Abstract: Clostridium histolyticum collagenase H (ColH) recognizes the Y-Gly of collagen and hydrolyzes it into small peptides. The high molecular weight ColH (116 kDa) secreting strain was successfully constructed by fusing colH gene with signal peptide sequence of outer membrane protein A. In this study, we found that the secretion of ColH was affected by the position of the signal peptide at the N-terminal, and the presence of excess amino acid fragments at the N-terminal significantly reduced the secretion function of the signal peptide-guided collagenase. Orthogonal experiment and single factor experiment were used to optimize the induction conditions and medium additives to improve the secretory expression. Under the conditions of inducing temperature of 25 ℃, the cell density (OD₆₀₀) of 0.9, IPTG concentration of 0.1 mmol/L, liquid volume of 20%, magnesium ion concentration of 10 mmol/L, and 2% glycine were added at 2.5 h after induction, the highest extracellular collagenase activity was 0.68 U/mL after induction for 20 h, which was 38.1 times of that of before optimization, and the secretory expression level was remarkably increased. Glycine added into the culture medium is a common strategy to promote the secretion of recombinant protein. Experimental results demonstrated that the amount and time of glycine added after induction showed the greatest influence on the secretion of collagenase. The addition of calcium and magnesium ions in the medium can promote the growth of E.coli. The results also showed that only the addition of magnesium ion can promote the secretion of ColH.