Team:Osaka/Project pga

Poly (Gamma-Glutamic Acid)

Overview

Poly (gamma-glutamic acid), also known as gamma polyglutamic acid (γ-PGA) or gamma polyglutamate, is a very interesting biopolymer that has attracted attention in recent years.
It is a sticky substance found in "natto", a traditional Japanese fermented food made from soybeans by Bacillus subtilis (formerly Bacillus natto).

In this sticky product secreted by B. subtilis, γ-PGA is contained.γ-PGA and its derivatives offer a wide range of unique applications including being used as thickener, humectant, drug carrier, biodegradable fibers, highly water absorbable hydrogels, biopolymer flocculants, and heavy metal absorber. ( Ing-Lung Shih et al,. 2001→)
Most famous products of PGA is highly water absorbable hydrogels. Its PGA is from B.natto. PGA fromB.natto have high ability to absorb and hold water. This character is strengthened by UV-crosslink among PGA chains and very useful to environmental problems.
Further more, PGA is very safe, not having toxicity to animals.

PGA STRUCTURE

( Ing-Lung Shih et al,. 2001→)

It is Dehydration-condensation reaction between gamma-position carboxyl group and alpha-position amino group of glutamate to compose PGA .
Glutamate have chirality. Although almost glutamate exist in L-form in nature, PGA don't have only L-form Glutamate but also D-form Glutamate.The ratio with L and D depends on some factors, species, culture condition and so on. These factors effect on the degree of polymerization.

POLY GLUTAMATE SYNTHETASE

Three genes ware required for a poly-gamma-glutamate synthetic system of B. subtilis IFO 3336 (B. natto), previously reported.The E. coli cloned these genes produced poly-gamma-glutamate extracellularly.( H. Misono et al,. 1999→)
Three genes, pgsB, pgsC, and pgsA, code three essential enzymes to creat and secrete PGA .Unfortunately, the roles of these enzymes are not confirmed, but the localizations are known, all extracellularly.

glutamate racemase is also important enzyme for this synthesis because PGA made of only L-glutamate is synthesized without this enzyme. Therefore, Co-expression of pgsB,C,A genes and racemase increased both the polymer production and D-glutamate content in the polymer.( H. Misono et al,. 1999→)

Bacillus subtilis has enzymes to biodegrade PGA; however, E. coli does not. Therefore, we want E. coli to produce PGA for greening of the desert.

STRATEGY / RESULT

Our plasmid containing genes for four enzymes had several point mutations and restriction sites. These plasmid were contributed. Therefore We tried to make these sequence optimized for BioBricks with Polymerase Chain Reaction (PCR).However, we faced many difficulties about PCR.Finally, we succeeded to integrate these genes to BioBricks!!

References

• Makoto Ashiuchi, Kenji Soda, and Haruo Misono Biochemical and Biophysical Research Communications 263, 6–12 (1999)
• Ashiuchi M., Misono H. Appl Microbiol Biotechnol 59:9–14 (2002)
• Tanaka, T., Fujita, K., Takenishi, S., and Taniguchi, M. J. Ferment. Bioeng. 84, 361–364. (1997)
• Akio Baba and Ikuya Shibata The Chemical Record, Vol. 5, 352–366 (2005)
• Ashiuchi M.; Kamei T.; Misono H. Journal of Molecular Catalysis B: Enzymatic 23 101–106 (2003)
• Ashiuchi M, Tani K, Soda K, Misono H. J. Biochem. 123, 1156-1163 (1998)
• Shih IL, Van YT. Bioresource Technology 79 207-225 (2001)
• 生化学第８０巻第４号，pp.３１６―３２３，２００８
• D-グルタミン酸とポリ--グルタミン酸合成システム