 expression and MMP-9 secretion were also detected by Biotrak MMP-9 activity assay and gelatin zymography assay. High cell density evidently enhanced αvß6 expression and promoted MMP-9 secretion compared with low density. CONCLUSION: Integrin αvß6 sustains and promotes tumor invasive growth in tumor progression via a self-perpetuating mechanism. Integrin ανß6-mediated MMP-9 secretion facilitates pericellular matrix degradation at high cell density, which provides the basis of invasive growth.