Novel use for the osmolyte trimethylamine N-oxide: retaining the psychrophilic characters of cold-adapted protease deseasin MCP-01 and simultaneously improving its thermostability.
The low thermostability of cold-adapted enzymes is a main barrier for their application. A simple and reliable method to improve both the stability and the activity of cold-adapted enzymes is still rare. As a protein stabilizer, the effect of trimethylamine N-oxide (TMAO) on a cold-adapted enzyme or protein has not been reported. In this study, effects of TMAO on the structure, activity, and stability of a cold-adapted protease, deseasin MCP-01, were studied. Deseasin MCP-01 is a new type of subtilase from deep-sea psychrotolerant bacterium Pseudoalteromonas sp. SM9913. Fluorescence and CD spectra showed that TMAO did not perturb the structure of MCP-01 and therefore kept the conformational flexibility of MCP-01. One molar TMAO improved the activity of MCP-01 by 174% and its catalytic efficiency (k(cat) /K(m)) by 290% at 0 degrees C. In the presence of 1 M TMAO, the thermostability (t(1/2)) of MCP-01 increased by two- to fivefold at 60 approximately 40 degrees C. Structural analysis with CD showed that 1 M TMAO could keep the structural thermostability of MCP-01 close to that of its mesophilic counterpart subtilisin Carlsberg when incubated at 40 degrees C for 1 h. Moreover, 1 M TMAO increased the melting temperature (T(m)) of MCP-01 by 10.5 degrees C. These results suggest that TMAO can be used as a perfect stabilizing agent to retain the psychrophilic characters of a cold-adapted enzyme and simultaneously improve its thermostability.
He
Hai-Lun
HL
The State Key Lab of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Jinan, 250100, China.
Chen
Xiu-Lan
XL
Zhang
Xi-Ying
XY
Sun
Cai-Yun
CY
Zou
Bai-Chen
BC
Zhang
Yu-Zhong
YZ
D000222:Adaptation, Physiological / Q000235:genetics*; D002384:Catalysis / Q000187:drug effects; D002942:Circular Dichroism; D003080:Cold Temperature; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability / Q000187:drug effects; D008744:Methylamines / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects*; D031161:Pseudoalteromonas / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D044366:Transition Temperature / Q000187:drug effects
D008744:Methylamines; D010450:Endopeptidases; C477696:protease MCP-01, Pseudoaltermonas; C005855:trimethyloxamine