Study On The Cloning, Expression And Properties Of Three Kinds Of Heparinase ?

Heparinase ??Hep ??specifically degrades heparin to oligosaccharide or unsaturated disaccharide and has been widely used in the preparation of low molecular weight heparin?LMWH?,detection and removal of heparin contaminants and structure analysis of heparin.In this study,a variety of bacteria containing the heparinase ? gene were screened to obtain highly expressed and stable heparinases.Three Heparinases from different organisms were identified,cloned and heterologously expressed in Escherichia coli expression systems.Then the recombinant heparinases were characterized,and the interaction between heparinases ? and substrate heparin was preliminarily investigated.The main research contents are as follows:1)Three heparinases from Bacteroides eggerthii VPI T5-42B-1,Bacteroides xylanisolvens SD CC 2a,and Bacteroides cellulosilyticus DSM 14838,named BeHep ?,BxHep ? and BcHep ?,and encoding 394,381 and 389 amino acids were identified,respectively.All the genes were cloned and inserted into pET-28a to construct the recombinant Heps ?,and then they were transformed into the host E.coli BL21?DE3?cells.2)Expression of the three recombinant Heps ? was optimized.The optimal conditions for expression of recombinant BeHep ? were as follows:0.1 mM of IPTG,temperature 30?,and the induction time was 6 h,the activity of the obtained crude BeHep ? was 5038 U·L^-1.After the purification of the crude enzyme with Ni-NTA,an obvious target protein band with a molecular weight of approximately 42.23 kDa was shown by SDS-PAGE.The specific activity of the purified BeHep ? was 480 U·mg^-1.The optimal expression of BxHep ? was also observed at the following conditions:0.2 mM of IPTG,temperature 25 ?,and the induction time was 7 h,the activity of the crude enzyme was 7144 U·L^-1.After the affinity adsorption of the protein molecules with the nickel column,an obvious protein band around 43.06 kDa was found by SDS-PAGE.The specific activity of the purified BxHep ? was 57.6 U·mg^-1.The optimal conditions for expression of recombinant BcHep ? were as follows:0.05 mM of IPTG,temperature 30 ?,and the induction time was 12 h,the obtained crude enzyme had a activity of 7005.4 U·L^-1.A single band around 42 kDa was found with SDS-PAGE after Ni-NTA purification,and the purified BcHep ? had a specific activity of 639.5 U·mg^-1.3)The characterization of the three expressed Heps ? was performed.BeHep ? showed the highest relative enzyme activity in a 40 mM pH 7.5 Tris-HCl buffer.The enzyme displayed the highest relative activity at 30? and remained 50% of the relative activity after 350 min incubation at this optimal temperature.The activity of BeHep ? was enhanced 69%by 1 mM Ca²⁺and inhibited completely by 1 mM Zn²⁺.The K_m and V_max values of the recombinant BeHep ? were calculated as 3.6 mg·mL^-1 and 647.93 U·mg^-1,respectively.The remaining activity of BeHep ? was 89.1%after being stored at 4? for one week.The characterization of the BxHep ? was also investigated.The optimal temperature of the BxHep ? was 35? and the optimal pH was 7.5.Ca²⁺and Mg²⁺could accelerate the enzymatic activity of the BxHep ? while that Zn²⁺and Co²⁺inhibited the enzymatic activity.The K_m and V_max of the recombinant BxHep ? were calculated as 0.79 mg·mL^-1 and 124.58 U·mg^-1,respectively.The t_1/2 value of BxHep ? was 597 min when incubated at 30?,and 158 min at 37?.Around 73%of the relative activity was left after stored one week at 4?.The enzymatic properties of BcHep ? was studied.The enzyme obtained the highest relative activity at 35? and pH 7.5.Ca²⁺,Mg²⁺,and Mn²⁺promoted the enzyme activity at different degrees,while Co²⁺and Zn²⁺decreased the enzyme activity.BcHep ? had a K_m of 0.17 mg·mL^-11 and a V_max of 740.58 U·mg^-1.The enzyme could remain the 50%relative activity after 300 and 59 min incubation at 30 and 37 ?,respectively.After 3 days of storage at 4 and-20?,BcHep ? could maintain a relative activity of 81%,and its half-life at 4?was about 10 days.4)Homologous modeling and molecular docking were conducted to analyze the interaction of Heps I and heparin.The interaction of the active site and substrate of BeHep ? suggested that the binding of heparin involved 6 highly conserved residues Lys99,Arg101,Gln241,Lys270,Asn275,and Lys292.And the length of the hydrogen bonds in BeHep ? was shorter than that in BsHep ? and BtHep ?,which indicated the higher catalytic activity of BeHep ?.Besides,BeHep ? exhibited the minimum conformational entropy than other three Heps I,which indicated that the conformation of BeHep ? was more stable than that of others.As for BxHep ?,heparin bound with the protein molecule through residues Asn25,Gln27,Arg88,Lys116,His156,Arg161,Gln228,Tyr356,Lys358,and Tyr362.The 13 hydrogen bonds formed between BxHep ? and heparin enhanced the stability of the protein-ligand,and the minimum conformational entropy of BxHep ? also proved the excellent stability of the enzyme.However,Arg88,His156,Tyr356,and Tyr362 in the active site might increase the steric hindrance thereby hinder the binding of BxHep ? and heparin,so the affinity of the enzyme to the substrate and the catalytic activity of BxHep ? were lower.This explained that the larger K_m value and the lower specific activity of BxHep ?.The binding of heparin to BcHep ? involved 9amino acid residues,Gln15,Lys74,Arg76,Lys104,Arg149,Gln208,Tyr336,Tyr342,and Lys338,and 11 hydrogen bonds were formed between the enzyme and the substrate.The absence of His residue which possessing a larger side chain in BcHep ? avoided the formation of additional steric hindrance.So the K_m value of BcHep ? was smaller and the specific activity was higher than that of BeHep ? and BxHep ?.