A comparative study of extraction techniques for maximum recovery of glutamate decarboxylase (GAD) from Aspergillus oryzae NSK

BMC Research Notes

Nazamid Saari²  Raja Noor Zaliha Raja Abd Rahman¹  Hasanah Mohd Ghazali²  Mohd Safuan Ab Kadir²  Audrey Lee Ying Yeng² 

[1] Faculty Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400, UPM Serdang, Selangor, Malaysia;Faculty of Food Science and Technology, University Putra Malaysia, 43400, Serdang, Selangor, D.E., Malaysia

关键词: Chemical permeabilization;    Enzymatic lysis;    Mechanical disruption;    Sonication;    γ-Amino-butyric acid;    Glutamate decarboxylase;    Aspergillus oryzae;   

Others  :  1140501
DOI  :  10.1186/1756-0500-6-526

 received in 2012-12-06, accepted in 2013-12-06,  发布年份 2013

【 摘 要 】

Background

γ-Amino butyric acid (GABA) is a major inhibitory neurotransmitter of the mammalian central nervous system that plays a vital role in regulating vital neurological functions. The enzyme responsible for producing GABA is glutamate decarboxylase (GAD), an intracellular enzyme that both food and pharmaceutical industries are currently using as the major catalyst in trial biotransformation process of GABA. We have successfully isolated a novel strain of Aspergillus oryzae NSK that possesses a relatively high GABA biosynthesizing capability compared to other reported GABA-producing fungal strains, indicating the presence of an active GAD. This finding has prompted us to explore an effective method to recover maximum amount of GAD for further studies on the GAD’s biochemical and kinetic properties. The extraction techniques examined were enzymatic lysis, chemical permeabilization, and mechanical disruption. Under the GAD activity assay used, one unit of GAD activity is expressed as 1 μmol of GABA produced per min per ml enzyme extract (U/ml) while the specific activity was expressed as U/mg protein.

Results

Mechanical disruption by sonication, which yielded 1.99 U/mg of GAD, was by far the most effective cell disintegration method compared with the other extraction procedures examined. In contrast, the second most effective method, freeze grinding followed by 10% v/v toluene permeabilization at 25°C for 120 min, yielded only 1.17 U/mg of GAD, which is 170% lower than the sonication method. Optimized enzymatic lysis with 3 mg/ml Yatalase^® at 60°C for 30 min was the least effective. It yielded only 0.70 U/mg of GAD. Extraction using sonication was further optimized using a one-variable-at-a-time approach (OVAT). Results obtained show that the yield of GAD increased 176% from 1.99 U/mg to 3.50 U/mg.

Conclusion

Of the techniques used to extract GAD from A. oryzae NSK, sonication was found to be the best. Under optimized conditions, about 176% of GAD was recovered compared to recovery under non optimized conditions. The high production level of GAD in this strain offers an opportunity to conduct further studies on GABA production at a larger scale.

【 授权许可】

   
2013 Lee Ying Yeng et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

【 参考文献 】

• [1]Krnjević K: Chemical nature of synaptic transmission in vertebrates. Physiol Rev 1974, 54:418-540.
• [2]Wong GT, Bottiglieri T, Snead C: GABA, γ-hydroxybutyric acid, and neurological disease. Ann Neurol 2003, 6:3-12.
• [3]Brandão ML, De Aguiar JC, Graeff FG: GABA mediation of the anti-aversive action of minor tranquilizers. Pharmacol Biochem Behav 1982, 16(Suppl 3):397-402.
• [4]Shimada M, Hasegawa T, Nishimura C, Kan H, Kanno T, Nakamura T, Matsubayashi T: Anti-hypertensive effect of gamma-aminobutyric acid (GABA)-rich Chlorella on high-normal blood pressure and borderline hypertension in placebo-controlled double blind study. Clin Exp Hypertens 2009, 31(Suppl 4):342-354.
• [5]Soltani N, Qiu H, Aleksic M, Glinka Y, Zhao F, Liu R, Li Y, Zhang N, Chakrabarti R, Ng T, Jin T, Zhang H, Lu WY, Feng ZP, Prud'homme GJ, Wang Q: GABA exerts protective and regenerative effects on islet beta cells and reverses diabetes. Proc Natl Acad Sci U S A 2011, 108(Suppl 28):11692-11697.
• [6]Ueno Y, Hayakawa K, Takahashi S, Oda K: Purification and characterization of glutamate decarboxylase from Lactobacillus brevis IFO 12005. Biosci Biotech Biochem 1997, 61(Suppl 7):1168-1171.
• [7]Tsuchiya K, Nishimura K, Iwahara M: Purification and characterization of glutamate decarboxylase from Aspergillus oryzae. Food Sci & Tech Res 2003, 9(Suppl 3):283-287.
• [8]Yang SY, Lin Q, Lu ZX, Lu FX, Bie XM, Zou XK, Sun LJ: Characterization of a novel glutamate decarboxylase from Streptococcus salivarius ssp. thermophilus Y2. J Chem Tech & Biotech 2008, 83:855-861.
• [9]Kato Y, Kato Y, Furukawa K, Hara S: Cloning and Nucleotide Sequence of the Glutamate Decarboxylase-encoding Gene gadA from Aspergillus oryzae. Biosci Biotech Biochem 2002, 66(Suppl 12):2600-2605.
• [10]Partial List of Enzyme Preparations That are Used in Foods, US Food and Drugs Administration http://www.fda.gov/food/ingredientspackaginglabeling/gras/enzymepreparations/ucm084292.htm webcite
• [11]FAO/WHO: Evaluation of certain food additives and contaminants. Thirtieth Report of the Joint FAO/WHO Expert Committee on Food Additives 1987, 15-16. http://whqlibdoc.who.int/trs/WHO_TRS_751.pdf webcite
• [12]Taubert J, Krings U, Berger RG: A comparative study on the disintegration of filamentous fungi. J Microbiol Meth 2000, 42:225-232.
• [13]Strigácová J, Chovanec P, Liptaj T, Hudecová D, Turský T, Simkovic M, Varecka L: Glutamate decarboxylase activity in Trichoderma viride conidia and developing mycelia. Arch Microbiol 2001, 175:32-40.
• [14]Kumar S, Punekar N: The metabolism of 4-aminobutyrate (GABA) in fungi. Mycol Res 1997, 101(Suppl 4):403-409.
• [15]Klimek-Ochab M, Brzezinska-Rodak M, Zymanczyk-Duda E, Lejczak B, Kafarski P: Comparative study of fungal cell disruption-scope and limitations of the methods. Folia Microbiol 2011, 56:469-475.
• [16]Belur PD, Mugeraya G, Nainegali B: Release of cell associated tannase of Serratia ficaria DTC by sonication, surfactants and solvents. Asian J Biotech 2011, 3:91-97.
• [17]Saptarshi SD, Lele SS: Application of evolutionary optimization technique in maximizing the recovery of L-asparaginase from E. caratovovora MTCC 1428. Glob J Biotech Biochem 2010, 5(Suppl 2):97-105.
• [18]Ho CW, Chewa TK, Ling TC, Kamaruddin S, Tan WS, Tey BT: Efficient mechanical cell disruption of Escherichia coli by an ultrasonicator and recovery of intracellular hepatitis B core antigen. Proc Biochem 2006, 41:1829-1834.
• [19]Breddam K, Beenfeldt T: Acceleration of yeast autolysis by chemical methods for production of intracellular enzymes. Appl Microbiol Biotechnol 1991, 35:323-329.
• [20]Ahmed H: Extraction of Protein. In Principles and Reactions of Protein Extraction, Purification, and Characterization. Edited by Vasta GR. Baltimore: CRC Press; 2005:17-21.
• [21]Chisti Y, Young MM: Disruption of microbial cells for intracellular products. Enz Microbiol Tech 1986, 8:196-204.
• [22]Middelberg APJ: Process-scale disruption of microorganisms. Biotech Adv 1995, 13(Suppl 3):491-551.
• [23]Ho CW, Tan WS, Yap WB, Ling TC, Tey BT: Comparative evaluation of different cell disruption methods for the release of recombinant hepatitis B core antigen from Escherishia coli. Biotechnol Bioproc Eng 2008, 13(Suppl 5):577-583.
• [24]Ab Kadir MS: Enhancement of γ-aminobutyric acid from Aspergillus oryzae in batch fermentation. Master thesis: Universiti Putra Malaysia, Food Science Department; 2011.
• [25]Rosetti V, Lombard A: Determination of glutamate decarboxylase by high performance liquid chromatography. J chroma 1996, 681:63-67.
• [26]Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal Biochem 1976, 72:248-254.
• [27]Kormanik PP, McGraw AC: Quantification of vesicular-arbuscular mycorrhizae in plant roots. In Methods and Principles of Mycorrhizal Research. Edited by Schenck NC. Minnesota: APS press; 1982:37-40.
• [28]Huang B, Lin W, Cheung PC, Wu J: Differential proteomic analysis of temperature-induced autolysis in mycelium of Pleurotus tuber-regium. Curr Microbiol 2011, 62(Suppl 4):1160-1167.
• [29]Gogate PR, Wilhelm AM, Pandit AB: Some aspects of the design of sonochemical reactors. Ultrason Sonochem 2003, 10:325-330.
• [30]Feliu JX, Villa VA, Cubarsi R: Optimized release of recombinant protein by ultrasonication of Escherichia coli. Biotechnol Bioeng 1998, 58(Suppl 5):36-40.
• [31]Dagbagli S, Goksungur Y: Optimization of β-galactosidase production using Kluyveromyces lactis NRRL Y-8279 by response surface methodology. Electron J Biotechnol 2008, 11:1-12.
• [32]Doulah MS: Mechanism of disintegration of biological cells in ultrasonic cavitation. Biotechnol Bioeng 1977, 19(Suppl 5):649-660.
• [33]Rachinskaya ZV, Karasyova EI, Metelitza DI: Inactivation of glucose-6-phosphate dehydrogenase in solution by low and high frequency ultrasound. Appl Biochem Microbiol 2004, 40:120-128.
• [34]Kuboi R, Umakoshi H, Takagi N, Komasawa I: Optimal disruption methods for the selective recovery of β-Galactosidase from Escherichia coli. J Ferment Bioeng 1995, 79(Suppl 4):335-341.
• [35]Bowman SM, Free SJ: The structure and synthesis of fungal cell wall. BioEssays 2006, 28(Suppl 8):799-808.