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Collagenase from Lysinibacillus sphaericus: Production, Optimization and Application in Adipose-Derived Stem Cell Isolation.

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Τίτλος: Collagenase from Lysinibacillus sphaericus: Production, Optimization and Application in Adipose-Derived Stem Cell Isolation.
Συγγραφείς: Wang ZZ; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, 214122, PR China., Xue D; Wuxi Children's Hospital, Wuxi, 214000, PR China. 337855521@qq.com., Qian JY; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, 214122, PR China. jackieqian@163.com., Wang K; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, 214122, PR China., Xu LF; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, 214122, PR China., Ma XD; Cytori Therapeutics LLC, Shanghai, 201802, PR China., Xie N; Cytori Therapeutics LLC, Shanghai, 201802, PR China., Su C; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, 214122, PR China., Gong JS; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, 214122, PR China., Xu ZH; College of Biomass Science and Engineering, Sichuan University, Chengdu, 610065, PR China., Shi JS; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, 214122, PR China.
Πηγή: Applied biochemistry and biotechnology [Appl Biochem Biotechnol] 2026 Oct; Vol. 198 (10), pp. 7448-7463. Date of Electronic Publication: 2026 Jun 13.
Τύπος έκδοσης: Journal Article
Γλώσσα: English
Στοιχεία περιοδικού: Publisher: Humana Press Country of Publication: United States NLM ID: 8208561 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1559-0291 (Electronic) Linking ISSN: 02732289 NLM ISO Abbreviation: Appl Biochem Biotechnol Subsets: MEDLINE
Imprint Name(s): Original Publication: Clifton, N.J. : Humana Press, c1981-
Ιατρικοί όροι (MeSH): Bacillaceae*/enzymology , Bacillaceae*/genetics , Stem Cells*/cytology , Collagenases*/biosynthesis , Collagenases*/isolation & purification , Collagenases*/chemistry , Collagenases*/genetics , Cell Separation*/methods , Adipose Tissue*/cytology, Bacillus subtilis/genetics ; Hydrogen-Ion Concentration
Περίληψη: Collagenase, predominantly found in microorganisms, has significant potential for tissue separation and primary cell extraction. In this study, a collagenase Col51 derived from Lysinibacillus sphaericus JN-51 was isolated from sludge samples collected from aquaculture markets and slaughterhouses. Col51 was expressed in Bacillus subtilis (SCK6) and purified using a Ni-NTA column. Enzymatic characterization revealed that the enzyme exhibits optimal activity at 37 °C and pH 7.5. The presence of 10 mM Mg²⁺ and Ca2⁺ enhances Col51 activity, whereas Zn²⁺ and Al³⁺ exert inhibitory effects. Through signal peptide optimization, Col51 expression increased from 53.5 U/mL to 164.8 U/mL in shake-flask experiments. Batch fermentation in a 7-L fermenter further enhanced enzyme activity, reaching a peak of 246.5 U/mL. In adipose stem cell isolation applications, Col51 demonstrated comparable separation efficiency to commercial collagenase I, with cell viability exceeding 90%, and cells exhibiting good morphology and stable growth characteristics. This study successfully identifies and optimizes Col51 collagenase from the non-pathogenic strain Lysinibacillus sphaericus, providing a novel tool enzyme for adipose stem cell isolation.
(© 2026. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)
Competing Interests: Declarations. Ethics Approval: This study was conducted in accordance with the Guidelines for the Euthanasia of Animals (2020 Edition, American Veterinary Medical Association, AVMA) and the ethical standards approved by the Laboratory Animal Center of Jiangnan University, reference number JN.No20250315b1040425[111]. This study utilized SPF-grade male Balb/c mice that were 8 weeks of age and weighed 18–25 g. All procedures were performed to minimize animal distress and suffering. Competing interests: The authors declare that they have no competing interests.
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Kubyshkin, V. (2019). Stabilization of the Triple Helix in Collagen Mimicking Peptides. Organic & Biomolecular Chemistry, 17(35), 8031–8047. (PMID: 10.1039/C9OB01646E)
Vincenti, M. P., Coon, C. I., Mengshol, J. A., Yocum, S., Mitchell, P., & Brinckerhoff, C. E. (1998). Cloning of the Gene for Interstitial Collagenase-3 (Matrix Metalloproteinase-13) from Rabbit Synovial Fibroblasts: Differential Expression with Collagenase-1 (Matrix Metalloproteinase-1). Biochemical Journal, 331, 341–346. (PMID: 10.1042/bj331034195124981219357)
Matsushita, O., Yoshihara, K., Katayama, S. I., Minami, J., & Okabe, A. (1994). Purification and Characterization of Clostridium Perfringens 120-Kilodalton Collagenase and Nucleotide Sequence of the Corresponding Gene. Journal of Bacteriology, 176(1), 149–156. (PMID: 10.1128/jb.176.1.149-156.19948282691205026)
Quintero Sierra, L. A., Biswas, R., Busato, A., Conti, A., Ossanna, R., Conti, G., Zingaretti, N., & Caputo, M. (2023). Cuppari. In Vitro Study of a Novel Vibrio Alginolyticus-Based Collagenase for Future Medical Application. Cells, 12(16), 2025. (PMID: 10.3390/cells121620253762683410453626)
Bacakova, L., Zarubova, J., Travnickova, M., Musilkova, J., Pajorova, J., Slepicka, P., Kasalkova, N. S., Svorcik, V., Kolska, Z., Motarjemi, H., et al. (2018). Stem Cells: Their Source, Potency and Use in Regenerative Therapies with Focus on Adipose-Derived Stem Cells - a Review. Biotechnology Advances, 36(4), 1111–1126. (PMID: 10.1016/j.biotechadv.2018.03.01129563048)
Ishii, K., Suzuki, N., Mabuchi, Y., Sekiya, I., & Akazawa, C. (2017). Technical Advantage of Recombinant Collagenase for Isolation of Muscle Stem Cells. Regenerative Therapy, 7, 1–7. (PMID: 10.1016/j.reth.2017.06.001302718466134918)
Wang, Z-Z., Wang, K., Xu, L-F., Su, C., Gong, J-S., Shi, J-S., Ma, X-D., Xie, N., & Qian, J-Y. (2024). Unlocking the Potential of Collagenases: Structures, Functions, and Emerging Therapeutic Horizons. BioDesign Research, 6, 0050. (PMID: 10.34133/bdr.00503938162311458858)
Zhang, X-Z., Cui, Z-L., Hong, Q., & Li, S-P. (2005). High-Level Expression and Secretion of Methyl Parathion Hydrolase in Bacillus Subtilis Wb800. Applied and Environmental Microbiology, 71(7), 4101–4103. (PMID: 10.1128/AEM.71.7.4101-4103.2005160008261169017)
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Gautam, M., & Wamik Azmi, G. J. N. (2019). Factorial Design Based Bench-Scale Production of Collagenase by Pseudomonas sp. Found in Protein Waste of Himalayan Region. Journal of Advances in Biology & Biotechnology, 21(2), 1–14. (PMID: 10.9734/jabb/2019/v21i230085)
Medina, P., & Baresi, L. (2007). Rapid Identification of Gelatin and Casein Hydrolysis Using TCA. Journal of Microbiological Methods, 69(2), 391–393. (PMID: 10.1016/j.mimet.2007.01.00517289191)
Suphatharaprateep, W., Cheirsilp, B., & Jongjareonrak, A. (2011). Production and Properties of Two Collagenases from Bacteria and Their Application for Collagen Extraction. New Biotechnology, 28(6), 649–655. (PMID: 10.1016/j.nbt.2011.04.00321549225)
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Grant Information: 2023YFA0914500 National Key Research and Development Program of China; 32301283 National Natural Science Foundation of China
Contributed Indexing: Keywords: Bacillus subtilis; Lysinibacillus sphaericus; Adipose-derived stem cells; Collagenase
Substance Nomenclature: EC 3.4.24.- (Collagenases)
SCR Organism: Lysinibacillus sphaericus
Entry Date(s): Date Created: 20260613 Date Completed: 20260923 Latest Revision: 20260923
Update Code: 20260924
DOI: 10.1007/s12010-026-05761-3
PMID: 42287536
Βάση Δεδομένων: MEDLINE
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  Data: Collagenase from Lysinibacillus sphaericus: Production, Optimization and Application in Adipose-Derived Stem Cell Isolation.
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  Data: <searchLink fieldCode="AU" term="%22Wang+ZZ%22">Wang ZZ</searchLink>; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, 214122, PR China.<br /><searchLink fieldCode="AU" term="%22Xue+D%22">Xue D</searchLink>; Wuxi Children's Hospital, Wuxi, 214000, PR China. 337855521@qq.com.<br /><searchLink fieldCode="AU" term="%22Qian+JY%22">Qian JY</searchLink>; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, 214122, PR China. jackieqian@163.com.<br /><searchLink fieldCode="AU" term="%22Wang+K%22">Wang K</searchLink>; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, 214122, PR China.<br /><searchLink fieldCode="AU" term="%22Xu+LF%22">Xu LF</searchLink>; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, 214122, PR China.<br /><searchLink fieldCode="AU" term="%22Ma+XD%22">Ma XD</searchLink>; Cytori Therapeutics LLC, Shanghai, 201802, PR China.<br /><searchLink fieldCode="AU" term="%22Xie+N%22">Xie N</searchLink>; Cytori Therapeutics LLC, Shanghai, 201802, PR China.<br /><searchLink fieldCode="AU" term="%22Su+C%22">Su C</searchLink>; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, 214122, PR China.<br /><searchLink fieldCode="AU" term="%22Gong+JS%22">Gong JS</searchLink>; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, 214122, PR China.<br /><searchLink fieldCode="AU" term="%22Xu+ZH%22">Xu ZH</searchLink>; College of Biomass Science and Engineering, Sichuan University, Chengdu, 610065, PR China.<br /><searchLink fieldCode="AU" term="%22Shi+JS%22">Shi JS</searchLink>; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, 214122, PR China.
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  Data: <i>Publisher: </i><searchLink fieldCode="PB" term="%22Humana+Press%22">Humana Press </searchLink><i>Country of Publication: </i>United States <i>NLM ID: </i>8208561 <i>Publication Model: </i>Print-Electronic <i>Cited Medium: </i>Internet <i>ISSN: </i>1559-0291 (Electronic) <i>Linking ISSN: </i><searchLink fieldCode="IS" term="%2202732289%22">02732289 </searchLink><i>NLM ISO Abbreviation: </i>Appl Biochem Biotechnol <i>Subsets: </i>MEDLINE
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  Data: <i>Original Publication</i>: Clifton, N.J. : Humana Press, c1981-
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  Data: <searchLink fieldCode="MM" term="%22Bacillaceae%22">Bacillaceae*</searchLink>/<searchLink fieldCode="MM" term="%22Bacillaceae+enzymology%22">enzymology</searchLink> <br /><searchLink fieldCode="MM" term="%22Bacillaceae%22">Bacillaceae*</searchLink>/<searchLink fieldCode="MM" term="%22Bacillaceae+genetics%22">genetics</searchLink> <br /><searchLink fieldCode="MM" term="%22Stem+Cells%22">Stem Cells*</searchLink>/<searchLink fieldCode="MM" term="%22Stem+Cells+cytology%22">cytology</searchLink> <br /><searchLink fieldCode="MM" term="%22Collagenases%22">Collagenases*</searchLink>/<searchLink fieldCode="MM" term="%22Collagenases+biosynthesis%22">biosynthesis</searchLink> <br /><searchLink fieldCode="MM" term="%22Collagenases%22">Collagenases*</searchLink>/<searchLink fieldCode="MM" term="%22Collagenases+isolation+%26+purification%22">isolation & purification</searchLink> <br /><searchLink fieldCode="MM" term="%22Collagenases%22">Collagenases*</searchLink>/<searchLink fieldCode="MM" term="%22Collagenases+chemistry%22">chemistry</searchLink> <br /><searchLink fieldCode="MM" term="%22Collagenases%22">Collagenases*</searchLink>/<searchLink fieldCode="MM" term="%22Collagenases+genetics%22">genetics</searchLink> <br /><searchLink fieldCode="MM" term="%22Cell+Separation%22">Cell Separation*</searchLink>/<searchLink fieldCode="MM" term="%22Cell+Separation+methods%22">methods</searchLink> <br /><searchLink fieldCode="MM" term="%22Adipose+Tissue%22">Adipose Tissue*</searchLink>/<searchLink fieldCode="MM" term="%22Adipose+Tissue+cytology%22">cytology</searchLink><br /><searchLink fieldCode="MH" term="%22Bacillus+subtilis%22">Bacillus subtilis</searchLink>/<searchLink fieldCode="MH" term="%22Bacillus+subtilis+genetics%22">genetics</searchLink> ; <searchLink fieldCode="MH" term="%22Hydrogen-Ion+Concentration%22">Hydrogen-Ion Concentration</searchLink>
– Name: Abstract
  Label: Abstract
  Group: Ab
  Data: Collagenase, predominantly found in microorganisms, has significant potential for tissue separation and primary cell extraction. In this study, a collagenase Col51 derived from Lysinibacillus sphaericus JN-51 was isolated from sludge samples collected from aquaculture markets and slaughterhouses. Col51 was expressed in Bacillus subtilis (SCK6) and purified using a Ni-NTA column. Enzymatic characterization revealed that the enzyme exhibits optimal activity at 37 °C and pH 7.5. The presence of 10 mM Mg²⁺ and Ca<superscript>2</superscript>⁺ enhances Col51 activity, whereas Zn²⁺ and Al³⁺ exert inhibitory effects. Through signal peptide optimization, Col51 expression increased from 53.5 U/mL to 164.8 U/mL in shake-flask experiments. Batch fermentation in a 7-L fermenter further enhanced enzyme activity, reaching a peak of 246.5 U/mL. In adipose stem cell isolation applications, Col51 demonstrated comparable separation efficiency to commercial collagenase I, with cell viability exceeding 90%, and cells exhibiting good morphology and stable growth characteristics. This study successfully identifies and optimizes Col51 collagenase from the non-pathogenic strain Lysinibacillus sphaericus, providing a novel tool enzyme for adipose stem cell isolation.<br /> (© 2026. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)
– Name: Abstract
  Label: Competing Interests
  Group: Ab
  Data: Declarations. Ethics Approval: This study was conducted in accordance with the Guidelines for the Euthanasia of Animals (2020 Edition, American Veterinary Medical Association, AVMA) and the ethical standards approved by the Laboratory Animal Center of Jiangnan University, reference number JN.No20250315b1040425[111]. This study utilized SPF-grade male Balb/c mice that were 8 weeks of age and weighed 18–25 g. All procedures were performed to minimize animal distress and suffering. Competing interests: The authors declare that they have no competing interests.
– Name: Ref
  Label: References
  Group: RefInfo
  Data: Birk, D. E., & Bruckner, P. (2005). Collagen suprastructures. Collagen: Primer in structure, processing and assembly. In: J. Brinckmann, H. Notbohm, & P.K. Muller (Eds), 247: 185–205.<br />Kubyshkin, V. (2019). Stabilization of the Triple Helix in Collagen Mimicking Peptides. Organic & Biomolecular Chemistry, 17(35), 8031–8047. (PMID: <searchLink fieldCode="PM" term="%2210%2E1039%2FC9OB01646E%22">10.1039/C9OB01646E)</searchLink><br />Vincenti, M. P., Coon, C. I., Mengshol, J. A., Yocum, S., Mitchell, P., & Brinckerhoff, C. E. (1998). Cloning of the Gene for Interstitial Collagenase-3 (Matrix Metalloproteinase-13) from Rabbit Synovial Fibroblasts: Differential Expression with Collagenase-1 (Matrix Metalloproteinase-1). Biochemical Journal, 331, 341–346. (PMID: <searchLink fieldCode="PM" term="%2210%2E1042%2Fbj331034195124981219357%22">10.1042/bj331034195124981219357)</searchLink><br />Matsushita, O., Yoshihara, K., Katayama, S. I., Minami, J., & Okabe, A. (1994). Purification and Characterization of Clostridium Perfringens 120-Kilodalton Collagenase and Nucleotide Sequence of the Corresponding Gene. Journal of Bacteriology, 176(1), 149–156. (PMID: <searchLink fieldCode="PM" term="%2210%2E1128%2Fjb%2E176%2E1%2E149-156%2E19948282691205026%22">10.1128/jb.176.1.149-156.19948282691205026)</searchLink><br />Quintero Sierra, L. A., Biswas, R., Busato, A., Conti, A., Ossanna, R., Conti, G., Zingaretti, N., & Caputo, M. (2023). Cuppari. In Vitro Study of a Novel Vibrio Alginolyticus-Based Collagenase for Future Medical Application. Cells, 12(16), 2025. (PMID: <searchLink fieldCode="PM" term="%2210%2E3390%2Fcells121620253762683410453626%22">10.3390/cells121620253762683410453626)</searchLink><br />Bacakova, L., Zarubova, J., Travnickova, M., Musilkova, J., Pajorova, J., Slepicka, P., Kasalkova, N. S., Svorcik, V., Kolska, Z., Motarjemi, H., et al. (2018). Stem Cells: Their Source, Potency and Use in Regenerative Therapies with Focus on Adipose-Derived Stem Cells - a Review. Biotechnology Advances, 36(4), 1111–1126. (PMID: <searchLink fieldCode="PM" term="%2210%2E1016%2Fj%2Ebiotechadv%2E2018%2E03%2E01129563048%22">10.1016/j.biotechadv.2018.03.01129563048)</searchLink><br />Ishii, K., Suzuki, N., Mabuchi, Y., Sekiya, I., & Akazawa, C. (2017). Technical Advantage of Recombinant Collagenase for Isolation of Muscle Stem Cells. Regenerative Therapy, 7, 1–7. (PMID: <searchLink fieldCode="PM" term="%2210%2E1016%2Fj%2Ereth%2E2017%2E06%2E001302718466134918%22">10.1016/j.reth.2017.06.001302718466134918)</searchLink><br />Wang, Z-Z., Wang, K., Xu, L-F., Su, C., Gong, J-S., Shi, J-S., Ma, X-D., Xie, N., & Qian, J-Y. (2024). Unlocking the Potential of Collagenases: Structures, Functions, and Emerging Therapeutic Horizons. BioDesign Research, 6, 0050. 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  Group: Grant
  Data: 2023YFA0914500 National Key Research and Development Program of China; 32301283 National Natural Science Foundation of China
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  Data: <i>Keywords: </i>Bacillus subtilis; Lysinibacillus sphaericus; Adipose-derived stem cells; Collagenase
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  Label: Substance Nomenclature
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  Data: EC 3.4.24.- (Collagenases)
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  Group: Su
  Data: Lysinibacillus sphaericus
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  Data: <i>Date Created: </i>20260613 <i>Date Completed: </i>20260923 <i>Latest Revision: </i>20260923
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  Data: 10.1007/s12010-026-05761-3
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        Value: 10.1007/s12010-026-05761-3
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      – Code: eng
        Text: English
    PhysicalDescription:
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        StartPage: 7448
    Subjects:
      – SubjectFull: Bacillus subtilis genetics
        Type: general
      – SubjectFull: Hydrogen-Ion Concentration
        Type: general
      – SubjectFull: Bacillaceae enzymology
        Type: general
      – SubjectFull: Bacillaceae genetics
        Type: general
      – SubjectFull: Stem Cells cytology
        Type: general
      – SubjectFull: Collagenases biosynthesis
        Type: general
      – SubjectFull: Collagenases isolation & purification
        Type: general
      – SubjectFull: Collagenases chemistry
        Type: general
      – SubjectFull: Collagenases genetics
        Type: general
      – SubjectFull: Cell Separation methods
        Type: general
      – SubjectFull: Adipose Tissue cytology
        Type: general
    Titles:
      – TitleFull: Collagenase from Lysinibacillus sphaericus: Production, Optimization and Application in Adipose-Derived Stem Cell Isolation.
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            – D: 01
              M: 10
              Text: 2026 Oct
              Type: published
              Y: 2026
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