Structure-based engineering of heparinase I with improved specific activity for degrading heparin

Abstract

Background Heparinase I from Pedobacter heparinus (Ph-HepI), which specifically cleaves heparin and heparan sulfate, is one of the most extensively studied glycosaminoglycan lyases. Enzymatic degradation of heparin by heparin lyases not only largely facilitates heparin structural analysis but also showed great potential to produce low-molecular-weight heparin (LMWH) in an environmentally friendly way. However, industrial applications of Ph-HepI have been limited by their poor yield and enzyme activity. In this work, we improve the specific enzyme activity of Ph-HepI based on homology modeling, multiple sequence alignment, molecular docking and site-directed mutagenesis. Results Three mutations (S169D, A259D, S169D/A259D) exhibited a 50.18, 40.43, and 122.05% increase in the specific enzyme activity and a 91.67, 108.33, and 75% increase in the yield, respectively. The catalytic efficiencies ( k cat / K m ) of the mutanted enzymes S169D, A259D, and S169D/A259D were higher than those of the wild-type enzyme by 275, 164, and 406%, respectively. Mass spectrometry and activity detection showed the enzyme degradation products were in line with the standards of the European Pharmacopoeia. Protein structure analysis showed that hydrogen bonds and ionic bonds were important factors for improving specific enzyme activity and yield. Conclusions We found that the mutant S169D/A259D had more industrial application value than the wild-type enzyme due to molecular modifications. Our results provide a new strategy to increase the catalytic efficiency of other heparinases. Electronic supplementary material The online version of this article (10.1186/s12896-019-0553-3) contains supplementary material, which is available to authorized users.

A highly active heparinase I from Bacteroides cellulosilyticus: Cloning, high level expression, and molecular characterization

“…The superimposition of BcHep-I and BtHep-I ( Fig 5C) showed that BcHep-I has a similar steric structure with BtHep-I and both enzymes bind calcium ion with Trp, Asn, Asp, and Glu residues. The substrate-binding involved many direct and water-mediated hydrogen bonds between the sulfate groups and the basic side chains within the active site [36,37]. The critical residues in active pockets of the Hep-I were shown in Fig 5D. These residues including Gln15, Lys74, Arg76, Lys104, Arg149, Gln208, Tyr336, Tyr342, and Lys338 connected the heparin by forming hydrogen bonds.…”

Rational protein engineering of thermostable heparinase I from Bacteroides thetaiotaomicron for highly efficient heparin degradation

“…Enzyme activity at the optimum pH was defined as 100%, and the relative enzyme activity of HepI was determined. Enzyme activity was measured at 25,30,35,40,45,50, and 55℃ to determine the optimum reaction temperature. Enzyme activity at the optimum temperature was defined as 100%, and the relative enzyme activity of HepI was determined.…”

“…The samples were successively mixed with the R1, R2, and R3 (buffers in the kit). The reaction was terminated by adding a citric acid solution; absorbance was measured at 405 nm [35].…”

Not Just Anticoagulation—New and Old Applications of Heparin

“…A sulfated glycosaminoglycan, heparin was named after its initial isolation from liver tissue a century ago [ 1 , 2 ]. It exists in the lung, vascular wall, intestinal mucosa, and so on.…”