تبدیل زیستی دیادزین به 3ʹ-ODI توسط تیروزیناز تثبیت شده در سطح اسپور

Introduction

Daidzein (4′, 7-dihydroxyisoflavone) is a natural isoflavone and a bioactive compound isolated from soybean (1). The protective effects of daidzein against various diseases such as breast and prostate cancer, diabetes and cardiovascular diseases have been investigated. The pharmaceutical mechanism of daidzein is because of the structural similarities to mammalian estrogens. Daidzein has antiproliferative effects and it is an antiangiogenesis compound which plays a remarkable role in the inhibition of the migration of cancer cells to prevent metastasis (2). Phenolic compounds, especially ortho-dihydroxylated phenolic compounds, have the potential to prevent cancer (3). 3ʹ-ODI (7, 3ʹ, 4ʹ-trihydroxyisoflavone) is one of the ortho-dihydroxylated isoflavones which is not present naturally (4). Anti-oxidative and anti-cancer effects of 3ʹ-ODI have been investigated in several researches (5, 6). CytP450 and tyrosinase were used for the bioconversion of daidzein, to 7, 3ʹ, 4ʹ-trihydroxyisoflavone (3ʹ-ODI) to increase the antioxidant effects (7). Cyt P450 can selectively introduce atomic oxygen into non-activated carbon-hydrogen bonds; however, the utilization of Cyt P450 in industry, because of their low efficiencies, low yields, and necessity for cofactor has several problems (8). Tyrosinase, a type III copper containing monoxygenase, is an applicable enzyme in cosmetic, drug development and pharmaceutical industries and also it has been used in variety of biotechnological applications such as bioremediation, biosensor, biomedicine, and biocatalysis (9, 10).

The most important reaction organized by tyrosinase is the bioconversion of L- tyrosine to L- DOPA (3,4-di hydroxy phenylalanine) which is a drug for Parkinson disease (11). Unlike the Cyt P450, tyrosinase is simply used in industry with high efficiencies and outputs (8). Due to the high utilization of the enzyme, it is necessary to immobilize the enzyme on appropriate support for the efficient usage (12). Spore surface display technique is a developmental genetic engineering method to express and immobilize proteins on the surface of Bacillus subtilis spores (13). According to the spore resistance to heat shocks, pH changes and UV radiation, genetically immobilized protein can be used in such extreme conditions. The spore surface display technique has been used to develop vaccines and biosensors successfully (14, 15).

In this study, we used the spore displayed tyrosinase as a stable and reusable enzyme for synthesis of 3ʹ-ODI from daidzein.

Materials and Methods

Bacterial Strain and Culture Condition: Bacillus subtilis DB104 (pSDJH-cotE-tyr) which was constructed in our previous study was used as an enzyme source. To collect the spores, vegetative cells were inoculated into Difco sporolation medium (DSM) and cultivated for 24h at 37°C in a shaker incubator (200rpm). This medium contained 0.8% w/v nutrient broth, 0.1% KCl, 0.025% MgSO₄.7H₂O, 1mM Ca(NO₃)₂, 0.01mM MnCl₂, and 0.01mM FeSO₄ in 1L distillated water, pH 7. The spores were then purified using renografin (sodium diatrizoate, S-4506, Sigma) gradient method (16).

Bioconversion Assay of Diadzein to 3ʹ-ODI: The purified spores (4 * 10⁸) were used as an enzyme source for the bioconversion of daidzein to 3ʹ-ODI. The reaction of 1mM daidzein (D7802 Sigma) was done in 50mM tris- HCl buffer pH 8 with 5mM ascorbic acid and 10µM CuSO₄ in 37˚C for 1 hour. HPLC (YL9100, Young Lin instrument) was used to monitor the concentrations of substrates and products. The experiments were repeated in triplicate.

Reusability of the Enzyme: To assay the enzyme reusability, the spores were washed three times with Tris-HCl buffer (pH 8), and the formation of the product was monitored by HPLC again. The HPLC graphs were analyzed by ChromNAV 2.0 HPLC software.

Results

Bioconversion of Daidzein to 3ʹ-ODI by Spore Displayed Tyrosinase:Bioconversion of daidzein to 3ʹ-ODI by spore displayed tyrosinase was monitored by HPLC. As it is shown in figure 1, after 60 min, almost all content of diadzein were converted to 3’-ODI. Tyrosinase transferred one hydroxyl group on daidzein to create 3ʹ-ODI (Fig. 1).

Reusability of Spore Displayed Tyrosinase: The reusability of tyrosinase-displaying spores was also investigated by washing them repeatedly with a buffer. Retained activity of immobilized tyrosinase was about 58% after three times washing with Tris-HCl buffer, pH 8 (Fig. 2).

Fig. 1- HPLC result of the spore displayed tyrosinase reaction for producing 3ʹ-ODI. (a) The peaks at retention time 12.5 min and 7.5 min are related to daidzein and 3ʹ-ODI, respectively. (b) Time profile of daidzein consumption and 3ʹ-ODI production.

Fig. 2- HPLC result of the reusability of spore displayed tyrosinase after three times washing