Cloning and Expression of Heparinase Gene from a Novel Strain Raoultella NX-TZ-3-15

Overview

Journal Appl Biochem Biotechnol

Specialties Biochemistry
Biotechnology

Date 2022 Jun 9

PMID 35679015

Authors

Yinyin Li

Yue Lin

Yingzi Jiang

Hafiza Mahreen Mehwish

Muhammad Shahid Riaz Rajoka

Liqing Zhao

Affiliations

Soon will be listed here.

Abstract

Heparin is a class of highly sulfated, acidic, linear, and complex polysaccharide that belongs to the heparin/heparan sulfate (HS) glycosaminoglycans family. Enzymatic depolymerization of heparin by heparinases is a promising strategy for the production of ultra-low molecular weight heparins (ULMWHs) as anticoagulants. In the present study, a novel heparinase-producing strain Raoultella NX-TZ-3-15 was isolated and identified from soil samples. Herein, the heparinase gene MBP-H1 was cloned to the pBENT vector to enable expression in Escherichia coli. The optimized conditions made the activity of recombinant heparinase reach the highest level (2140 U/L). The overexpressed MBP-H1 was purified by affinity chromatography and a purity of more than 90% was obtained. The condition for biocatalysis was also optimized and three metal ions Ca, Co, and Mg were utilized to activate the reaction. In addition, the kinetics regarding the new fusion heparinase was also determined with a V value of 11.29 μmol/min and a K value of 31.2 μmol/L. In short, due to excellent K and V, the recombinant enzyme has great potential to be used in the clinic in medicine and industrial production of low or ultra-low molecule weight heparin.

Citing Articles

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Lu D, Wang L, Ning Z, Li Z, Li M, Jia Y Sci Rep. 2023; 13(1):20112.

PMID: 37978313 PMC: 10656541. DOI: 10.1038/s41598-023-47493-y.

References

Capila I, Hernaiz M, Mo Y, Mealy T, Campos B, Dedman J . Annexin V--heparin oligosaccharide complex suggests heparan sulfate--mediated assembly on cell surfaces. Structure. 2001; 9(1):57-64. DOI: 10.1016/s0969-2126(00)00549-9. View

Satish L, Santra S, Tsurkan M, Werner C, Jana M, Sahoo H . Conformational changes of GDNF-derived peptide induced by heparin, heparan sulfate, and sulfated hyaluronic acid - Analysis by circular dichroism spectroscopy and molecular dynamics simulation. Int J Biol Macromol. 2021; 182:2144-2150. DOI: 10.1016/j.ijbiomac.2021.05.194. View

Bergwik J, Kristiansson A, Larsson J, Ekstrom S, Akerstrom B, Allhorn M . Binding of the human antioxidation protein α-microglobulin (A1M) to heparin and heparan sulfate. Mapping of binding site, molecular and functional characterization, and co-localization in vivo and in vitro. Redox Biol. 2021; 41:101892. PMC: 7900767. DOI: 10.1016/j.redox.2021.101892. View

Lin J, Zheng L, Liang Q, Jiang L, Wei Z . Preparation and characterization of partial de-O-sulfation of heparin oligosaccharide library. Carbohydr Res. 2021; 499:108226. DOI: 10.1016/j.carres.2020.108226. View

Singh V, Haque S, Kumari V, El-Enshasy H, Mishra B, Somvanshi P . Isolation, Purification, and Characterization of Heparinase from Streptomyces variabilis MTCC 12266. Sci Rep. 2019; 9(1):6482. PMC: 6482181. DOI: 10.1038/s41598-019-42740-7. View

Hovingh P, Linker A . The disaccharide repeating-units of heparan sulfate. Carbohydr Res. 1974; 37(1):181-92. DOI: 10.1016/s0008-6215(00)87073-1. View

Kumar N, Bentolila A, Domb A . Structure and biological activity of heparinoid. Mini Rev Med Chem. 2005; 5(5):441-7. DOI: 10.2174/1389557053765538. View

Merilahti P, Karelehto E, Susi P . Role of Heparan Sulfate in Cellular Infection of Integrin-Binding Coxsackievirus A9 and Human Parechovirus 1 Isolates. PLoS One. 2016; 11(1):e0147168. PMC: 4718452. DOI: 10.1371/journal.pone.0147168. View

Walenga J, Lyman G . Evolution of heparin anticoagulants to ultra-low-molecular-weight heparins: a review of pharmacologic and clinical differences and applications in patients with cancer. Crit Rev Oncol Hematol. 2013; 88(1):1-18. DOI: 10.1016/j.critrevonc.2013.06.007. View

10.

Viskov C, Just M, Laux V, Mourier P, Lorenz M . Description of the chemical and pharmacological characteristics of a new hemisynthetic ultra-low-molecular-weight heparin, AVE5026. J Thromb Haemost. 2009; 7(7):1143-51. DOI: 10.1111/j.1538-7836.2009.03447.x. View

11.

Ji Y, Wang Y, Zeng W, Mei X, Du S, Yan Y . A heparin derivatives library constructed by chemical modification and enzymatic depolymerization for exploitation of non-anticoagulant functions. Carbohydr Polym. 2020; 249:116824. DOI: 10.1016/j.carbpol.2020.116824. View

12.

Fu L, Suflita M, Linhardt R . Bioengineered heparins and heparan sulfates. Adv Drug Deliv Rev. 2015; 97:237-49. PMC: 4753095. DOI: 10.1016/j.addr.2015.11.002. View

13.

Guo J, Li K, Ning C, Liu X . Improved cellular bioactivity by heparin immobilization on polycarbonate film via an aminolysis modification for potential tendon repair. Int J Biol Macromol. 2019; 142:835-845. DOI: 10.1016/j.ijbiomac.2019.09.136. View

14.

Tripathi C, Banga J, Mishra V . Microbial heparin/heparan sulphate lyases: potential and applications. Appl Microbiol Biotechnol. 2012; 94(2):307-21. DOI: 10.1007/s00253-012-3967-6. View

15.

Sepuru K, Rajarathnam K . Structural basis of chemokine interactions with heparan sulfate, chondroitin sulfate, and dermatan sulfate. J Biol Chem. 2019; 294(43):15650-15661. PMC: 6816105. DOI: 10.1074/jbc.RA119.009879. View

16.

Galliher P, Cooney C, Langer R, Linhardt R . Heparinase production by Flavobacterium heparinum. Appl Environ Microbiol. 1981; 41(2):360-5. PMC: 243699. DOI: 10.1128/aem.41.2.360-365.1981. View

17.

Yang V, Linhardt R, Bernstein H, Cooney C, Langer R . Purification and characterization of heparinase from Flavobacterium heparinum. J Biol Chem. 1985; 260(3):1849-57. View

18.

Ahn M, Shin K, Kim D, Jung E, Toida T, Linhardt R . Characterization of a Bacteroides species from human intestine that degrades glycosaminoglycans. Can J Microbiol. 1998; 44(5):423-9. DOI: 10.1139/cjm-44-5-423. View

19.

Kim B, Kim W, Kim Y, Linhardt R, Kim D . Purification and characterization of a novel heparinase from Bacteroides stercoris HJ-15. J Biochem. 2000; 128(2):323-8. DOI: 10.1093/oxfordjournals.jbchem.a022756. View

20.

Yoshida E, Sakai K, Tokuyama S, Miyazono H, Maruyama H, Morikawa K . Purification and characterization of heparinase that degrades both heparin and heparan sulfate from Bacillus circulans. Biosci Biotechnol Biochem. 2002; 66(5):1181-4. DOI: 10.1271/bbb.66.1181. View