Abstract: Immobilized Lactobacillus helveticus proteinase was prepared using chitosan as carrier and glutaraldehyde as cross-linking agent by adsorption and cross-linking method. The effects of condensate, chitosan concentration, enzyme amount, cross-linking time, and glutaraldehyde concentration on immobilization efficiency of Lactobacillus helveticus proteinase as indicated by the activity of immobilized proteinase were explored based on single factor experiments. The optimal immobilization conditions were determined by response surface analysis to be a mixture of NaOH and methanol at a ratio of 3:1 as condensate at a dose of 4 g/100 mL, 2.89 g/100 mL chitosan concentration, 2.95 mg enzyme amount, 1 h cross-linking time and 0.4 g/100 mL glutaraldehyde concentration.

Key words: Lactobacillus helveticus proteinase, immobilization, enzyme activity