Cloning, Expression, and Purification of a GDSL-like Lipase/Acylhydrolase from a Native Lipase-Producing Bacterium, Lactobacillus fermentum

Background: Lipase enzymes are of great importance in various industries. Currently, extensive efforts have been focused on exploring new lipase producer microorganism as well as genetic and protein engineering of available lipases to improve their functional features. Methods: For screening lipase-producing lactobacilli, isolated strains were inoculated onto tributyrin agar plates. Molecular identification of lipase-producing Lactobacilli was performed by sequencing the 16Sr DNA gene, and a phylogenetic tree was constructed. The LAF_RS05195 gene, encoding lipase protein in L. fermentum isolates, was identified using specific primers, amplified by PCR (918 bp) and cloned into the pET28a (+) vector. The recombinant proteins were expressed 2, 4, 6, and 12 hours after induction with IPTG and assessed using the SDS polyacrylamide gel electrophoresis (SDS-PAGE). Enzymatic activity of the purified recombinant protein was measured at 410 nm in the presence of ρ-NPA and ρ-NPP. Results: Among five identified native lipase-producing isolates, one isolate showed 98% similarity with Enterococcus species. The other four isolates indicated 98% similarity to L. fermentum. After purification steps with Ni-NTA column, a single protein band of about 34 kDa was detected on SDS-PAGE gel. The enzymatic activity of purified recombinant protein alongside ρ-NPA and ρ-NPP was measured to be 0.6 U/ml and 0.2 U/ml, respectively. Conclusion: In the present research, a novel lipase/esterase from L. fermentum was cloned and expressed. The novel lipase/esterase has the merit to be further studied due to its substrate specificity.

Microencapsulation of Omeprazole by Lactobacillus acidophilus ATCC 4356 Surface Layer Protein and Evaluation of its Stability in Acidic Condition.

The present study introduces a novel method for encapsulation of the acid-labile drug called Omeprazole using Lactobacillus acidophilus (L. acidophilus) ATCC 4356 S-layer protein. Before preparing the Omeprazole suspension, a series of preliminary studies were performed on the Omeprazole powder. For this purpose, some parameters such as melting point, IR spectrum, UV spectrum, and the particle size of Omeprazole powder were investigated. The size reduction process was done in order to achieve an ideal formulation. Ultimately, the resulting powder had an average particle size of 35.516 µm and it was almost uniform. After calculating the amount of S-layer protein required for complete covering of drug particles, the effect of different factors on the drug coating process was investigated with one factor at a time method. Then stability of coated Omeprazole was evaluated in acetate buffer (pH 5). Finally, the maximum coat of drug particles was determined using S- layer protein of Lactobacillus acidophilus ATCC 4356 at 25 °C for 2 h, shaking rate of 100 rpm and ratio of 2:1 for S-layer protein amount/Omeprazole Surface in Tris hydrochloride buffer medium (50 mM, pH 8). The coating of Omeprazole by the S-layer protein decreased the drug decomposition rate up to 2.223.

Characteristics of surface layer proteins from two new and native strains of Lactobacillus brevis.

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 ß-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.