EXPLORATION AND DEVELOPMENT FOR ECTOINE PRODUCTION BY HALOPHILIC BACTERIA ISOLATED FROM THE MUD CRATER OF “BLEDUG KUWU” PURWODADI CENTRAL JAVA

• Main Author: Putu Parwata, I
• Format: Dissertations
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/36141
• Institution: Institut Teknologi Bandung

Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) is compatible organic molecules widely used in many fields, such as pharmaceuticals, cosmetics, and biotechnological applications. This molecule can protect cell’s biomolecules, such as proteins and cell membrane, from various environmental stresses like osmotic pressure, heating, freezing, dryness, UV rays, or contact with toxic materials. An increase of commercial demand of ectoine as an active biocompound has led a number of effort to improve the production of this molecule from microorganisms. The development of ectoine production is carried out both from microbes that are naturally capable of producing ectoine as well as from those that are unable to produce ectoine by applying genetic engineering techniques. In addition, exploration of microbes that have superior ability to produce ectoine become one of the research trends today. One of the potential ectoine-producing bacteria is halophile, which is the class of extremophile that favor to live in hypersaline environment. The aim of ectoine production by this type of bacteria is as one of the protection strategies against high osmotic pressure exerted by hypersaline environment in order to prevent difusion of liquid out of the cell. Several local halophilic bacteria have been isolated from brine samples obtained from the mud crater of "Bledug Kuwu" located at Kuwu Village, Kradenan District, Grobogan Regency, Central Java. The results of the preliminary study showed that the halophilic bacteria exhibited relatively high tolerance to salt levels (0.5-30% [w/v]), which is the important characteristic for ectoine producing bacteria. The present study is aimed to further explore the potency of those local halophilic bacteria in producing ectoine. This study was conducted in four steps: the first one is screening the potential ectoine-producing bacteria in terms of their tolerance against high level of salt, the growth and ectoine yield per dry cell weight. The second step was the optimization of ectoine production yield from the best halophilic bacterium obtained from the first step. The third step was producing ectoine from recombinant E. coli, which has been transformed to carry the ectoine gene cluster (ectABC) isolated from the best halophilic bacterium. The ectoine production by the recombinant cell was subjected to the optimization. The last step was application of ectoine as a stabilizer for protein. The results of the ectoine production test of five halophilic bacteria BK-AB12, BK-AG13, BK-AB18, BK-AG18, and BK-AG25 gave the respective yield of 33, 65; 8,21; 10.31; 9.71; and 61.73 mg/g cdw. The highest ectoine yield was thus produced by BK-AG25 isolate and hence it was selected for further study. The identification of the bacterial isolate by ribotyping method was phylogenetically closest to Halomonas elongata. Afterwards, it was labeled as Halomonas elongata BK-AG25. The optimization of ectoine production by H. elongata BK-AG25 was conducted by two-stage cultivation method. The first cultivation stage was intended to increase the yield of bacterial biomass (cell density), while the second one was aimed to enhance ectoine production yield. There were four parameters, i.e. the levels of glucose, (NH4)2SO4, MgSO4 and NaCl as well as the incubation temperatures, which were optimized by response surface methodology (RSM) in the first cultivation stage. The result of optimization gave the regression model suggesting that the highest yield of biomass can be achieved when H. elongata BK-AG25 is cultivated at 37.4 °C in MM63 medium containing 8.9% [w/v] NaCl, 1.1% [w/v] glucose, 0.37% [w/v] (NH4)2SO4 and 0.04% [w/v] MgSO4. The bacterial cultivation at such conditions gave biomass about 4.92 ? 0.028 mg/mL. In the second stage of cultivation, H. elongata BK-AG25 was inoculated in the optimized MM63 medium but containing higher NaCl concentration to stimulate ectoine biosynthesis. The production yield of ectoine was optimized by RSM, which targeted NaCl concentration and temperature of cultivation as optimization parameters. The obtained regression model suggest the highest yield of ectoine production by H. elongata BK-AG25 can be achieved when it is cultivaled in MM63 medium containing 18% [w/v] NaCl at 33 °C. At these conditions, the yield of ectoine production was about 1.17 ? 0.015 g/L and the bacterial productivity was about 179.9 ? 8.52 mg ectoine/g cdw (cell dry weight). The production yield was further optimized by targeting glucose concentration and incubation time. The optimization successfully improved the yield of ectoine production up to 1.57 g/L with a bacterial productivity of about 269 mg ectoine/g cdw when the cultivation was conducated at glucose concentration of 0.8% [w/v] and an incubation time about 35 hours. After the condition of ectoine biosynthesis by H. elongata BK-AG25 was optimized, the next stage is to optimize ectoine extraction using the osmotic shock process and the "bacterial milking". The bacterial inoculum was grown by two- stage cultivation optimized above. The bacterial culture was then transferred aseptically into sterile water containing a lower level of salt to stimulate osmotic downshock in order to drive ectoine excretion. By this way about 80% of ectoine was excreted out of the cell. The survival rate of bacteria was relatively high, which was about 70%. One trial of osmotic downshock exerted by pure distilled (0% NaCl) siginificantly lowered the survival rate of the bacterial cell to only about 9%. After osmotic downshock, the cells were reinoculated in fress MM63 medium containing high level of salt (osmotic upshock) to stimulate ectoine biosynthesis. The osmotic upshock and downshock were repeated several times to produce high level of ectoine. Such proccess is known as “bacterial milking”. Our experimental data showed that four cycles of "bacterial milking" by H. elongata BK-AG25 were successfully produced about 2.26 g/L of ectoine. Besides producing ectoine by the wild type H. elongata BK-AG25, in this study, ectoine was also produced by recombinant cell of nonhalophile bacteria carrying genes cluster encoding enzymes involved in ectoine biosynthesis. The ectoine gene cluster of H. elongata BK-AG25 (ectABC) was successfully amplified with a total length of 2,438 base pairs (bp), consisting of ectA (579 bp), ectB (1,266 bp) and ectC (414 bp), which were constructed as one operon. The operon was inserted into the expression vector pET30a(+) and was transfered into E. coli BL21 (DE3). The recombinant cell was then grown in LB medium and the expression of the operon was induced by IPTG. The expression results showed that all genes in ectABC operon were successfully expressed by the recombinant cells but in different levels, in which ectA and ectB genes were strongly expressed, while ectC was weakly expressed. The production of ectoine by the recombinant E. coli was than tested using MM63 medium. The results showed that the recombinant E. coli was able to produce ectoine and most of them (> 70%) were excreted in to the medium. Incubation of the recombinant cell for 14 hours after induction using IPTG enabled the bacteria produced around 0.23 g/L extracellular ectoine with the productivity of about 69 mg ectoine/g cdw. Up to present, this is the first report on the expression of the ectoine gene cluster of Halomonas elongata under the control of T7 promoter in E. coli BL21. Ectoine production by the recombinant E. coli was then optimized. Optimization of the levels of glucose and NaCl in MM63 medium as well as the incubation temperature produced a regression model for the concentration of intracellular and extracellular ectoine produced by the recombinant. The regression model suggested an optimal extracellular ectoine production by the recombinant E. coli at glucose level of 0.92% [w/v] and NaCl level of 0.28% [w/v] at 34 °C. The experimental results at these conditions yield about 0.37 + 0.027 g/L extracellular ectoine. Meanwhile, about 0.05 + 0.005 g/L of intracellular ectoine was produced by the recombinant cell at NaCl level of 1.78% [w/v] and 32 °C. Further optimization was targeting optical density and IPTG concentration, resulted regression model for the concentration of extracellular ectoine and the bacterial produtivity. The regression model predicted that the optimum concentration of extracellular ectoine produced by the recombinant cells at the initial OD value of 0.74 and the final concentration of IPTG of 0.62 mM. In addition, the optimum bacterial productivity was predicted at the initial OD of 0.3 and the final concentration of IPTG of 1.5 mM. The experimental results at these optimum conditions were enable the bacteria to produce about 0.71 ? 0.03 g/L of extracellular ectoine with the bacterial productivity of 376 ? 2.3 mg ectoine/g cdw. Furthermore, the production of ectoine from the recombinant E. coli against the incubation time showed that the optimum ectoine concentration of 0.75 g/L with the bacterial productivity of 418 mg ectoine/g cdw were produced by the bacteria after 12 hours of incubation. Ectoine produced by H. elongata BK-AG25 was applied as a stabilizer of lipase. The experimental results showed that ectoine was able to maintain and increase the catalytic activity of lipase against deleterious effect of high temperature and methanol. Addition of 60-150 mM ectoine could increase lipase activity up to 20% after heating for 1 hour at temperature below 80 °C. Meanwhile, the activity of lipase containing 40-125 mM ectoine was succesfully increased up to 50% after incubated for 1 hour in methanol with the level up to 78% [v/v].