Intrinsic Properties Analysis of Multiproteases System from Marine Bacteria by Inhibitor-Subsatrate Immersion Zymography

Dan Liu; Cui-Ling Wu; Xing-Hao Yang; Ri-Bang Wu; Jiang Zhang; Jia-Heng Huang; Bin-Qiang Liao; Fei Bian; Hai-Lun He

doi:doi:10.11648/j.ajbio.20160403.11

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Intrinsic Properties Analysis of Multiproteases System from Marine Bacteria by Inhibitor-Subsatrate Immersion Zymography

Dan Liu, Cui-Ling Wu, Xing-Hao Yang, Ri-Bang Wu, Jiang Zhang, Jia-Heng Huang, Bin-Qiang Liao, Fei Bian, Hai-Lun He

Published in American Journal of BioScience (Volume 4, Issue 3)

Received: 20 June 2016 Accepted: Published: 21 June 2016

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Abstract

Based on digital image analysis techniques and inhibitor-substrate immersing zymography, intrinsic properties of each active component in the enzymatic system secreted by marine bacteria were studied. This method provides an easy way to characterize the proteases in situ, which can be further verified by Mass spectrometry. Compared to the Folin phenol method, a traditional method used to determine proteases activities, the inhibitor-substrate immersing zymography method coupled with digital image analysis used in this study could determine caseinolytic activity and measure gelatinolytic activity at the same time. The effect on activities of extracellular proteases by inhibitor (phenylmethylsulfonyl fluoride or 1, 10-Phenanthroline) can be quantified by gray value changes of the corresponding band after electrophoretic separation. Because of its high throughput, great sensitivity, and convenient operation, inhibitor-substrate immersing zymography can be used to demonstrate the natural diversity of protein hydrolases and multienzyme expression systems. Thus, it is an effective approach to study the functional proteomics of proteases secreted by marine bacteria.

Published in	American Journal of BioScience (Volume 4, Issue 3)
DOI	10.11648/j.ajbio.20160403.11
Page(s)	20-27
Creative Commons	This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.
Copyright	Copyright © The Author(s), 2024. Published by Science Publishing Group

Keywords

Gital Image Analysis, Inhibitor-Substrate Immersing Zymography, Extracellular Protease, Multienzyme, Marine Bacteria

References

[1]	Barrett, A. J., N. D. Rawlings, and J. F. Woessner (2003) The handbook of proteolytic enzymes, 2^nd ed. Academic Press.
[2]	Rao, M. B., M. S. Tanksale, M. S. Ghatge, and V. V. Deshpande (1998) Molecular and biotechnological aspects of microbial proteases. Microbiol. Mol Biol. Rev. 62: 597-635.
[3]	Rawlings, N. D., A. J. Barrett, and A. Bateman (2014) Using the MEROPS database for proteolytic enzymes and their inhibitors and substrates. Curr. Protoc. Bioinformatics. 48: 1.25.1-1.25.33.
[4]	Foukis, A., P. Y. Stergiou, L. G. Theodorou, M. Papagianni, and E. M. Papamichael (2012) Purification, kinetic characterization and properties of a novel thermo-tolerant extracellular protease from Kluyveromyces marxianus IFO 0288 with potential biotechnological interest. Bioresour. Technol. 123: 214-220.
[5]	Vary, P. S., R. Biedendieck, T. Fuerch, F. Meinhardt, M. Rohde, W. D. Deckwer, and D. Jahn (2007) Bacillus megaterium-from simple soil bacterium to industrial protein production host. Appl. Microbiol. Biotechnol. 76: 957-967.
[6]	Hunter, E. M., H. J. Mills, and J. E. Kostka (2006) Microbial community diversity associated with carbon and nitrogen cycling in permeable shelf sediments. Appl. Environ. Microbiol. 72: 5689-5701.
[7]	Ran, L. Y., H. N. Su, M. Y. Zhou, L. Wang, X. L. Chen, B. B. Xie, X. Y. Song, M. Shi, Q. L. Qin, X. Pang, B. C. Zhou, Y. Z. Zhang, and X. Y. Zhang (2014) Characterization of a novel subtilisin-like protease myroicolsin from deep sea bacterium Myroides profundi D25 and molecular insight into its collagenolytic mechanism. J. Biol. Chem. 289: 6041-6053.
[8]	Van Dijl, J. M. and M. Hecker (2013) Bacillus subtilis: from soil bacterium to super-secreting cell factory. Microb. Cell. Fact. 12: 3.
[9]	Zacaria, J., A. P. L. Delamare, S. O. P. Costa, and S. Echeverrigaray (2010) Diversity of extracellular proteases among Aeromonas determined by zymogram analysis. J. Appl. Microbiol. 109: 212-219.
[10]	Massaoud, M. K., J. Marokházi, A. Fodor, and I. Venekei (2010) Proteolytic enzyme production by strains of the insect pathogen xenorhabdus and characterization of an early-log-phase-secreted protease as a potential virulence factor. Appl. Environ. Microbiol. 76: 6901-6909.
[11]	Li, X. and H. Y. Yu (2012) Purification and characterization of novel organic-solvent-tolerant β-amylase and serine protease from a newly isolated Salimicrobium halophilum strain LY20. FEMS. Microbiol. Lett. 29: 204-211.
[12]	Liew, S. M., S. T. Tay, and S. D. Puthucheary (2013) Enzymatic and molecular characterisation of leucine aminopeptidase of Burkholderia pseudomallei. BMC. Microbiol. 13: 110.
[13]	Liu, D., X. H. Yang, J. F. Huang, R. B. Wu, C. L. Wu, H. L. He, and H. Li (2015) In situ demonstration and characteristic analysis of the protease components from marine bacteria using substrate immersing zymography. Appl. Biochem. Biotechnol. 175: 489-501.
[14]	He, H. L., J. Guo, X. L. Chen, B. B. Xie, X. Y. Zhang, Y. Yu, B. Chen, B. C. Zhou, and Y. Z. Zhang (2012) Structural and functional characterization of mature forms of metalloprotease E495 from arctic Sea-Ice bacterium pseudoalteromonas sp. SM495. PLoS. ONE. 7: e35442.
[15]	Zhou, M. Y., X. L. Chen, H. L. Zhao, H. Y. Dang, X. W. Luan, X. Y. Zhang, H. L. He, B. C. Zhou, and Y. Z. Zhang (2009) Diversity of both the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the South China sea. Microb. Ecol. 58: 582-590.
[16]	Shevchenko, A., M. Wilm, O. Vorm, and M. Mann (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68: 850-858.
[17]	Zhang, X. M., N. Liu, F. Yang, J. H. Li, L. S. Wang, G. J. Chen, and P. J. Gao (2012) In situ demonstration and quantitative analysis of the intrinsic properties of glycoside hydrolases. Electrophoresis. 33: 280-287.
[18]	Xie, B. B., Y. L. Shu, Q. L. Qin, J. C. Rong, X. Y. Zhang, X. L. Chen, M. Shi, H. L. He, B. C. Zhou, and Y. Z. Zhang (2012) Genome sequences of type strains of seven species of the marine bacterium Pseudoalteromonas. J. Bacteriol. 194: 2746-2747.
[19]	Haddar, A., R. Agrebi, A. Bougatef, N. Hmidet, A. Sellami-Kamoun, and M. Nasri (2009) Two detergent stable alkaline serine-proteases from Bacillus mojavensis A21: purification, characterization and potential application as a laundry detergent additive. Bioresour. Technol. 100: 3366-3373.
[20]	Sinha, R., A. K. Srivastava, and S. K. Khare (2014) Efficient proteolysis and application of an alkaline protease from halophilic Bacillus sp. EMB9. Prep. Biochem. Biotechnol. 44: 680-696.
[21]	Marokházi, J., K. Lengyel, S. Pekár, G. Felföldi, A. Patthy, L. Gráf, A. Fodor, and I. Venekei (2004) Comparison of proteolytic activities produced by entomopathogenic Photorhabdus bacteria: strain- and phase-dependent heterogeneity in composition and activity of four enzymes. Appl. Environ. Microbiol. 70: 7311-7320.
[22]	Rui, H., Q. Liu, Q. Wang, Y. Ma, H. Liu, C. Shi, and Y. Zhang (2009) Role of alkaline serine protease, asp, in vibrio alginolyticus virulence and regulation of its expression by luxO-luxR regulatory system. J. Microbiol. Biotechnol. 19: 431-438.
[23]	Miyoshi, I. M., Wakae, H., Tomochika, K. I., and Shinoda, S. 1997. Functional domains of a zinc metalloprotease from Vibrio vulnificus. J. Bacteriol. 179 (23): 7606–7609.
[24]	Sinsabaugh, R. L. and J. J. Shah (2010) Integrating resource utilization and temperature in metabolic scaling of riverine bacterial production. Ecology. 91: 1455-1465.
[25]	Wang, Y., A. Nakajima, K. Hosokawa, A. B. Soliev, I. Osaka, R. Arakawa, and K. Enomoto (2012) Cytotoxic prodigiosin family pigments from Pseudoalteromonas sp. 1020R isolated from the Pacific coast of Japan. Biosci. Biotechnol. Biochem. 76: 1229-1232.

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APA Style

Dan Liu, Cui-Ling Wu, Xing-Hao Yang, Ri-Bang Wu, Jiang Zhang, et al. (2016). Intrinsic Properties Analysis of Multiproteases System from Marine Bacteria by Inhibitor-Subsatrate Immersion Zymography. American Journal of BioScience, 4(3), 20-27. https://doi.org/10.11648/j.ajbio.20160403.11

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ACS Style

Dan Liu; Cui-Ling Wu; Xing-Hao Yang; Ri-Bang Wu; Jiang Zhang, et al. Intrinsic Properties Analysis of Multiproteases System from Marine Bacteria by Inhibitor-Subsatrate Immersion Zymography. Am. J. BioScience 2016, 4(3), 20-27. doi: 10.11648/j.ajbio.20160403.11

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AMA Style

Dan Liu, Cui-Ling Wu, Xing-Hao Yang, Ri-Bang Wu, Jiang Zhang, et al. Intrinsic Properties Analysis of Multiproteases System from Marine Bacteria by Inhibitor-Subsatrate Immersion Zymography. Am J BioScience. 2016;4(3):20-27. doi: 10.11648/j.ajbio.20160403.11

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@article{10.11648/j.ajbio.20160403.11,
  author = {Dan Liu and Cui-Ling Wu and Xing-Hao Yang and Ri-Bang Wu and Jiang Zhang and Jia-Heng Huang and Bin-Qiang Liao and Fei Bian and Hai-Lun He},
  title = {Intrinsic Properties Analysis of Multiproteases System from Marine Bacteria by Inhibitor-Subsatrate Immersion Zymography},
  journal = {American Journal of BioScience},
  volume = {4},
  number = {3},
  pages = {20-27},
  doi = {10.11648/j.ajbio.20160403.11},
  url = {https://doi.org/10.11648/j.ajbio.20160403.11},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ajbio.20160403.11},
  abstract = {Based on digital image analysis techniques and inhibitor-substrate immersing zymography, intrinsic properties of each active component in the enzymatic system secreted by marine bacteria were studied. This method provides an easy way to characterize the proteases in situ, which can be further verified by Mass spectrometry. Compared to the Folin phenol method, a traditional method used to determine proteases activities, the inhibitor-substrate immersing zymography method coupled with digital image analysis used in this study could determine caseinolytic activity and measure gelatinolytic activity at the same time. The effect on activities of extracellular proteases by inhibitor (phenylmethylsulfonyl fluoride or 1, 10-Phenanthroline) can be quantified by gray value changes of the corresponding band after electrophoretic separation. Because of its high throughput, great sensitivity, and convenient operation, inhibitor-substrate immersing zymography can be used to demonstrate the natural diversity of protein hydrolases and multienzyme expression systems. Thus, it is an effective approach to study the functional proteomics of proteases secreted by marine bacteria.},
 year = {2016}
}

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TY  - JOUR
T1  - Intrinsic Properties Analysis of Multiproteases System from Marine Bacteria by Inhibitor-Subsatrate Immersion Zymography
AU  - Dan Liu
AU  - Cui-Ling Wu
AU  - Xing-Hao Yang
AU  - Ri-Bang Wu
AU  - Jiang Zhang
AU  - Jia-Heng Huang
AU  - Bin-Qiang Liao
AU  - Fei Bian
AU  - Hai-Lun He
Y1  - 2016/06/21
PY  - 2016
N1  - https://doi.org/10.11648/j.ajbio.20160403.11
DO  - 10.11648/j.ajbio.20160403.11
T2  - American Journal of BioScience
JF  - American Journal of BioScience
JO  - American Journal of BioScience
SP  - 20
EP  - 27
PB  - Science Publishing Group
SN  - 2330-0167
UR  - https://doi.org/10.11648/j.ajbio.20160403.11
AB  - Based on digital image analysis techniques and inhibitor-substrate immersing zymography, intrinsic properties of each active component in the enzymatic system secreted by marine bacteria were studied. This method provides an easy way to characterize the proteases in situ, which can be further verified by Mass spectrometry. Compared to the Folin phenol method, a traditional method used to determine proteases activities, the inhibitor-substrate immersing zymography method coupled with digital image analysis used in this study could determine caseinolytic activity and measure gelatinolytic activity at the same time. The effect on activities of extracellular proteases by inhibitor (phenylmethylsulfonyl fluoride or 1, 10-Phenanthroline) can be quantified by gray value changes of the corresponding band after electrophoretic separation. Because of its high throughput, great sensitivity, and convenient operation, inhibitor-substrate immersing zymography can be used to demonstrate the natural diversity of protein hydrolases and multienzyme expression systems. Thus, it is an effective approach to study the functional proteomics of proteases secreted by marine bacteria.
VL  - 4
IS  - 3
ER  -

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Author Information

Dan Liu

School of Life Sciences, State Key Laboratory of Medical Genetics, Central South University, Changsha, China
Cui-Ling Wu

School of Life Sciences, State Key Laboratory of Medical Genetics, Central South University, Changsha, China
Xing-Hao Yang

School of Life Sciences, State Key Laboratory of Medical Genetics, Central South University, Changsha, China
Ri-Bang Wu

School of Life Sciences, State Key Laboratory of Medical Genetics, Central South University, Changsha, China
Jiang Zhang

School of Life Sciences, State Key Laboratory of Medical Genetics, Central South University, Changsha, China
Jia-Heng Huang

School of Life Sciences, State Key Laboratory of Medical Genetics, Central South University, Changsha, China
Bin-Qiang Liao

School of Life Sciences, State Key Laboratory of Medical Genetics, Central South University, Changsha, China
Fei Bian

Biotechnology Research Center, Shandong Academy of Agricultural Sciences, Jinan, China
Hai-Lun He

School of Life Sciences, State Key Laboratory of Medical Genetics, Central South University, Changsha, China

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APA Style

Dan Liu, Cui-Ling Wu, Xing-Hao Yang, Ri-Bang Wu, Jiang Zhang, et al. (2016). Intrinsic Properties Analysis of Multiproteases System from Marine Bacteria by Inhibitor-Subsatrate Immersion Zymography. American Journal of BioScience, 4(3), 20-27. https://doi.org/10.11648/j.ajbio.20160403.11

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ACS Style

Dan Liu; Cui-Ling Wu; Xing-Hao Yang; Ri-Bang Wu; Jiang Zhang, et al. Intrinsic Properties Analysis of Multiproteases System from Marine Bacteria by Inhibitor-Subsatrate Immersion Zymography. Am. J. BioScience 2016, 4(3), 20-27. doi: 10.11648/j.ajbio.20160403.11

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AMA Style

Dan Liu, Cui-Ling Wu, Xing-Hao Yang, Ri-Bang Wu, Jiang Zhang, et al. Intrinsic Properties Analysis of Multiproteases System from Marine Bacteria by Inhibitor-Subsatrate Immersion Zymography. Am J BioScience. 2016;4(3):20-27. doi: 10.11648/j.ajbio.20160403.11

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@article{10.11648/j.ajbio.20160403.11,
  author = {Dan Liu and Cui-Ling Wu and Xing-Hao Yang and Ri-Bang Wu and Jiang Zhang and Jia-Heng Huang and Bin-Qiang Liao and Fei Bian and Hai-Lun He},
  title = {Intrinsic Properties Analysis of Multiproteases System from Marine Bacteria by Inhibitor-Subsatrate Immersion Zymography},
  journal = {American Journal of BioScience},
  volume = {4},
  number = {3},
  pages = {20-27},
  doi = {10.11648/j.ajbio.20160403.11},
  url = {https://doi.org/10.11648/j.ajbio.20160403.11},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ajbio.20160403.11},
  abstract = {Based on digital image analysis techniques and inhibitor-substrate immersing zymography, intrinsic properties of each active component in the enzymatic system secreted by marine bacteria were studied. This method provides an easy way to characterize the proteases in situ, which can be further verified by Mass spectrometry. Compared to the Folin phenol method, a traditional method used to determine proteases activities, the inhibitor-substrate immersing zymography method coupled with digital image analysis used in this study could determine caseinolytic activity and measure gelatinolytic activity at the same time. The effect on activities of extracellular proteases by inhibitor (phenylmethylsulfonyl fluoride or 1, 10-Phenanthroline) can be quantified by gray value changes of the corresponding band after electrophoretic separation. Because of its high throughput, great sensitivity, and convenient operation, inhibitor-substrate immersing zymography can be used to demonstrate the natural diversity of protein hydrolases and multienzyme expression systems. Thus, it is an effective approach to study the functional proteomics of proteases secreted by marine bacteria.},
 year = {2016}
}

Copy | Download

TY  - JOUR
T1  - Intrinsic Properties Analysis of Multiproteases System from Marine Bacteria by Inhibitor-Subsatrate Immersion Zymography
AU  - Dan Liu
AU  - Cui-Ling Wu
AU  - Xing-Hao Yang
AU  - Ri-Bang Wu
AU  - Jiang Zhang
AU  - Jia-Heng Huang
AU  - Bin-Qiang Liao
AU  - Fei Bian
AU  - Hai-Lun He
Y1  - 2016/06/21
PY  - 2016
N1  - https://doi.org/10.11648/j.ajbio.20160403.11
DO  - 10.11648/j.ajbio.20160403.11
T2  - American Journal of BioScience
JF  - American Journal of BioScience
JO  - American Journal of BioScience
SP  - 20
EP  - 27
PB  - Science Publishing Group
SN  - 2330-0167
UR  - https://doi.org/10.11648/j.ajbio.20160403.11
AB  - Based on digital image analysis techniques and inhibitor-substrate immersing zymography, intrinsic properties of each active component in the enzymatic system secreted by marine bacteria were studied. This method provides an easy way to characterize the proteases in situ, which can be further verified by Mass spectrometry. Compared to the Folin phenol method, a traditional method used to determine proteases activities, the inhibitor-substrate immersing zymography method coupled with digital image analysis used in this study could determine caseinolytic activity and measure gelatinolytic activity at the same time. The effect on activities of extracellular proteases by inhibitor (phenylmethylsulfonyl fluoride or 1, 10-Phenanthroline) can be quantified by gray value changes of the corresponding band after electrophoretic separation. Because of its high throughput, great sensitivity, and convenient operation, inhibitor-substrate immersing zymography can be used to demonstrate the natural diversity of protein hydrolases and multienzyme expression systems. Thus, it is an effective approach to study the functional proteomics of proteases secreted by marine bacteria.
VL  - 4
IS  - 3
ER  -

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