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Applied and Environmental Microbiology, July 2004, p. 4249-4255, Vol. 70, No. 7
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.7.4249-4255.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Enzymatic Synthesis of High-Molecular-Mass Poly-{gamma}-Glutamate and Regulation of Its Stereochemistry

Makoto Ashiuchi,1* Kazuya Shimanouchi,1 Hisaaki Nakamura,1 Tohru Kamei,1 Kenji Soda,2 Chung Park,3 Moon-Hee Sung,3 and Haruo Misono1

Department of Bioresources Science, Kochi University, Nankoku, Kochi 783-8502,1 Department of Biotechnology, Kansai University, Suita, Osaka 564-8680, Japan,2 BioLeaders Corporation, Joong-gu, Daejeon 301-212, Korea3

Received 28 November 2003/ Accepted 4 April 2004

For the first time, we succeeded in synthesizing in vitro poly-{gamma}-glutamate (PGA) with high molecular masses (>1,000 kDa) by the use of enzyme-associated cell membranes from Bacillus subtilis subsp. chungkookjang. The activity for PGA synthesis, however, was readily lost in the presence of critical concentrations of detergents tested in micelles. The optimum pH for the reaction was found to be ~7.0. We examined the effects of some divalent cations on PGA synthesis and found that Mg2+ was essential in catalysis and that Zn2+ additionally boosted the activity. In contrast, Fe2+ and Ca2+ acted as inhibitors. Mn2+ did not apparently influence the in vitro formation of PGA. DL-Glutamate (D isomer content, 60 to 80%) apparently served as the best substrate; D-Glutamate was preferable to the L isomer as a substrate. When D- and L-glutamate were used for the reaction, the elongated chains of PGAs were composed of the D- and L-isomers, respectively. Our results suggest that the stereochemical properties of enzymatically synthesized PGAs substantially depend on the stereochemistry (DL ratio) of glutamate as the substrate. Furthermore, genetic analysis indicated that all the pgsB, -C, and -A gene products, which are responsible for PGA production by B. subtilis cells, were also indispensable for enzymatic PGA synthesis.

* Corresponding author. Mailing address: Department of Bioresources Science, Kochi University, Nankoku, Kochi 783-8502, Japan. Phone: 81-88-864-5215. Fax: 81-88-864-5200. E-mail: ashiuchi{at}cc.kochi-u.ac.jp.

Applied and Environmental Microbiology, July 2004, p. 4249-4255, Vol. 70, No. 7
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.7.4249-4255.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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