Enhancement of production of l-methioninase after optimizing culture condition of Pseudomonas stutzeri using artificial neural network

, , ,

Research Articles | Published:

Print ISSN : 0970-4078.
Online ISSN : 2229-4473.
Pub Email: contact@vegetosindia.org
Doi: 10.1007/s42535-021-00330-x
First Page: 453
Last Page: 464
Views: 1624

Keywords: l-Methioninase, Response surface methodology (RSM), Artificial neural network (ANN)


l-Methioninase isolated from microbial strain has currently great demand in pharmaceutical and food sectors. In this study, Pseudomonas stutzeri was explored as new novel bacterial strain for producing l-methioninase. The optimization of its cultural condition would be crucial for scale up of its production at large extent. Response surface methodology (RSM) was first applied to obtain central composite design data and artificial neural networking (ANN) was further applied to analyze these data. ANN had analyzed these data more accurately as compared to RSM model with lower value of absolute average deviation (0.25% ANN < 1.22% RSM), low value of mean square error (MSE = 3.6%). The model was verified by carrying experiments at optimum parameters like inoculum volume (1 ml), pH of media (7.5), incubation period (20 h), incubation temperature (37 °C) and agitation speed (110 rpm). The experimental activity of l-methioninase was obtained as 285.63 U/l. The study of growth and production kinetic at these optimum cultural conditions showed non-growth associated behavior with improved production of l-methioninase.

                        l-Methioninase, Response surface methodology (RSM), Artificial neural network (ANN)

*Get Access

(*Only SPR Members can get full access. Click Here to Apply and get access)



Abdel-Fattah YR, Olama ZA (2002) l-Asparaginase production by Pseudomonas aeruginosa in solid-state culture: evaluation and optimization of culture conditions using factorial designs. Process Biochem 38(1):115–122

Bas D, Boyaci IH (2007) Modeling and optimization I: usability of response surface methodology. J Food Eng 78(3):836–845

Cellarier E, Durando X, Vasson MP, Farges MC, Demiden A, Maurizis JC, Chollet P (2003) Methionine dependency and cancer treatment. Cancer Treat Rev 29(6):489–499

Clausen T, Huber R, Laber B, Pohlenz HD, Messerschmidt A (1996) Crystal structure of the pyridoxal-5′-phosphate dependent cystathionine βlyase from Escherichia coli at 183 Å. J Mol Biol 262(2):202–224

El-Sayed AS (2011) Purification and characterization of a new l-methioninase from solid cultures of Aspergillus flavipes. J Microbiol 49(1):130–140

El-Sayed AS, Shouman SA, Nassrat HM (2012) Pharmacokinetics, immunogenicity and anticancer efficiency of Aspergillus flavipes l-methioninase. Enzyme Microb Technol 51(4):200–210

Frey DD, Engelhardt F, Greitzer EM (2003) A role for" one-factor-at-a-time" experimentation in parameter design. Res Eng Design 14(2):65–74

Ghanem KM, Al-Garni SM, Al-Makishah NH (2010) Statistical optimization of cultural conditions for chitinase production from fish scales waste by Aspergillus terreus. Afr J Biotechnol 9(32):5135–5146

Goyer A, Collakova E, Shachar-Hill Y, Hanson AD (2007) Functional characterization of a methionine γ-lyase in Arabidopsis and its implication in an alternative to the reverse trans-sulfuration pathway. Plant Cell Physiol 48(2):232–242

Gubskaya AV, Khan IJ, Valenzuela LM, Lisnyak YV, Kohn J (2013) Investigating the release of a hydrophobic peptide from matrices of biodegradable polymers: an integrated method approach. Polymer 54(15):3806–3820

Hui-Chuan Y, Huang S-M, Lin W-M, Kuo C-H, Shieh C-J (2019) Comparison of artificial neural networks and response surface methodology towards an efficient ultrasound-assisted extraction of chlorogenic acid from Lonicera japonica. Molecules 24:2304. https://doi.org/10.3390/molecules24122304

Iyer P, Singhal RS (2008) Production of glutaminase (EC from Zygosaccharomyces rouxii: statistical optimization using response surface methodology. Bioresour Technol 99(10):4300–4307

Johnston M, Jankowski D, Marcotte P, Tanaka H, Esaki N, Soda K, Walsh C (1979) Suicide inactivation of bacterial cystathionine gamma-synthase and methionine gamma-lyase during processing of l-propargylglycine. Biochemistry 18(21):4690–4701

Khalaf SA, El-Sayed AS (2009) l-Methioninase production by filamentous fungi: I-screening and optimization under submerged conditions. Curr Microbiol 58(3):219–226

Khan F, Tanaka M, Ahmad SR (2015) Fabrication of polymeric biomaterials: a strategy of tissue engineering and medical devices. J Mater Chem 3:8224–8249

Kharayat B, Singh P (2018) Microbial production of l-methioninase and its biotechnology application. Int J Recent Sci Res 9(8C):28439–28446

Kharayat B, Singh P (2019) Study of media optimization and kinetic modeling of l-methioninase from Pseudomonas stutzeri. Vegetos 32(3):370–380

Kim HO, Lim JM, Joo JH, Kim SW, Hwang HJ, Choi JW, Yun JW (2005) Optimization of submerged culture condition for the production of mycelial biomass and exopolysaccharides by Agrocybe cylindracea. Bioresour Technol 96(10):1175–1182

Kreis W, Hession C (1973) Isolation and purification of l-methionine-α-deamino-γ-mercaptomethane-lyase (l-methioninase) from Clostridium sporogenes. Can Res 33(8):1862–1865

Kudou D, Misaki S, Yamashita M, Tamura T, Takakura T, Yoshioka T, Yagi S, Hoffman RM, Takimoto A, Esaki N, Inagaki K (2007) Structure of the antitumour enzyme l-methionine γ-lyase from Pseudomonas putida at 1.8 Å resolution. J Biochem 141(4):535–544

Kumar RS, Ananthan G, Prabhu AS (2014) Optimization of medium composition for alkaline protease production by Marinobacter sp. GA CAS9 using response surface methodology—a statistical approach. Biocatal Agric Biotechnol 3(2):191–197

Lalucat J, Bennasar A, Bosch R, Garcıa-Valdes E, Norberto J (2006) Palleroni biology of Pseudomonas stutzeri. Microbiol Mol Biol Rev 70(2):510–547

Li XY, Liu ZQ, Chi ZM (2008) Production of phytase by a marine yeast Kodamaea ohmeri BG3 in an oats medium: optimization by response surface methodology. Bioresour Technol 99(14):6386–6390

Malinowska E, Krzyczkowski W, Łapienis G, Herold F (2009) Improved simultaneous production of mycelial biomass and polysaccharides by submerged culture of Hericium erinaceum: optimization using a central composite rotatable design (CCRD). J Ind Microbiol Biotechnol 36(12):1513–1527

Morales-Gonzalez M, Martinez BS, Ramirez-Rodriguez L, Gomez JEC, Diaz LE (2018) Optimization of l-asparaginase activity of actinobacteria isolated from Guaviare river sediments in Colombia. Trop J Pharm Res 17(11):2199–2206

Najafi AR, Rahimpour MR, Jahanmiri AH, Roostaazad R, Arabian D, Ghobadi Z (2010) Enhancing biosurfactant production from an indigenous strain of Bacillus mycoides by optimizing the growth conditions using a response surface methodology. Chem Eng J 163(3):188–194

Nakayama T, Esaki N, Tanaka H, Soda K (1988a) Chemical modification of cysteine residues of l-methionine γ-lyase. Agric Biol Chem 52(1):177–183

Nakayama T, Esaki N, Tanaka H, Soda K (1988b) Specific labeling of the essential cysteine residue of l-methionine. Gamma-lyase with a cofactor analog, N-(bromoacetyl) pyridoxamine phosphate. Biochemistry 27(5):1587–1591

Nikulin A, Revtovich S, Morozova E, Nevskaya N, Nikonov S, Garber M, Demidkina T (2008) High-resolution structure of methionine γ-lyase from Citrobacter freundii. Acta Crystallogr D Biol Crystallogr 64(2):211–218

Oskouie SFG, Tabandeh F, Yakhchali B, Eftekhar F (2008) Response surface optimization of medium composition for alkaline protease production by Bacillus clausii. Biochem Eng J 39(1):37–42

Pinnamaneni N, Funderburgh JL (2012) Concise review: stem cells in the corneal stroma. Stem Cells 30(6):1059–1063

Pradhan B, Dash SK, Sahoo S (2013) Screening and characterization of extracellular l-asparaginase producing Bacillus subtilis strain hswx88, isolated from Taptapani hotspring of Odisha, India. Asian Pac J Trop Biomed 3(12):936–941

Ranalli G, Alfano G, Belli C, Lustrato G, Colombini MP, Bonaduce I, Zanardini E, Abbruscato P, Cappitelli F, Sorlini C (2005) Biotechnology applied to cultural heritage: biorestoration of frescoes using viable bacterial cells and enzymes. J Appl Microbiol 98:73–83

Rao CS, Sathish T, Ravichandra P, Prakasham RS (2009) Characterization of thermo-and detergent stable serine protease from isolated Bacillus circulans and evaluation of eco-friendly applications. Process Biochem 44(3):262–268

Sato D, Yamagata W, Harada S, Nozaki T (2008) Kinetic characterization of methionine γ- lyases from the enteric protozoan parasite Entamoeba histolytica against physiological substrates and trifluoromethionine, a promising lead compound against amoebiasis. FEBS J 275(3):548–560

Sharma B, Singh S, Kanwar SS (2014) l-Methioninase: a therapeutic enzyme to treatmalignancies. Biomed Res Int. https://doi.org/10.1155/2014/506287

Singh P, Shera SS, Banik J, Banik RM (2013) Optimization of cultural conditions using response surface methodology versus artificial neural network and modeling of l-glutaminase production by Bacillus cereus MTCC 1305. Bioresour Technol 137:261–269

Tan Y, Sun X, Xu M, An Z, Tan X, Tan X, Hoffman RM (1998) Polyethylene glycol conjugation of recombinant methioninase for cancer therapy. Protein Expr Purif 12(1):45–52

Tanaka H, Esaki N, Soda K (1985) A versatile bacterial enzyme: l-Methionine γ-lyase. Enzyme Microb Technol 7(11):530–537

Thompson J, Morrison G (1951) Determination of organic nitrogen. Control of variables in the use of Nessler’s reagent. Anal Chem 23(8):1153–1157

Vatankhah E, Semnani D, Prabhakaran MP, Tadayon M, Razavi S, Ramakrishna S (2014) Artificial neural network for modeling the elastic modulus of electrospun polycaprolactone/gelatin scaffolds. Acta Biomater 10(2):709–721



The authors would like to thank Department of Bioscience and Biotechnology, Banasthali Vidyapith, Rajasthan, India, to provide lab facility and research support to carry out this research work.

Author Information

Kharayat Bhawana
Department of Bioscience and Biotechnology, Banasthali Vidyapith, Tonk, India

Singh Priyanka
Advance Science and Technology, NIET, NIMS, Jaipur, India

Shera Shailendra Singh
School of Biochemical Engineering, Indian Institute of Technology (BHU), Varanasi, India

Banik Rathindra Mohan
School of Biochemical Engineering, Indian Institute of Technology (BHU), Varanasi, India