Lactobacillus reuteri KCTC3594로부터 분리한 두 종류의 서로 다른 글루타미네이즈의 특성 분석

Lactobacillus reuteri KCTC3594로부터 분리한 두 종류의 서로 다른 글루타미네이즈의 특성 분석
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Glutaminase plays an important role in glutamine degradation, catalyzing the hydrolytic deamination of L-glutamine to L-glutamic acid and ammonia. Glutaminases have been found in several microorganisms including lactobacilli. Lactobacillus spp. are known as GRAS (generally recognized as safe) bacteria widely used in food industry and primary member of probiotics. In this study, individual glutaminase gene, glu1 and glu2, was cloned from L. reuteri KCTC3594 and subsequently introduced into L. paracasei KLB58 which has no glutaminase gene using glass bead transformation method, thereby expecting increased glutaminase activity and contributing to the value of L. paracasei KLB58 as a producer of a natural flavor. In addition, to compare the enzymatic properties of two glutaminases from L. reuteri KCTC3594, the effects of different reaction conditions such as temperature, pH and salt concentration were investigated. When cloned into L. paracasei KLB58 two glutaminase genes showed glutaminase activity using L-glutamine as the substrate, but differed in biochemical properties. Glu2 showed an optimal activity at 40℃ and 10% (w/v) NaCl, with a higher affinity for L-glutamine than Glu1 which had optimal activity at 20-30℃ and 0% (w/v) NaCl. However their responses to pH were similar. The optimum pH for both Glu1 and Glu2 was observed at 5.0. In addition, to investigate the role of glutaminase in acidic stress, cell survivability was measured under various pH conditions. As cell culture pH decreased, cell viabilities of L. reuteri KCTC3594, L. paracasei KLB58::Glu1 and L. paracasei KLB58::Glu2 were decreased slightly, while that of L. paracasei KLB58 which intrinsically had no glutaminase activity was decreased significantly. These results suggest that NH4+ hydrolyzed from glutamine by glutaminase activity neutralized H+ present in acidic condition and thereby prevented cells from acid stress. Taken together, two glutaminases from L. reuteri were successfully expressed in str
1. INTRODUCTION 1 1.1 Lactobacillus spp. 1 1.2 Probiotics 2 1.3 Natural flavor 3 1.4 Glutaminase 4 2. OBJECTIVES 5 3. MATERIALS AND METHODS 6 3.1 Bacterial strains, plasmids, culture media, and growth conditions 6 3.2 Cloning of the second glutaminase gene 8 3.3 Transformation 10 3.3.1 Making protoplasts 10 3.3.2 Glass bead transformation 11 3.4 Plasmid stability 12 3.5 Comparison with wild type and transformants 14 3.5.1 Glutaminase enzyme activity assay 14 3.5.2 Acid stress test 16 4. RESULTS AND DISCUSSION 18 4.1 Sequence analysis 18 4.2 Confirmation of transformation 20 4.3 Plasmid stability 25 4.4 Comparison with wild type and transformants 27 4.4.1 Glutaminase enzyme activity assay 27 4.4.2 Acid stress test 33 5. CONCLUSION 35 6. REFERENCES 37
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College of Natural Science(자연과학대학) > Ocean Sciences (해양과학) > Theses(해양과학 석박사 학위논문)
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