RESUMO
Nitrile hydratase (NHase), an excellent bio-catalyst for the synthesis of amide compounds, was composed of two heterologous subunits. A thermoalkaliphilic NHase NHCTA1 (Tm = 71.3°C) obtained by in silico screening in our study exhibited high flexibility of α-subunit but excellent thermostability, as opposed to previous examples. To gain a deeper structural insight into the thermostability of NHCTA1, comparative molecular dynamics simulation of NHCTA1 and reported NHases was carried out. By comparison, we speculated that ß-subunit played a key role in adjusting the flexibility of α-subunit and the different conformations of linker in "α5-helix-coil ring" supersecondary structure of ß-subunit can affect the interaction between ß-subunit and α-subunit. Mutant NHCTA1-α6 C with a random coil linker and mutant NHCTA1-αßγ with a truncated linker were therefore constructed to understand the impact on NHCTA1 thermostability by varying the supersecondary structure. The varied thermostability of NHCTA1-α6 C and NHCTA1-αßγ (Tmα6C = 74.4°C, Tmαßγ = 65.6°C) verified that the flexibility of α-subunit adjusted by ß-subunit was relevant to the stability of NHCTA1. This study gained an insight into the NNHCTA1 thermostability by virtual dynamics comparison and experimental studies without crystallization, and this approach could be applied to other industrial-important enzymes.
RESUMO
A Gram-stain-negative, rod-shaped and motile bacterial strain, designated A9T, was isolated from the surface of rock collected from the shore of Nvshan lake in Mingguang, Anhui province, China. Phylogenetic analysis based on 16S rDNA sequence data showed that strain A9T was affiliated with the genus Massilia and showed the highest sequence similarities to Massilia plicata KCTC 12344T (98.8â%) and Massilia lurida CGMCC 1.10822T (97.9â%). The major fatty acids (>5â%) were summed feature 3 (C16â:â1ω7c and/or C15â:â0 iso 2-OH), C16â:â0 and C18â:â1ω7c. Strain A9T contained Q-8 as the predominant ubiquinone and diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and an unidentified aminophospholipid as the predominant polar lipids. The DNA G+C content was 69.9 mol%. Mean DNA-DNA relatedness values between strain A9T and its closest phylogenetic relatives, M. plicata KCTC 12344T and M. lurida CGMCC 1.10822T, were 38.8â% and 23.23â%, respectively. On the basis of the results obtained in this study, strain A9T is considered to represent a novel species of the genus Massilia, for which the name Massilia buxea sp. nov. is proposed. The type strain is A9T (=DSM 103547T=CGMCC 1.15931T=KCTC 52429T).