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Purification and characterization of fibrinolytic enzyme from a bacterium isolated from soil

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Abstract

A novel extracellular enzyme with strong fibrinolytic activity, produced by Bacillus tequilensis, which was isolated from the soil of Zhuhai City (China) was purified and characterized. The enzyme was secreted by cultured B. tequilensis in solid state and purified at a high efficiency using the combination of salting out, ion exchange chromatography, and size exclusion chromatography. The enzyme was estimated to have a molecular weight of approximately 27 kDa, pI of 8.9 ± 0.1, to stable at pH 5.0–12.0 and up to 50 °C; the optimum pH and temperature are 10.5 and 45 °C (2373.59 ± 54.81 U/mg), respectively. The fibrinolytic activity was enhanced by K+, Na+, Mg2+, Mn2+, Ca2+, and Ba2+ and inhibited by Cu2+, Zn2+, and Fe3+. Moreover, the activity was slightly enhanced by PMSF and EDTA at low concentrations and inhibited by β-mercaptoethanol. The N-terminal amino acid sequence is AQSVPYGISQI. The enzyme has a higher enzymatic activity than most other fibrinolytic enzymes. The high thermal stability indicated that it is easy to preserve and could be activated under high-temperature conditions.

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Abbreviations

CGMCC:

General Microbiological Culture Collection Center

PMSF:

Phenyl methyl sulfonyl fluoride

EDTA:

Ethylene diamine tetra acetic acid

IAA:

Iodoacetic acid

CVD:

Cardiovascular disease

SDS:

Sodium dodecyl sulfate

APSAC:

Anisoylated plasminogen streptokinase activator complex

pI:

Isoelectric point

DI:

Deionized water

BCA:

Bicinchoninic acid

BSA:

Bovine serum albumin

QFF:

Q sepharose fast flow ion exchange chromatography

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

PVDF:

Polyvinylidene difluoride membrane

PTH:

Phenylthiohydantoins

MW:

Molecular weight

kDa:

Kilodaltons

IEF:

Iso-electric focusing

t-PA:

Tissue plasminogen activator

PMF:

Peptide mass fingerprint

(NH4)2SO4 ppt:

Ammonium sulfate precipitation

MgCl2 :

Magnesium chloride

Na2CO3 :

Sodium carbonate

NaHCO3 :

Sodium bicarbonate

NaCl:

Sodium chloride

NaOH:

Sodium hydroxide

KCl:

Potassium chloride

MgSO4 :

Magnesium sulfate

MnCl2 :

Manganese chloride

CaCl2 :

Calcium chloride

CuCl2 :

Copper chloride

ZnCl2 :

Zinc chloride

Ba(SO4)2 :

Barium sulfate

FeCl3 :

Iron chloride

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Acknowledgements

The authors thank a research Grant no. 201319 by Beijing Normal University-Hong Kong Baptist University United International College for providing financial support for this project.

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Authors and Affiliations

  1. Food Science and Technology Program, Beijing Normal University-Hong Kong Baptist University United International College, 28 Jinfeng Road, Tangjiawan, Zhuhai, 519085, Guangdong, China

    Xiong Xin, Ranga Rao Ambati & Bo Lei

  2. State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, 224 Waterloo Road, Kowloon Tong, Hong Kong Special Administrative Region, China

    Xiong Xin & Zongwei Cai

  3. Zhuhai Key Laboratory of Agriculture Product Quality and Food Safety, 28, Jinfeng Road, Tangjiawan, Zhuhai, 519085, Guangdong, China

    Ranga Rao Ambati, Zongwei Cai & Bo Lei

  4. Department of Biotechnology, Vignan’s Foundation for Science, Technology and Research University (Deemed to be University), Guntur, Andhra Pradesh, 5222, India

    Ranga Rao Ambati

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  1. Xiong Xin
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  2. Ranga Rao Ambati
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  3. Zongwei Cai
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Correspondence to Bo Lei.

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Xin, X., Ambati, R.R., Cai, Z. et al. Purification and characterization of fibrinolytic enzyme from a bacterium isolated from soil. 3 Biotech 8, 90 (2018). https://doi.org/10.1007/s13205-018-1115-4

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