Which ph resulted in maximum lipase activity

There were no adverse effects after either mock or LipL protein immunization. Splenocytes from mice immunized with LipL Ms were isolated 2—3 weeks after the last immunization and stimulated with LipL Ms protein in a well plate for 12 hours. These results suggested that LipL could elicit significantly strong cell immune responses and particularly a cytotoxic effect in mice.

Primarily, we attempted to express the members of the Lip family of M. All of the recombinant proteins formed inclusion bodies and failed to renaturate. To obtain natural and biochemically active products, a robust and acetamide-inducible expression vector for the host bacterium M. The total lipase activities were assayed using the cell lysate supernatants from the 10 recombinant M. Interestingly, we found that the highest lipase activity was observed with the strain overexpressing the lipL gene Fig 1A , which was markedly higher than that obtained for the positive control LipY, the most intensively studied lipase in M.

Therefore, we focused on the LipL protein in the subsequent studies. Meanwhile, experiments with LipY were performed as the positive control S4 Fig. The results showed that LipL could not only hydrolyze short-chain esterases but also had a rather high activity against long-chain lipids Fig 1B.

Both of these characteristics reflect the adaptation of mycobacteria to the host environment. In addition, five of these proteins had the S-x-x-K motif, which is conserved in the carboxylesterase VIII family [ 32 ], and four had the GGG motif, which had been reported in certain esterases [ 33 ]. Among the 10 proteins, while some had one or two of the three motifs, LipL had all three motifs. To determine which motif was responsible for the lipase activity of LipL, a site-directed mutagenesis experiment was performed.

In addition, the key amino acid residues of the active center of the lipase were determined through the site-directed mutagenesis.

Through homologous modeling, we found that the GGG motif formed the pocket structure on the three dimensional surface Fig 4E and 4F. A previous study revealed that the GGG motif was considered an oxyanion hole motif, which is consistent with the findings of our modeling study [ 33 ]. We revealed the overall topological organization of LipL through homology modeling, and the predicted active center was consistent with the experimental results of the site-directed mutagenesis experiment.

This finding provides information that has important reference value for further detailed analysis of the structure and function of LipL. Although the LipL protein had no predicted signal peptides according to the SignalP 3. Regarding non-classical secretion mechanisms, important secretory proteins, such as ESAT-6 and CFP, also contain no signal sequence in their N-termini.

The result from the Western blot analysis of the mycobacterial subcellular fractions showed that LipL was located on the cell wall and cell membrane, suggesting the likelihood of the secretion of LipL. Given its subcellular location in mycobacteria, we thus hypothesized that LipL could induce host humoral or cell-mediated immune responses.

The reason might be due to the LipL E could not correctly fold via resolution from an inclusion body, which suggested that the immunogenicity of LipL was mostly derived from spatial epitopes and that a probable linear conformation of LipL E could not appropriately react with LipL-specific antibodies. This finding also suggested that the use of the M. Similarly, the M. The sera samples from TB patients used in our study represented a heterogeneous population, including newly infected cases and relapsed cases.

The immune response profiles of LipL in the two groups were assessed. A previous report emphasized a lack of sufficient immune responses against many M. Surprisingly, in our study, the newly infected patients group 1 responded better against LipL than the relapsed patients group 2. This difference may be attributed to the difference in immunogenicity among M. Alternatively, it is also likely that the group 2 patients could not induce sufficient immune responses against TB, resulting in relapse.

Taken together, the results showed that the M. In addition, LipL was up-regulated in M. We speculated that LipL provides a potential target for drug screening. The LipL protein was successfully expressed in and purified from M. The key amino acid residues of the active center of LipL were determined through site-directed mutagenesis and homologous modeling experiments.

Proteins were eluted with elution buffer at a gradient concentration of imidazole. Lanes: 1, eluate eluted with mM imidazole; 2, eluate eluted with mM imidazole; 3, eluate eluted with mM imidazole; M, molecular mass markers. B LipL protein was confirmed by Western blot. The protein band reacted with His-tag antibodies. Lanes: 1—2, eluate eluted with mM imidazole; 3—4: eluate eluted with mM imidazole; M, molecular mass markers.

We thank P. Hairong Huang for the generous gift of M. Conceived and designed the experiments: SL LC. Wrote the paper: JC LC. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper and its Supporting Information files.

Introduction Tuberculosis TB is an infectious disease caused by bacteria in the Mycobacterium tuberculosis complex, which includes M. Gene amplification and plasmids construction The parent vector pMV kind gift from Lu Yu was a promoterless Escherichia coli -mycobacteria shuttle plasmid. Download: PPT. Table 2. Expression and purification of Lip family proteins from M.

Lipase assay Lipase activity was assayed by measuring the amount of p -nitrophenol p -NP released from p -NP ester substrate with varying lengths of fatty acids [ 16 ]. Subcellular fractionation of mycobacteria and immunoblotting The subcellular fractionation of M. Study population and serological characterization Sera samples were obtained from 51 TB patients who were admitted to the Heilongjiang Provincial Hospital for Prevention and Treatment of Tuberculosis, Harbin, China, and 45 clinically healthy donors.

Substrate specificity of LipL Lipases, which hydrolyze long-chain esters, are considered much more crucial for the pathogenicity of M.

Effects of detergents on LipL activity We next assessed the effect of various detergents on LipL lipase activity. Fig 4. Subcellular location of LipL in mycobacteria To investigate the subcellular location of LipL in mycobacteria, subcellular fractions prepared from M. Fig 5. Subcellular localization, the humoral and cell-mediated immune responses of LipL. Immunogenicity of LipL The results of the subcellular localization experiment suggested that LipL was mainly located in the cell membrane and cell wall.

Conclusions The LipL protein was successfully expressed in and purified from M. Supporting Information. S1 Checklist. S1 Fig. S2 Fig. Subcellular localization of LipL in M. S3 Fig. Cell-mediated immune response of LipL. S4 Fig. The lipase activity of LipY. Acknowledgments We thank P. References 1. A new evolutionary scenario for the Mycobacterium tuberculosis complex.

Genomic analysis of smooth tubercle bacilli provides insights into ancestry and pathoadaptation of Mycobacterium tuberculosis. Nat Genet. World Health Organization. Global Tuberculosis Report. Geneva: World Health Organization; Re-annotation of the genome sequence of Mycobacterium tuberculosis H37Rv.

Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. A monoacylglycerol lipase from Mycobacterium smegmatis Involved in bacterial cell interaction. J Bacteriol. Intracellular lipophilic inclusions of mycobacteria in vitro and in sputum.

Induction of a novel class of diacylglycerol acyltransferases and triacylglycerol accumulation in Mycobacterium tuberculosis as it goes into a dormancy-like state in culture. Kaufmann SHE. How can immunology contribute to the control of tuberculosis? Nat Rev Immunol. Foamy macrophages from tuberculous patients' granulomas constitute a nutrient-rich reservoir for M.

PLoS Pathog. Identification and structural characterization of an unusual mycobacterial monomeromycolyl-diacylglycerol. Dependence between protease production and pH has been previously demonstrated Mc Ewen and Young, An increase in air flow rate produced an increase of pH at the beginning of cultivation. Thus, it is possible to suggest that procedures that lead to increase of external pH would favor protease production by Yarrowia lipolytica. A reduction in initial pH of the culture medium was observed when either stirring speed or air flow rate was increased.

In spite of this, no deleterious effects lysis, for instance on cell growth, were observed. Despite of pH decrease, cell growth is observed at the beginning of cultivation, even at rpm.

This decrease can be due to olive oil hydrolysis, caused by cell-bound enzyme Pereira-Meirelles et al. The observation that cultivation of yeast cells with triacylglycerols and exogenously supplied lipase results in extensive incorporation of free fatty acids Dyer et al.

Furthermore, peptone degradation, which results in amino acid release, can also explain the decrease in pH, as observed by Corzo and Revah These findings are supported by the detection of extracellular proteases from the beginning of cultivation.

After this initial decrease, pH stabilization occurs as nutrients are metabolized by the cells. Thus, it is possible to say that alterations in pH profiles reflect metabolic changes. This hypothesis is corroborated by the observation that when lipid metabolism was slower, decreases in pH were smaller, whereas at higher stirring speeds or air flow rates, when lipid consumption rates were higher, the decrease in pH was faster. The increase in pH at the late stationary phase at a constant air flow or after diauxie at a constant stirring speed can result from consumption of acid molecules present in the medium, as verified by Corzo and Revah , and from ammonia released through amino acid deamination.

Although drastic changes in the pH of the medium were observed, no correlation was found with maximum lipase levels, since high levels of lipase activity were obtained from pH 4 to 8 at rpm. These results are in agreement with previous findings of Novotny et al. Thus, this paper fills some gaps in the literature concerning the effect of oxygen on lipase production and on lipid metabolism by this NCY.

Relationship among lipid consumption, cell growth and lipase production was presented. It was observed that the most pronounced effect of oxygen on lipase production was determined by stirring speed rather than by air flow rate. Data presented herein will be used to support further work employing experimental design where other variables will be investigated.

The authors are very grateful to Dr. Maria Alice Zarur Coelho for help on k L a measurement. Abrir menu Brasil. Brazilian Journal of Chemical Engineering. Abrir menu. Alonso E. Oliveira G. Dellamora-Ortiz F. Pereira-Meirelles About the authors. Lipase; Yarrowia; Stirring; Aeration; Air flow.

Baillargeon, M. Charney, J. Chen, J. Corzo, G. Dellamora-Ortiz, G. M, Martins. Destain, J. Dyer, J. Eggert, T. Elibol, M. Finogenova, T. P, Morgunov, I. Flores, C. Freire, D. Frings, C. Gombert, A. Gulati, R. Hagler, A.

Herrero, A. Madzak, C. Use the simulation to help you answer this question if you are not sure. What do you think would happen if you decreased the temperature of incubation to 10 deg. There should be less digestion at a lower temperature due to the fact that the enzyme is most efficient at 37 deg. C body temperature Activity 4 Explain the difference in activity between tubes 1 and 2. Tube 1 had bile salts and tube 2 had deionized water — tube 2 showed a slightly higher enzyme reaction.

Can we determine if fat hydrolysis has occurred in tube 6? Which pH resulted in maximum lipase activity? Activity 5 While swallowing a mouthful of water from a paper cup, consciously note the movement of your tongue during the process. Record your observations. Repeat the swallowing process while your laboratory partner watches the externally visible movements of your larynx.

What do these movements accomplish? I'm Belinda!