SCIENCE@home

Characterization of maltose‐binding protein–fused heparinases with enhanced thermostability by application of rigid and flexible linkers

Xi Wu,Z. Yun,Nan Su,Lin Zhao,Hui Zhang,Mengyan Zhang,Qi Wu,Chong Zhang,Xin-Hui Xing

Published 2024 in Biotechnology and applied biochemistry

ABSTRACT

Heparinases, including heparinases I–III (HepI, HepII, and HepIII, respectively), are important tools for producing low‐molecular‐weight heparin, an improved anticoagulant. The poor thermostability of heparinases significantly hinders their industrial and laboratory applications. To improve the thermostability of heparinases, we applied a rigid linker (EAAAK)5 (R) and a flexible linker (GGGGS)5 (F) to fuse maltose‐binding protein (MBP) and HepI, HepII, and HepIII from Pedobacter heparinus, replacing the original linker from the plasmid pMAL‐c2X. Compared with their parental fusion protein, MBP‐fused HepIs, HepIIs, and HepIIIs with linkers (EAAAK)5 or (GGGGS)5 all displayed enhanced thermostability (half‐lives at 30°C: 242%–464%). MBP‐fused HepIs and HepIIs exhibited higher specific activity (127%–324%), whereas MBP‐fused HepIIIs displayed activity similar to that of their parental fusion protein. Kinetics analysis revealed that MBP‐fused HepIIs showed a significantly decreased affinity toward heparin with increased Km values (397%–480%) after the linker replacement, whereas the substrate affinity did not change significantly for MBP‐fused HepIs and HepIIIs. Furthermore, it preliminarily appeared that the depolymerization mechanism of these fusion proteins may not change after linker replacement. These findings suggest the superior enzymatic properties of MBP‐fused heparinases with suitable linker designs and their potential for the bioproduction of low‐molecular‐weight heparin.

PUBLICATION RECORD

CITATION MAP

EXTRACTION MAP

CLAIMS

  • No claims are published for this paper.

CONCEPTS

  • No concepts are published for this paper.

REFERENCES

Showing 1-45 of 45 references · Page 1 of 1

CITED BY

Showing 1-5 of 5 citing papers · Page 1 of 1