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

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Collagenases are crucial for tissue separation and cell extraction.
• Microbial collagenases offer potential for biotechnological applications.
• Lysinibacillus sphaericus is a source of novel enzymes.

Purpose of the Study:

• To isolate and characterize a novel collagenase (Col51) from Lysinibacillus sphaericus.
• To optimize Col51 expression and enzyme activity.
• To evaluate Col51's efficacy in adipose stem cell isolation.

Main Methods:

• Isolation of collagenase Col51 from Lysinibacillus sphaericus.
• Expression in Bacillus subtilis and purification using Ni-NTA chromatography.
• Enzymatic characterization including optimal temperature, pH, and ion effects.
• Signal peptide optimization and batch fermentation for enhanced expression.
• Application in adipose stem cell isolation and assessment of cell viability and morphology.

Main Results:

• Col51 exhibited optimal activity at 37°C and pH 7.5.
• Mg²⁺ and Ca²⁺ enhanced Col51 activity; Zn²⁺ and Al³⁺ inhibited it.
• Signal peptide optimization increased expression from 53.5 U/mL to 164.8 U/mL.
• Batch fermentation achieved a peak enzyme activity of 246.5 U/mL.
• Col51 demonstrated comparable adipose stem cell separation efficiency to commercial collagenase I with >90% cell viability.

Conclusions:

• Col51 collagenase from Lysinibacillus sphaericus is a novel, effective enzyme.
• Optimization strategies significantly enhanced Col51 production.
• Col51 is a promising tool for adipose stem cell isolation with high cell viability and stable growth characteristics.