Enzymatic Synthesis of Theanine in the Presence of L-glutaminase Produced by Trichoderma koningii
Applied Food Biotechnology,
Vol. 4 No. 2 (2017),
5 April 2017
Page pp. 113-121
Background and Objective: Since ancient times, it has been said that drinking green tea brings relaxation. The substance that is responsible for a sense of relaxation is theanine. Theanine (γ-glutamylethylamide) is a unique non-protein amino acid. It gives an umami taste and a unique flavor to the tea, and has many physiological and pharmaceutical effects such as anti-tumor, anti-cancer, neuro-protective, anti-hypertensive and anti-obesity effects; it may further help in relaxation and increase focus. So this compound is essential for human body; however, it is not synthesized in the body and should be administrated orally. In the present study, the enzymatic biosynthesis of theanine was examined in the presence of Ethylamine and L-glutamine, and for the first time the enzyme was produced by the fungal strain Trichoderma koningii.
Material and Methods: At first, solid state fermentation was carried out for the production of L-glutaminase by the fungal strain Trichoderma koningii using sesamum oil cake as the solid substrate. Then the biosynthesis of theanine was performed in the presence of extracted enzyme solution, and ethylamine and L-glutamine as substrates. The concentration of effective parameters, namely L-glutamine and ethylamine, and the volume of enzyme solution on theanine production were evaluated based on the response surface methodology coupled with central composite design. 16 experiments were designed by the design expert software and carried out to examine the changes of theanine concentration with changes in the concentration of ethylamine and L-glutamine and the volume of enzyme solution.
Results and Conclusion: This investigation indicated simultaneous synthesis of theanine as well as hydrolysis of L-glutamine and L-glutamic acid. Selected independent variables (including ethylamine concentration, L-glutamine concentration and enzyme solution volume) were effective on theanine concentration. Increase of enzyme solution volume had a significant effect on theanine concentration. The highest theanine concentration (43 mM) was obtained at the ethylamine concentration of 0.9 M, L-glutamine concentration of 0.3 M and enzyme solution of 3 ml.
Conflict of interest: The authors declare no conflict of interest.
- ▪ Enzymatic biosynthesis ▪ L-glutaminase ▪ L-Theanine ▪ Response surface methodology ▪ Trichoderma koningii
How to Cite
Bharadwaz A, Bhattacharjee C. Extraction of polyphenols
from dried tea leaves. IJSER. 2012; 3.
Sakato, Y. The chemical constituents of tea. III. A new amide theanine. J. Agri. Chem. 1949; 23: 262-267.
FDA confirms GRAS status of Suntheanine. Available from: nutraingredients-USA.com. [accessed 22 March 2007].
Juneja LR, Chu D, Okubo T, Nagato Y, Yokogoshi H. Ltheanine: A unique amino acid of green tea and its relaxation effect in humans. Food Sci Technol. 1999; 10: 199-204. doi:10.1016/S0924-2244(99)00044-8.
Lu K, Gray MA, Oliver C, Liley DT, Harrison BJ,
Bartholomeusz CF, Phan L, Nathan PJ. The acute effects of
L-theanine in comparison with alprazolam on anticipatory
anxiety in humans. Human Psychopharmacology: Clinical
and Experimental, 2004; 19: 457-465. doi:10.1002/hup.611.
Haskell CF, Kennedy DO, Milne AL, Wesnes KA, Scholey
AB. The effects of l-theanine, caffeine and their combination on cognition and mood. Biol Psychol J, 2008; 77: 113-122.doi: 10.1016/j.biopsycho.2007.09.008.
Liu Q, Duan H, Luan J, Yagasaki K, Zhang G. Effects of
theanine on growth of human lung cancer and leukemia cells as well as migration and invasion of human lung cancer cells. Cytotechnol J. 2009; 59: 211-217. doi: 10.1007/s10616-009-9223-y.
Yokogoshi H, Kato Y, Sagesaka YM, Takihara-Matsuura T,
Kakuda T, Takeuchi N. Reduction effect of theanine on blood pressure and brain 5-hydroxyindoles in spontaneously hypertensive rats. Biosci Biotechnol Biochem J. 1995; 59:615-618, doi: 10.1271/bbb.59.615.
Zheng G, Sayama K, Okubo T, Juneja LR, Oguni I. Antiobesity effects of three major components of green tea, catechins, caffeine and theanine, in mice. in vivo. 2004; 18:55-62.
Egashira N, Hayakawa K, Osajima M,Mishima K, Iwasaki K, Oishi R, Fujododa M. Involvement of GABA A receptors in the neuroprotective effect of theanine on focal cerebral
ischemia in mice. J Pharmacol Sci. 2007; 105: 211-214, doi:
Deng WW, Ogita S and Ashihara H. Biosynthesis of theanine (γ -ethylamino-l-glutamic acid) in seedlings of Camellia sinensis. Phytochem Lett. 2008; 1: 115-119. doi:10.1016-j.phytol.2008.06.002.
Mu W, Zhang T, Bo Jian. An overview of biological production of L-theanine. g. Biotech Advances, 2015; 33: 335-342,doi: 10.1016/j.biotechadv.2015.04.004
Parmjit P S.; Kaur R, Singla G, Sangwan R S. Bio-processing of agro-industrial wastes for production of food-grade enzymes: progress and prospects. Appl Food Biotechnol, 2016; 3: 208-227.
Yamamoto, S., M. Wakayama, and T. Tachiki. Theanine
production by coupled fermentation with energy transfer
employing Pseudomonas taetrolens Y-30 glutamine
synthetase and baker’s yeast cells. Biosci Biotechnol
Biochem. 2005; 69: 784-789. doi: 10.1271/bbb.69.784.
Suzuki H, Izuka S, Miyakawa N, Kumagai H. Enzymatic
production of theanine, an “umami” component of tea, from glutamine and ethylamine with bacterial γ-glutamyltranspeptidase. Enzyme Microb Technol. 2002: 31: 884-889. doi:10.1016/S0141-0229(02)00213-2.
Wang H Q, Yao Z, Zhao Z, Sun Y, Wei P, Quyang P.
Enzymatic synthesis of theanine with L-glutamine-Zn (II)
complexes. Biotech Biopro Eng. 2012; 17: 1135-1139, doi:
Tachiki T, Yamada T, Ueda M, Naemura Y, Imamura N,
Hamada Y, Shiode J. Purification and some properties of
glutaminase from Pseudomonas nitroreducens IFO 12694.
Biosci Biotech Bioch J. 1996; 60: 1160-1164. doi: 10.1271-
Tachiki T, Yamada T,Mizuno K, Ueda M, Shiode J, Fukami H. γ-Glutamyl transfer reactions by glutaminase from Pseudomonas nitroreducens IFO12694 and their application for the syntheses of theanine and γ-glutamylmethylamide. Biosci Biotechnol Biochem. 1998; 62: 1279-1283. doi: 10.-1271-bbb.62.1279.
Matsuura S, Chiba M, Tomon E, Tsunoda T. Synthesis of
amino acid using a flow-type microreactor containing
enzyme–mesoporous silica microsphere composites. RSC
Advances J, 2014; 4: 9021-9030. doi: 10.1039/c3ra45315d.
Elshafei A M, Hassan M M, Ali N H, Abouzeid M A,
Mahmoud DA, Elghonemy D H. Purification, kinetic
properties and antitumor activity of L-glutaminase from
penicillium brevicompactum NRC 829. Br Microbiol Res J.
; 4: 97-115. doi: 10.9734/bmrj/2014/5098.
Pallem C, Manipati S, Somalanka S R. Process optimization of L-glutaminase production by trichoderma koningii under solid state fermentation. IJABPT. 2010; 1: 1168-1174.
Imada A, Igarasi S, Nakahama K and Isono M. Asparaginase and glutaminase activities of microorganisms. Gen Microbiol J. 1973; 76: 85-99. doi: 10.1099/00221287-76-1-85.
Bezerra, M A, Santelli R E, Oliviera E P, Villar L S, Escaleria L A. Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta. 2008; 76: 965-977. doi: 10.1016/j.talanta.2008.05.019.
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