Characterization of 36-kDa Dimeric Methotrexate-degrading Enzyme from Variovorax paradoxus, its Performance and Stability of its Cross-linked Aggregates

IF 1 4区生物学 Q4 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Applied Biochemistry and Microbiology Pub Date : 2023-08-12 DOI:10.1134/S0003683823040129

C. K. Sahu, V. K. Bayineni, J. S. Hampapura, C. M. Hussain, R.-K. Kadeppagari

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Abstract

In the present study, methotrexate (MTX)-degrading 36-kDa dimer was characterized from the 0–80% ammonium sulphate precipitate of Variovorax paradoxus cell lysate. This enzyme showed optimum activity at 35°C and pH 6.5 with K_M value of 199.6 µM for MTX which are different from those of 46-kDa dimer. Micro- or nano-sized cross-linked aggregates of new enzyme showed better stability in the presence of serum than the native soluble form. Addition of an endogenic carrier protein, human serum albumin (HSA) to the enzyme aggregates further improved their serum stability. Nanoaggregates showed better serum stability over microaggregates. Nanoaggregates of the enzyme degraded the MTX faster than soluble form and microaggregates due to their lower K_M values on MTX, whereas microaggregates were slower than soluble enzyme itself due to their higher K_M values. Hence, cross-linked aggregates of MTX- degrading enzyme isolated from V. paradoxus shown better performance in their nanoform compared to the microform. Nanoaggregates of enzyme revealed the highest functionality and serum stability that makes them more suitable for therapeutic applications.

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36-kDa二聚甲氨蝶呤降解酶的表征、性能及其交联聚集体的稳定性

在本研究中，从异卵弧菌细胞裂解液的0-80%硫酸铵沉淀中表征了甲氨蝶呤(MTX)降解36-kDa二聚体。该酶在35°C和pH 6.5条件下对MTX具有最佳活性，KM值为199.6µM，与46-kDa二聚体不同。新酶的微或纳米级交联聚集体在血清中表现出比天然可溶性形式更好的稳定性。在酶聚合体中加入内源性载体蛋白——人血清白蛋白(HSA)进一步提高了其血清稳定性。纳米聚集体比微聚集体表现出更好的血清稳定性。由于其对MTX的KM值较低，酶的纳米团聚体降解MTX的速度比可溶性形式和微团聚体快，而微团聚体由于其较高的KM值而比可溶性酶本身慢。因此，从悖论弧菌中分离出的MTX-降解酶的交联聚集体在纳米形态下比在显微形态下表现出更好的性能。酶的纳米聚集体显示出最高的功能和血清稳定性，使它们更适合于治疗应用。

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来源期刊

Applied Biochemistry and Microbiology 生物-生物工程与应用微生物

CiteScore

1.70

自引率

12.50%

发文量

审稿时长

6-12 weeks

期刊介绍： Applied Biochemistry and Microbiology is an international peer reviewed journal that publishes original articles on biochemistry and microbiology that have or may have practical applications. The studies include: enzymes and mechanisms of enzymatic reactions, biosynthesis of low and high molecular physiologically active compounds; the studies of their structure and properties; biogenesis and pathways of their regulation; metabolism of producers of biologically active compounds, biocatalysis in organic synthesis, applied genetics of microorganisms, applied enzymology; protein and metabolic engineering, biochemical bases of phytoimmunity, applied aspects of biochemical and immunochemical analysis; biodegradation of xenobiotics; biosensors; biomedical research (without clinical studies). Along with experimental works, the journal publishes descriptions of novel research techniques and reviews on selected topics.