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

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.