Alcaligenes Eutrophus and Its Role in Biodegradable Polymer Production: Difference between revisions
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<br>Maurice Lemoigne (mid 1920s) was the first person who discovered monopolyester of identified poly(3-hydroxybutyrate) or PHB. It took a while until the discovery was known to public because it was originally written in French. Later on, PHB was chemically identified by Staudinger (1920s). However, PHB did not get the attention from public and scientists as the prototypical biodegradable thermoplastic until thirty years later. Imperial Chemical Industries Ltd. (1980s) led was the first one to do intensive studies on PHB in multiple research areas including genetic engineering, biotechnology, and the enzymes responsible for biosynthesis and biodegradation. PHB is a reverse polymer found in bacteria and it can produce both the monomer and polymer of [R]-3-hydroxybutyric acid | <br>Maurice Lemoigne (mid 1920s) was the first person who discovered monopolyester of identified poly(3-hydroxybutyrate) or PHB. It took a while until the discovery was known to public because it was originally written in French. Later on, PHB was chemically identified by Staudinger (1920s). However, PHB did not get the attention from public and scientists as the prototypical biodegradable thermoplastic until thirty years later. Imperial Chemical Industries Ltd. (1980s) led was the first one to do intensive studies on PHB in multiple research areas including genetic engineering, biotechnology, and the enzymes responsible for biosynthesis and biodegradation. PHB is a reverse polymer found in bacteria and it can produce both the monomer and polymer of [R]-3-hydroxybutyric acid | ||
<br> | <br> | ||
<br>Synthesis in Alcaligenes eutrophus<br> | |||
<br>In A. eutrophus, the synthesis of PHB involves glucose as a carbon source and three enzymes; 3-ketothiolase, acetoacetyl-CoA reductase, and PHA syntase [1]. The first enzyme, 3-ketothiolase, is responsible in carrying the condensation reaction of acetyl-CoA into acetoacetyl-CoA. Then, acetoacetyl-CoA reductase reduces acetoacetly-CoA into R(-)-3-hydroxybutynl-CoA, with NADPH serves as the reducing agent. R(-)-3-hydroxybutynl-CoA is then polymerized into PHB with the aid of PHA synthase. A (Figure X). eutrophus can produce PHB as much as 80% of their dry body weight when grown in sugar-rich but nitrogen/ phosphate-deprived media. | |||
Alcaligenes eutrophus synthesizes different kind of polymers base on the carbon source they are exposed to in the media. For example, having propionic acid, instead of glucose, as the main carbon source leads to the production of a polymer containing 3-hydroxybutyrate a (3HB) and 3-hydroxyvalerate (3HV) instead of PHB (Figure X). On the other hand, completely substituting glucose with valeric acid leads to higher production of polymer in A. eutrophus— 90% of the dry body weight.<br> | |||
<br>Physical Characteristics: Benefits and disadvantages<br> | |||
<br>PHB is a rigid homopolymer and is a fragile thermoplastic. It has 100% stereospecificity with the asymmetric D(-) molecule and therefore is highly crystalline[2]. Unlike many other biodegradable polymers, PHB is resistant to moisture, impermeable to oxygen, and insoluble in water [1], which makes it particularly beneficial for the food packaging industry. However, PHB’s melting point (175o C) is so close to its degrading temperature (185o C) which is why it is so fragile and hence hard to mold [2]. The fragility of PHB can be lowered by integrating co-monomer 3-hydroxyvalerate or mixing PHB with more elastic polymer. The PHB copolymer containing 3-hydroxyvalerate unit P(3HB-co-3HV) is one of the developed forms of PHB with better mechanical functions: the more the 3-hydroxyvalerate unit makes PHB tougher, more elastic (i.e. lower young’s modulus), its elongation to break increases, lowers its melting point, but does not change its degrading temperature. Hence, the physical properties of PHB can be controlled by the amount of integrated 3-hydroxyvalerate during fermentation.<br> | |||
==Section 2== | ==Section 2== | ||
Revision as of 03:50, 26 April 2011
Introduction
By [Angel Mogie]
General
Alcaligenes eutrophus (sometimes referred as Ralstonia metallidurans) is a gram negative bacillus with an optimum-growth temperature of 30oC [1]. It is a non-spore forming, obligate aerobic, and facultatively chemolithoautotrohpic bacterium that can thrive in environments containing mM concentrations of some toxic heavy metals such as Zinc, Cadmium, Cobalt, Lead, Mercury, Nickle, and Chromium [2]. In the presence of nitrate, A. eutrhopus can grow anaerobically. It naturally synthesizes polyhydroxybutyrate (PHB), a specific type of polyhydroxyalkanoates (PHA) that is used in the manufacture of biodegradable plastic.
The Enzymes: phbC-phbA-phbB
There are three regulatory enzymes involved in the bioproduction of PHB in Alcaligenes eutrophus: 3-ketothiolase, acetoacetyl-CoA reductase, and PHA syntase, encoded by by genes phbA, phbB, and phbC respectively. The molecular genetics and mapping of these enzymes were done in E. coli using the A. eutrophus strains of H16, 11599, 1159981, PHB 2, PHB3, PHB19.
In 1989, Sinskey and Peoples identified the location of phbA and phbB genes on plasmid pAeT29 of E. coli and that their expressions were under the control of Alcaligenes eutrophus promoter [1]. Later in the same year, the two researchers mapped the location phbC— phbC gene is located upstream from phbA-phbB. Additionally, they also observed a reduced activities in both thiolase and reeducate. Their results suggest that, in PHB2 and PHB3 strains, all three genes are expressed from the same promoter located upstream from phbC [1]
Moreover, Sinskey and Peoples also propose that Alcaligenes eutrophus has at least two P-ketothiolase and acetoace- tyl-CoA reductase enzymes. Thiolase and reductase enzymes, when interact with either enoyl-CoA hydratase or epimerase or both, can lead to the formation of D(-)-3-hydroxybutyryl-CoA. In E.coli the expression of phbC alone results in diminished amount of PHB synthesis and insignificant rate of PHB polymerase activity. The absence of both phbA and phB genes of A. eutrophus leads to no synthesis of D(-)-3-hydroxybutyryl-CoA. Only when the three A. eutrophus genes, phbC-phbA-phbB, are present in E. coli > 50% of PHB production was observed. They also figured that PHB production is inhibited by the presence of nitrogen. Moreover, they propose that the interaction of thiolase and/ or reducatse with the polymerase is necessary for the polymerase to function.
Heavy Metals Resistance
Among the many hydrogen-oxidizing bacteria, the Alcaligenes are characterized by their ability in forming two hydrogenases; an NAD-reducing hydrogenase found in the cytoplasm and a membrane-bound hydrogenase [1]. The hydrogen-oxidizing trait of A. eutrophus is encoded by a 450 kb plasmid, which was identified through a plasmid transfer to Hox- cells lacking the plasmid. Two gene encoding the heavy-metal resistance are located within two large megaplasmids (pMOL28=180 kbp and pMOL30=240 kbp). These two megaplasmid also carry genes that express cation-efflux pumps for both bacterial inner and outer membranes.
PHB
Discovery
Maurice Lemoigne (mid 1920s) was the first person who discovered monopolyester of identified poly(3-hydroxybutyrate) or PHB. It took a while until the discovery was known to public because it was originally written in French. Later on, PHB was chemically identified by Staudinger (1920s). However, PHB did not get the attention from public and scientists as the prototypical biodegradable thermoplastic until thirty years later. Imperial Chemical Industries Ltd. (1980s) led was the first one to do intensive studies on PHB in multiple research areas including genetic engineering, biotechnology, and the enzymes responsible for biosynthesis and biodegradation. PHB is a reverse polymer found in bacteria and it can produce both the monomer and polymer of [R]-3-hydroxybutyric acid
Synthesis in Alcaligenes eutrophus
In A. eutrophus, the synthesis of PHB involves glucose as a carbon source and three enzymes; 3-ketothiolase, acetoacetyl-CoA reductase, and PHA syntase [1]. The first enzyme, 3-ketothiolase, is responsible in carrying the condensation reaction of acetyl-CoA into acetoacetyl-CoA. Then, acetoacetyl-CoA reductase reduces acetoacetly-CoA into R(-)-3-hydroxybutynl-CoA, with NADPH serves as the reducing agent. R(-)-3-hydroxybutynl-CoA is then polymerized into PHB with the aid of PHA synthase. A (Figure X). eutrophus can produce PHB as much as 80% of their dry body weight when grown in sugar-rich but nitrogen/ phosphate-deprived media.
Alcaligenes eutrophus synthesizes different kind of polymers base on the carbon source they are exposed to in the media. For example, having propionic acid, instead of glucose, as the main carbon source leads to the production of a polymer containing 3-hydroxybutyrate a (3HB) and 3-hydroxyvalerate (3HV) instead of PHB (Figure X). On the other hand, completely substituting glucose with valeric acid leads to higher production of polymer in A. eutrophus— 90% of the dry body weight.
Physical Characteristics: Benefits and disadvantages
PHB is a rigid homopolymer and is a fragile thermoplastic. It has 100% stereospecificity with the asymmetric D(-) molecule and therefore is highly crystalline[2]. Unlike many other biodegradable polymers, PHB is resistant to moisture, impermeable to oxygen, and insoluble in water [1], which makes it particularly beneficial for the food packaging industry. However, PHB’s melting point (175o C) is so close to its degrading temperature (185o C) which is why it is so fragile and hence hard to mold [2]. The fragility of PHB can be lowered by integrating co-monomer 3-hydroxyvalerate or mixing PHB with more elastic polymer. The PHB copolymer containing 3-hydroxyvalerate unit P(3HB-co-3HV) is one of the developed forms of PHB with better mechanical functions: the more the 3-hydroxyvalerate unit makes PHB tougher, more elastic (i.e. lower young’s modulus), its elongation to break increases, lowers its melting point, but does not change its degrading temperature. Hence, the physical properties of PHB can be controlled by the amount of integrated 3-hydroxyvalerate during fermentation.
Section 2
Include some current research, with at least one figure showing data.
Section 3
Include some current research, with at least one figure showing data.
Conclusion
Overall text length at least 3,000 words, with at least 3 figures.
References
Edited by student of Joan Slonczewski for BIOL 238 Microbiology, 2011, Kenyon College.
