In this work, some important characteristics of surface layer (S-layer) proteins extracted from two new and native Lactobacillus strains, L.brevis KM3 and L.brevis KM7, were investigated. The presence of S-layer on the external surface of L.brevis KM3 was displayed by thin sectioning and negative staining. SDS-PAGE analysis were shown same dominant protein bands approximately around 48kDa for both S-layer proteins. Moreover, the S-layer reappeared when LiCl treated cells were allowed to grow again. Protein secondary structure and thermal behavior were evaluated by using circular dichroism (CD) and differential scanning calorimetry (DSC), respectively. Both S-layer proteins had high content of β-sheet and low amount of α-helix. The thermograms of lyophilized S-layer proteins of L.brevis KM3 and L.brevis KM7 showed one transition peak at 67.9°C and 59.14°C, respectively. To determine monodispersity of extracted S-layer proteins, dynamic light scattering (DLS) was used. The results indicated that the main population of S-layer molecules in two tested lactobacillus strains were composed of monomer with an expected diameter close to 10nm. Furthermore, Zeta potential measurements were showed positive potential for both S-layer proteins, as expected. Our results could be used as the basis for biotechnological applications of these two new S-layer proteins.

Background and Objectives: Due to the widespread use of lipase enzymes in various industries, finding native lipase pro- ducing microorganisms is of great value and importance. In this study, screening of lipase-producing lactobacilli from native dairy products was performed. Materials and Methods: Qualitative evaluation of lipolytic activity of lipase-producing lactobacilli was performed in differ- ent media containing olive oil. A clear zone observation around the colonies indicated the lipolytic activity. The strain with the highest enzymatic activity was identified. Determination of optimal pH and temperature of lipase activity was measured by spectrophotometry using p-nitrophenyl acetate (ρ-NPA) substrate. Partial purification of lipase enzyme was performed using 20-90% saturation ammonium sulfate. Eventually, lipase was immobilized by physical adsorption on chitosan beads. Results: Among screened lipolytic bacterial strains, one sample (5c isolate) which showed the highest enzymatic activity (5329.18 U/ml) was close to Lactobacillus fermentum. During characterization, the enzyme showed maximum activity in Tris-HCl buffer with pH 7, while remaining active over a temperature range of 5°C to 40°C. The results of the quantitative assay demonstrated that the fraction precipitated in ammonium sulfate at 20% saturation has the highest amount of lipolytic activity, with a specific activity of 22.0425 ± 3.6 U/mg. Purification folds and yields were calculated as 8.73 and 44%, respec- tively. Eventually, the enzyme was immobilized by physical adsorption on chitosan beads with a yield of 56.21%. Conclusion: The high efficiency of enzyme immobilization on chitosan beads indicates the suitability of this method for long-term storage of new lipase from native 5c isolate.

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