Determination of the initial rate of enzymatic reactions in soils

Yıl 2022, Cilt: 10 Sayı: 2, 177 - 185, 27.12.2022

Fariz Mikailsoy , Erhan Erdel

https://doi.org/10.33409/tbbbd.1200435

Öz

The enzymatic activity of the soil can be used as a diagnostic indicator of soil fertility and quality. In order to understand this feature of enzymes, the kinetics of enzymes should be examined. As is known, the practical determination of the reaction rate in kinetic studies is carried out on the basis of determining the initial rate of the enzymatic reaction.In the study, analysis were conducted with different concentrations (3, 6, 9, 12, 15, 18, 21, 24, 30 %) of H2O2 to calculate kinetic parameters (Vmax, KM, Vmax/KM, KSEE, [S]opt ve υ0,max) of catalase enzyme to calculate the kinetic parameters of the soil catalase enzyme (Vmax, KM, Vmax/KM, KSEE, [S]opt and υ0,max). In this regard, first the values for velocity (υ) were calculated by using the analysis results. Then, different models expressing the relation product – substrate: [P]=f([S]) were determined. Based the model selection criteria (R2, R2adj, σ, A, D, UII, AIC), the most proper model showing the relation [P]=f([S]) was determined. Initial velocity was calculated with the formula υ0=d([P])/dt│t=0 using the selected model. All calculations in the study were made with the STATISTICA-10 package program. Results showed that, it was determined that as the substrate (H2O2) concentration increased, the product (O2) decreased, and it was also observed that the substrate inhibited the catalase enzyme reaction. In order to determine the initial velocity, it was found that the most suitable model according to the model selection criteria was Binomial-1. The kinetic parameters were calculated using the initial rate values according to low and high concentration values of the substrate. In addition, the highest initial velocity (υ0,max =5.3508 ml O2/1 min 5g soil) was reached at the optimum value of the substrate (12.2957 %).

Anahtar Kelimeler

Soil, enzyme, catalase, kinetic parameters, initial rate

Topraklarda enzimatik reaksiyonların başlangıç hızının belirlenmesi

Öz

Toprağın enzimatik aktivitesi, toprakların verimliliğinin, kalitesinin tanısal bir göstergesi olarak kullanılabilir. Enzimlerin bu özelliğinin anlamlandırılması için enzimlerin kinetiğinin incelenmesi gerekmektedir. Bilindiği gibi, kinetik araştırmalarda reaksiyon hızının pratik olarak tespit edilmesi, enzimatik reaksiyonun başlangıç hızının belirlenmesi temelinde gerçekleştirilir. Çalışmada, toprağın katalaz enziminin kinetik parametrelerini (Vmax, KM, Vmax/KM, KSEE, [S]opt ve υ0,max) hesaplamak için H2O2 substratının farklı konsantrasyonlarında (3, 6, 9, 12, 15, 18, 21, 24, 30 %) analizler yapılmıştır. Bu parametrelerin hesaplanması için analiz sonuçları kullanılarak önce hız (υ) değerleri hesaplanmıştır. Sonra ürün – substrat: [P]=f([S]) bağıntısını ifade eden farklı modeller belirlenmiştir. Daha sonra model seçim kriterlerine (R2, R2adj, σ, A, D, UII, AIC) göre [P]=f([S]) bağıntısını yansıtan en uygun model belirlenmiştir. Belirlenen modele göre başlangıç hız: υ0=d([P])/dt│t=0 formulü ile hesaplanmıştır. Çalışmadaki tüm hesaplamalar STATISTICA-10 paket programı ile yapılmıştır. Araştırma sonuçlarında; substrat (H2O2) konsantrasyonu arttıkça, ürünün (O2’nin) azaldığı tespit edilmiş, substratın katalaz enzim reaksiyonunu engellediği (inhibe ettiği) gözlenmiştir. Başlangıç hızın belirlenmesi için, model seçim ölçütlerine göre en uygun modelin Binom-1 olduğu bulunmuştur. Substratın düşük ve yüksek konsantrasyon değerlerine karşılık gelen başlangıç hız değerleri kullanılarak kinetik parametreler hesaplanmıştır. Ayrıca, substratın optimum değerinde (12.2957 %) en yüksek başlangıç hıza (υ0,max =5.3508 ml O2/1 dak 5g toprak) ulaşılmıştır.

Anahtar Kelimeler

Toprak, enzim, katalaz, kinetik parametreler, başlangıç hız.

Kaynakça

• Aliev SA, Gadzhiyev DA, Mikaylov FD, 1981. Kinetic indexes of catalase activity in the main soil groups of Azerbaijan. Soviet Soil Science, 13, 29-35.
• Beck TH, 1971. Die messung der katalaseaktivitaet von Böden. Zeitschrift für Pflanzenernährung und Bodenkunde 130.1. 68-81(in German).
• Blake GR, 1965. Bulk density. In: Black, C.A. (ed.), Methods of Soil Analysis. Part II, American Society of Agronomy. Madison, Wisconsin, 374-390.
• Briggs GE, Haldane JBS, 1925.A note on the kinetics of enzyme action. Bio-chem J. 19(2): 338–339. https://doi.org/10.1042/bj0190338
• Cornish-Bowden A, 1976. Principles of Enzyme Kinetics. Butterworths, London and Mir Publishing, Moscow, 190 p.
• Edwards VH, 1970. The influence of high substrate concentration on microbial kinetics. Biotechnol Bioeng, 12, pp: 679–712. https://doi.org/10.1002/bit.260120504
• Gee GW, Bauder JW, 1986. Particle-size analysis. Methods of Soil Analysis. Part 1. Physical and Minerological Methods. Second Edition. Agronomy, 9, 383-441.
• Haldane JBS, 1930. Enzymes, London, Longmans Green, 28–53, 74–92.
• Hegyi G, Kardos J, Kovács M, Málnási-Csizmadia A, Nyitray L, Pál G, Radnai L, Reményi A, Venekei I, 2013. Introduction to practical biochemistry. Eötvös Loránd University.
• Keleti T, 1986. Basic Enzyme Kinetics, Akademiai Kiadb, Budapest.
• Khabirov IK, KuvatovYuG, 1990. Kinetics and Thermodynamics of the Hydrolysis Reaction of 14c-Labelled Carbamide in the Pre-Urals Soils, Soviet Soil Science, 22(8), 83 – 94.
• Khaziev FKh, Agafarova YaM, 1976. Michaelis Constants of Soil Ferments, Soviet Soil Science, 8, 149 – 157.
• Lineweaver H, Burk D, 1934. The determination of enzyme disso-ciation constants, J. Am Chem Soc., 56(3), 658 – 666. https://doi.org/10.1021/ja01318a036
• Mclean EO, 1982. Soil ph and lime requirement. Methods of soil analysis part 2. Chemical and microbiological properties second edition. Agronamy, 9, 199-224. https://doi.org/10.2134/agronmonogr9.2.2ed.c12
• Michaelis L, Menten ML, 1913. Die Kinetik der Invertinwirkung. Biochem. Z. 49 (352), 333–369.
• Mikailsoy FD, 2022. Influence of high substrate concentrations on catalase reaction in loam soil (Igdir Province, Turkey). I. Theoretical foundations of the kinetics of enzymatic soil reactions. Soil science and agrochemistry, 3, 60-86. https://doi.org/10.51886/1999-740Х_2022_3_60
• Panikov NS, Ksenzenko SM, 1982. Study of phosphohydrolase inhibition in sod-podzolic soil. Soviet Soil Science, 11, 43-49.
• Rhoades JD, 1983. Soluble salts. Methods of Soil Analysis: Part 2 Chemical and Microbiological Properties 9: 167-179. https://doi.org/10.2134/agronmonogr9.2.2ed.c10
• Tabatabai MA, Bremner JM, 1971. Michaelis constants of soil enzymes. Soil Biol. Biochem., 3(4), 317-323. https://doi.org/10.1016/0038-0717(71)90041-1
• Tazdaït D, Abdi N, Grib H, Lounici H, Pauss A, Mameri N, 2013. Comparison of different models of substrate inhibition in aerobic batch biodegradation of malathion. Turk. J. Eng. Environ. Sci., 37, 221–230. https://doi.org/10.3906/muh-1211-7
• Tusat E, Mikailsoy F, 2018. An investigation of the criteria used to select the polynomial models employed in local GNSS/leveling geoid determination studies. Arab J Geosci 11, 801. https://doi.org/10.1007/s12517-018-4176-0
• Walkley A, Black LA, 1934. An examination of the degtjareff method for determining soil organic matter, and a proposed modification of the chromic acid titration method. Soil Science, 37 (1), 29–38. doi: https://doi.org/10.1097/00010694-193401000-00003.
• Williams VR, Williams HB, 1973. Basic physical chemistry for the life sciences. Second Edition. Freeman and Company, San Francisco, 524.
• Yano T, Nakahara T, Kamiyama S, Yamada K, 1966. Kinetic studies on microbial activities in concentrated solutions. I. Effect of excess sugars on oxygen uptake rate of a cell-free respiratory system. Agricultural and Biological Chemistry, 30, 43-48. https://doi.org/10.1080/00021369.1966.10858549
• Yano D, Suzuki T, 2018. Kinetic Analyses of the Substrate Inhibition of Paramecium Arginine Kinase. Protein J 37, 581–588. https://doi.org/10.1007/s10930-018-9798-2
• Yun Kİ, Han TS, 2020. Relationship between enzyme concentration and Michaelis constant in enzyme assays, Biochimie, 176: 12–20. https://doi.org/10.1016/j.biochi.2020.06.002