Isolation,Identification And Crystal Structure Study Of SPB From Serratia Sp.FS14

Previous study showed that there may be a protese with unique V-shaped thermostable property from the culture supernatant of Serratia sp.FS14.To identify this unique V-shaped thermostable protease,the serralysin gene mutant FS14?serralysin was first constructed by homologus recombinatation.The result showed that there was a unique V-shaped thermostable protease still existed in the culture supernatant of mutant FS14?serralysin.A 50 kDa protein with unique V-shaped thermostable protease activity was then purified from the culture supernatant of FS14?serralysin.Protease charaterization results showed that it is a alkaline metalloprotease with optimum pH at 8.0 and optimum reaction temperature at 37 °C.It is stable at 4 to 50 °C,has some but increasing protease activities after pretreated at 70 to 90 °C for 10 min respectively;however,it almost loses protease activity after pretreated at 60 °C.Protein identification showed that this 50 kDa protease is a serralysin family protease,it was then named as serralysin-like protease B(SPB).To further confirm the V-shaped thermostable protrease is the SPB,the double gene deletion mutant FS14?serralysin?SPB was constructed.The protease assay showed that no protease activity could be found in the culture supernatant of the FS14?serralysinASPB.The gene of SPB with its own promotor and encoding gene of his-tag at the C-terminal was later amplified and expressed in FS14AserralysinASPB.The purified recombinant SPB showed similar characteristics with the SPB purified from FS14?serralysin.Furthermore,a mutant E176A of SPB was also constructed.The protease activity assay showed that the mutant E176A lost its protease activity.This implies that the E176 is essential for the protease activity of SPB.All results above confirm that the SPB is the protease with unique V-shaped thermostable property.To dissect the mechanism underlying the unusual V-shaped thermostable phenomenon of the SPB,SPB proteins pretreated with and without 100 mM EDTA were treated at 4 to 90 °C respectively,the residual proteins analysis implied that SPB protein could digest itself above 50 °C and the optimum temperature for the self-digestion is around 60 °C,the self-digestion is the reason for the unusual thermostable phenomenon.To dissect the structural basis for the unique thermostable property of SPB,SPB was purified from the culture supernatant of FS14,and then used for crystallization screening by vapor diffusion method at 4 and 22 °C,crystals were obtained in the condition of 0.1 M Tris pH8.0,28%PEG4000 at 4 °C.A complete 2.3 A diffraction dataset was collected and the crystal structure of SPB was also solved by molecular replacement.The structure comparison showed that the overall structure of SPB shares similarity with the structure of the serralysin from FS14,but there are still some differences among them.These differences may be the reason for the unique thermostable property of SPB.