| Psychrophilics refer to microorganism that can thrive in permanent cold environment or in liquid water below 0℃.Although the living conditions in cold environment are very harsh,psychrophilics have unique low temperature adaptability.Psychrophilics can reduce the stress response brought by the outside world through a series of psychrophilic mechanisms,including the maintenance of protein function.Existing studies have shown that the secretion of enzymes that can perform normal catalytic functions under extreme low temperature conditions is a key prerequisite for psychrophilics to adapt to living conditions.Therefore,understanding the adaptation mechanism of enzymes secreted by psychrophilics is the most direct and effective way to study the low temperature adaptation mechanism of psychrophilics.In this study,the isolation,purification and growth characteristics of bacteria from low temperature environment were firstly studied.Taking a psychrophilic bacterium Pseudoalteromonas sp.BSw20308 derived from the Arctic Chukchi Sea screened as the research object.The enzymatic properties and structural dynamics of itsβ-glucosidase were studied.In order to explore the low temperature adaptation mechanism of psychrophilics at the biophysical and biochemical levels.In this study,a total of 34 microorganisms with cryophilic properties were obtained by isolating and culturing strains of soil samples from Cang Shan,Dali,Yunnan Province,combined with screening laboratory conserved strains.A GH1familyβ-glucosidase gene（pBgl）was amplified from psychrophilic bacterium Pseudoalteromonas sp.BSw20308 by PCR.Cloning,heterologous expression and enzymatic properties research were performed in Escherichia coli BL21（DE3）.The results showed that the optimum temperature for the enzymatic reaction ofβ-glucosidase（pBGL）was 35°C.Under the optimal conditions,the catalytic efficiency（kcat/Km）measured with cellobiose as substrate was 180.58±19.52 s-1m M-1.pBGL showed more than 60%relative activity at 5℃,maintained more than80%relative activity at 10-40℃,and only had less than 20%relative activity at50℃,indicating that the enzyme is a cold active enzyme.Further,molecular dynamics（MD）simulations were used to compare the differences in structural dynamic properties of pBGL and its homologous mesophilicβ-glucosidase（mBGL）,as well as their effects on temperature adaptation.The results showed that pBGL exhibited larger-scale conformational fluctuations,fewer intermolecular interactions,higher global and local conformational flexibility than mBGL.pBGL has a wider,rougher,and more conformational entropy free energy landscape（FEL）.These differences in structural dynamic behavior will be more conducive to pBGL’s adaptation to low temperature environments.Meanwhile,pBGL has a larger number of protein-solvent hydrogen bonds（HBs）than mBGL.The stronger interaction between pBGL and water molecules makes it have higher conformational flexibility than mBGL,and the solvent plays an important role in the low temperature adaptation mechanism of cold active enzymes.This study not only elucidated the low temperature adaptation mechanism of pBGL,but also further revealed the cold adaptation strategy of psychrophilic enzymes. |
