Enterobacter cloacae kompleks sp. V1 suşu tarafından üretilen L-asparaginaz enziminin aktivitesi üzerine ortam bileşiminin etkisi

Year 2020, Volume: 77 Issue: 1, 59 - 68, 01.03.2020

Erdal Öğün

Abstract

Amaç: Bu araştırmada, Van Gölü Havzasından toplanan toprak örneklerinden izole edilen bakteri izolatlarının, L-asparaginaz enzimini üretme kabiliyetleri ve L-asparaginaz enziminin aktivitesi üzerine ortam bileşiminin etkisinin araştırılması amaçlanmıştır. Ayrıca, L-asparaginaz enzimi yönünden pozitif izolatların, fenotipik ve 16S rDNA diziliş analizine dayalı olarak bakteriyel taksonomideki yerlerinin belirlenmesi hedeflenmiştir.Yöntem:Toprak örneklerinden bakterilerin izolasyonunda ve kültüre alınmasında Luria-Bertani LB Agar besiyeri kullanılmıştır. L-asparaginaz üreten izolatların taranmasında %0.5 oranında L-asparagin ve %0,001 oranında fenol kırmızısı bulunan M9 minimal tuz ortamı kullanılmıştır. Pozitif izolatlar tarafından üretilen, enzimin aktivitesi 436 nm’de spektrofotometrik yöntemle belirlenmiştir. L-asparaginaz üreten izolatın teşhisi, standart mikrobiyolojik yöntemler ve 16S rDNA diziliş analizine göre yapılmıştır.Bulgular: Toplam 10 izolat arasında sadece birinin medium containing L-asparagine and phenol red. The L-asparaginase enzyme produced by this isolate was incubated at a temperature of 35 ºC 16.02 IU/mL at a pH of 8.0 15.25 IU/mL for a 48 hour incubation period 16.02 IU / mL, in the presence of mannitol as a carbon source 15.69 IU/mL, in the presence of yeast extract 14.55 IU/mL and arginine amino acid 17.63 IU/mL as a source of organic nitrogen. Morphological, physiological and biochemical properties of this isolate were determined. In addition, L-asparaginase positive isolate was found to belong to E. cloacae complex strain according to 16S rDNA sequencing analysis and its status in bacterial taxonomy was determined.Conclusion: This research; In our country, it is one of the first studies about screening of L-asparaginase producing bacteria isolated from nature. In the treatment of ALL, L-asparaginase E. cloacae complex sp. V1 strain, positive for the antineoplastic L-asparaginase enzyme is extremely important for our national gene sources. This strain has been found to have an activity that could potentially be used in the industrial production of the enzyme L-asparaginase.L-asparagin ve fenol kırmızısı içeren M9 minimal tuz ortamında aşikar bir şekilde L-asparaginaz yönünden pozitif olduğu gözlenmiştir. Bu izolatın ürettiği L-asparaginaz enziminin 48 saatlik inkübasyon aralığında 16,02 IU/mL, 35 ºC sıcaklıkta 16,02 IU/mL, pH 8,0 değerinde 15,25 IU/mL, karbon kaynağı olarak mannitol varlığında 15,69 IU/mL, organik azot kaynağı olarak yeast ekstrat varlığında 14,55 IU/mL ve arginin amino asitinin varlığında 17,63 IU/mL aktivite oluşturduğu tespit edilmiştir. Ayrıca bu izolatın morfolojik, fizyolojik ve biyokimyasal özellikleri belirlenmiştir. İlave olarak L-asparaginaz pozitif izolatın, 16S rDNA diziliş analizine göre Enterobacter cloacae kompleks suşuna mensup olduğu tespit edilerek bakteriyel taksonomideki durumu belirlenmiştir.Sonuç: Bu araştırma; ülkemizde doğadan izole edilen ve L-asparaginaz üreten bakterilerin taranması ile ilgili ilk araştırmalar arasındadır. ALL tedavisinde kullanılan, L-asparaginaz enziminin E. cloacae kompleks sp. V1 suşunun, antineoplastik L-asparaginaz enzimi yönünden pozitif bulunmas, milli gen kaynaklarımız açısından son derece önemlidir. Bu suşun, L-asparaginaz enziminin endüstriyel üretiminde potansiyel olarak kullanılabilecek bir aktiviteye sahip olduğu tespit edilmiştir

Keywords

Enterobacter cloacae kompleks, L-asparaginaz, Van Gölü Havzası

Effect of media composition on the activity of L-asparaginase enzyme produced by Enterobacter cloacae complex sp. V1 strain

Abstract

Objective: In this study, it was aimed to investigate the effect of medium composition on the ability of producing L-asparaginase enzyme and the activity of L-asparaginase enzyme isolated from soil samples collected from Van Lake Basin. In addition, it was aimed to determine the location of L-asparaginase positive isolates in bacterial taxonomy based on phenotypic and 16S rDNA sequencing analysis.Methods: Luria-Bertani LB Agar medium was used to isolate and cultivate bacteria from soil samples. L-asparaginase-producing isolates were screened for M9 minimal salt medium with 0.5% L-asparagine and 0.001% phenol red. The activity of the enzyme produced by positive isolates was determined by spectrophotometric method at 436 nm. The identification of the L-asparaginase producing isolate was performed according to standard microbiological methods and 16S rDNA sequencing analysis.Results: Of the 10 isolates, only one was clearly positive for L-asparaginase in the M9 minimal salt ortamda L-asparagin amino asitini L-aspartat ve L-asparaginaz EC3.5.1.1 enzimi, ekstraselüler medium containing L-asparagine and phenol red. The L-asparaginase enzyme produced by this isolate was incubated at a temperature of 35 ºC 16.02 IU/mL at a pH of 8.0 15.25 IU/mL for a 48 hour incubation period 16.02 IU / mL, in the presence of mannitol as a carbon source 15.69 IU/mL, in the presence of yeast extract 14.55 IU/mL and arginine amino acid 17.63 IU/mL as a source of organic nitrogen. Morphological, physiological and biochemical properties of this isolate were determined. In addition, L-asparaginase positive isolate was found to belong to E. cloacae complex strain according to 16S rDNA sequencing analysis and its status in bacterial taxonomy was determined.Conclusion: This research; In our country, it is one of the first studies about screening of L-asparaginase producing bacteria isolated from nature. In the treatment of ALL, L-asparaginase E. cloacae complex sp. V1 strain, positive for the antineoplastic L-asparaginase enzyme is extremely important for our national gene sources. This strain has been found to have an activity that could potentially be used in the industrial production of the enzyme L-asparaginase

Keywords

Enterobacter cloacae complex, L-asparaginase, Lake Van Basin

References

• 1. Shrivastava A, Khan AA, Khurshid M, Kalam, MA, Jain SK, Singhal PK. Recent developments in L-asparaginase discovery and its potential as anticancer agent. Crit Rev Oncol Hematol, 2016; 100(4): 1-10.
• 2. Sanawer S, Ali S, Mohsin T, Nasir A. Production, purification and advance applications of L-asparaginase. IJSRST, 2017; 3 (4): 351.
• 3. Dearfield KL, Abernathy CO, Ottley MS, Brantner JH, Hayes PF. Acrylamide: its metabolism, developmental and reproductive effects, genotoxicity, and carcinogenicity. Mutat Res Genet Toxicol Environ Mutagen, 1988; 195(1): 45-77.
• 4. Mahajan RV, Mihooliya KN, Saran S, Saxena RK. L-asparaginase from Bacillus sp. RKS-20: process optimization and application in the inhibition of acrylamide formation in fried foods. Proteins and Proteomics, 2014; 5(2): 15-132.
• 5. Pieters R, Hunger SP, Boos J, Rizzari C, Silverman L, Baruchel et al. L-asparaginase treatment in acute lymphoblastic leukemia. Cancer, 2011; 117(2), 238-49.
• 6. Aydın S, Geçkil H, Çaylak E, Kılıç N. Mikroorganizmaların kanser tedavisinde kullanımı. Fırat Tıp Derg, 2004; 9(2): 30-4.
• 7. Yadav S, Verma SK, Singh J, Kumar A. Industrial production and clinical application of L-asparaginase: a chemotherapeutic agent. IJMRPS, 2014; 8(1): 54-60.
• 8. Resnick AD, Magasanik B. L-asparaginase of Klebsiella aerogenes. Activation of its synthesis by glutamine synthetase. J Biol Chem, 1976; 251(9): 2722-8.
• 9. Mukherjee J, Joeris K, Riechel P, Scheper T. A simple method for the isolation and purification of L-asparaginase from Enterobacter aerogenes. Folia Microbiol, 1999; 44(1): 15-8.
• 10. Geckil H, Gencer S. Production of L-asparaginase in Enterobacter aerogenes expressing Vitreoscilla hemoglobin for efficient oxygen uptake. Appl Microbiol Biotechnol, 2004; 63(6): 691-7.
• 11. Baskar G, Muthukumaran C, Viruthagiri T, Reganathan S. Optimization of operating conditions for the production of L-asparaginase by Enterobacter aerogenes MTCC 2823 using central composite design. IJBST, 2009; 2(2): 32-6.
• 12. Erva RR, Goswami AN, Suman P, Vedanabhatla R, Rajulapati SB. Optimization of L-asparaginase production from novel Enterobacter sp., by submerged fermentation using response surface methodology. Prep Biochem Biotechnol , 2017; 47(3): 219-28.
• 13. Sharma A, Husain I. Optimization of medium components for extracellular glutaminase free asparaginase from Enterobacter cloacae. Int J Curr Microbiol App Sci, 2015; 4(1): 296-309.
• 14. Mezzatesta ML, Gona F, Stefani S Enterobacter cloacae complex: clinical impact and emerging antibiotic resistance. Future Microbiol, 2012; 7(7): 887-902.
• 15. Goldman E, Green, LH. Practical handbook of microbiology. In Practical Handbook of Microbiology, Third Ed. CRC Pres, 2015.
• 16. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
• 17. Gulati R, Saxena RK, Gupta R. A rapid plate assay for screening L-asparaginase producing microorganisms. J Appl Microbiol, 1997; 24(1), 23-6.
• 18. Bisswanger H. Enzyme assays. Perspectives in Science, 2014; 1(1-6): 41-55.
• 19. Imada A, Igarasi S, Nakahama K, Isono M. Asparaginase and glutaminase activities of microorganisms. Microbiology, 1973; 76(1), 85-99.
• 20. Brenner DJ, Farmer III JJ. Family I. Enterobacteriaceae. In: Brenner DJ, Krieg NR, Staley JT, Garrity GM, Bone DR, Vos P, et al, eds. Bergey’s Manual of Systematic Bacteriology, 2nd edn, New York, NY: Springer, 2005: 587–607.
• 21. Green MR, Sambrook J. Isolating DNA from Gramnegative bacteria. 2017 Cold Spring Harb Protoc; 2017; (3) : 2017(1).
• 22. Nikiforov YE, Howles PN. Polymerase chain reaction. In Ricardo VL, eds. Morphology Methods: Cell and Molecular Biology Techniques. New York: Humana Press, 2001:181-207.
• 23. Posada, D. jModelTest: phylogenetic model averaging. Molecular biology and evolution, 2008; 25(7): 1253-56.
• 24. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S. et al. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol, 2012; 61(3): 539-42.
• 25. Shukla D, Shrivastav, VK, Jana AM, Shrivastav A. Exploration of the potential L-asparaginase producing bacteria from the soil of Gwalior (India). Int J Curr Microbiol Appl Sci, 2014; 3(5): 665-72.
• 26. Prasad Talluri VSSL, Bhavana M, Siva Kumar K, Anil Kumar P, Rajagopal, SV. Myroides gitamensis sp. nov., L-asparaginase producing bacteria isolated from slaughter house soil sample in Visakhapatnam India. J Microb Biochem Technol, 2014; 6(3): 144- 7.
• 27. Singh Y, Gundampati RK, Jagannadham MV, Srivastava SK. Extracellular L-asparaginase from a protease-deficient Bacillus aryabhattai ITBHU02: purification, biochemical characterization, and evaluation of antineoplastic activity in vitro. Appl Biochem Biotechnol, 2013; 171(7): 1759-74.
• 28. Wakil SS, Adelegan AA. Screening, production and optimization of L-asparaginase from soil bacteria isolated in Ibadan, South-western Nigeria. Aust. J. Basic Appl Sci, 2015; 11: 39-51.
• 29. Reddy ER, Babu RS, Chandrasai PD, Madhuri P. Neural network modeling and genetic algorithm optimization strategy for the production of L-asparaginase from Novel Enterobacter sp. Int J Pharm Sci Res, 2017; 9(2): 124-130.
• 30. Shaheduzzaman M, Rahman MS, Nur IT. (2016). Influence of temperature on the growth of fecal coliform. SJ Microbiol, 2016; 6(1): 20-3.
• 31. Grimont PA, Grimont F. Enterobacter, In: Brenner LDJ, Krieg NR, Staley JT, Garrity GM (eds) Bergey’s manual of systematic bacteriology, 2nd edn, vol. 2, Springer, USA, 2005: 661-670.
• 32. Paauw A, Caspers MP, Schuren FH, Leverstein-van Hall MA, Delétoile A, Montijn RC. Genomic diversity within the Enterobacter cloacae complex. PLoS one, 2008; 3(8): e3018.