To elucidate the strategy of low temperature adaptation for a cold-adapted family 8 xylanase, the thermal and chemical stabilities, thermal inactivation, thermodependence of activity and conformational flexibility, as well as the thermodynamic basis of these processes, were compared with those of a thermophilic homolog. Differential scanning calorimetry, fluorescence monitoring of guanidine hydrochloride unfolding and fluorescence quenching were used, among other techniques, to show that the cold-adapted enzyme is characterized by a high activity at low temperatures, a poor stability and a high flexibility. In contrast, the thermophilic enzyme is shown to have a reduced low temperature activity, high stability and a reduced flexibility. These findings agree with the hypothesis that cold-adapted enzymes overcome the quandary imposed by low temperature environments via a global or local increase in the flexibility of their molecular edifice, with this in turn leading to a reduced stability. Analysis of the guanidine hydrochloride unfolding, as well as the thermodynamic parameters of irreversible thermal unfolding and thermal inactivation shows that the driving force for this denaturation and inactivation is a large entropy change while a low enthalpy change is implicated in the low temperature activity. A reduced number of salt-bridges are believed to be responsible for both these effects. Guanidine hydrochloride unfolding studies also indicate that both family 8 enzymes unfold via an intermediate prone to aggregation. Study holds ProTherm entries: 16152, 16153, 16154, 16155, 16156, 16157, 16158, 16159, 16160 Extra Details: i) 500 mM 3-(1-pyridinio)-1-propanesulfonate was added in the experiment.,ii) DSC scan rate 0.2 K/min family 8 glycosyl hydrolases; xylanase; thermodynamics; psychrophiles; thermophiles
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:47 p.m.
|Number of data points||18|
|Proteins||Endo-1,4-beta-xylanase ; Endo-1,4-beta-xylanase ; Cellulose 1,4-beta-cellobiosidase (reducing end) CelS|
|Assays/Quantities/Protocols||Experimental Assay: dHcal ; Experimental Assay: Tm|
|Libraries||Mutations for sequence MSTKLYGDVNDDGKVNSTDAVALKRYVLRSGISINTDNADLNEDGRVNSTDLGILKRYILKEIDTLPYKNG ; Mutations for sequence DTVITTNQTGTNNGYYYSFWTDGGGSVSMNLASGGSYGTSWTNCGNFVAGKGWANGARRTVNYSGSFNPSGNAYLTLYGWTANPLVEYYIVDNWGTYRPTGTYKGTVTSDGGTYDVYQTTRVNAPSVEGTKTFNQYWSVRQSKRTGGSITAGNHFDAWARYGMPLGSFNYYMIMATEGYQSSGSSSISVS|