Supplementary Materialsijms-20-01112-s001. biotransformation regarding substrates and products of low water solubility. have been previously explained in detail [17]. In the scope of this work, the use of the crude, wild-type enzyme for the production of quercetin and rutinose has been tested. Besides the production of rutinosidase (day time 6; rutinosidase activity 0.31 U/mL), the co-production of -l-rhamnosidase (0.30 U/mL) and -d-glucosidase (0.49 U/mL) during fermentation were observed. This made the use of the crude enzyme unfeasible, mainly due to the parallel cleavage of rutinose and additional unwanted reactions. Moreover, the crude wild-type enzyme is not stable (probably due to the presence of proteases in the Verbenalinp medium), which was also shown in the checks with rutin bioconversion (observe Section 2.2.1). In summary, without enzyme purification, which is not feasible economically at a large level, the crude wild-type enzyme cannot be used for this process. 2.1.2. Production of Recombinant Rutinosidase from in KM71H was scaled up in 3-L laboratory bioreactors. Glycerol was depleted after approximately 20 h, during which the exponential growth of biomass was observed (Number 3). Then, two methanol pulses of 3 g/L were added at hour 21 and hour 32. Fed-batch methanol feeding was started at hour 35, according to the optimized protocol explained by [19]. During the fed-batch phase, methanol was given based on the actual degree of dissolved air rather than exceeded a focus of 3 g/L, which is normally toxic because of this MutS stress. The focus of methanol began to boost slightly by the end from the fermentation as well as the development of biomass steadied, which indicated which the biomass Verbenalinp reached the fixed stage as well as the fermentation was terminated. SDS-PAGE electrophoresis (Amount S1, Supplementary Materials) demonstrated a music group of approx. 66 kDa, representing the created rutinosidase. The lack of various other significant protein rings demonstrates the fantastic advantage of managed fermentation, which creates no various other extracellular proteins compared to the desired rutinosidase. The overall productivity of the produced rutinosidase was 5.69 mgprotL?1h?1. Open in a separate window Number 3 Fed-batch cultivation of expressing rutinosidase with methanol feeding according to the actual level of dissolved oxygen. Conditions: 1.5 L BSM, 5% inoculum, 30 C, pH 5.0, DO 20%, stirring cascade 50C1000 rpm; after 35 h fixed at 600 rpm. 2.2. Bioconversion of Rutin to Quercetin 2.2.1. Bioconversion of Rutin to Quercetin Using Wild-Type RutinosidaseThe 1st tests of a larger-scale conversion of rutin to quercetin were performed with the wild-type enzyme. The reaction was successfully accomplished at analytical level with the purified wild-type rutinosidase (up to 100 g/L rutin). However, for any practical large-scale biotechnological software protein purification is not economically feasible. Consequently we also tested crude rutinosidase (centrifuged medium from your cultivation of produced in has a temp optimum of 50 C at pH 3.5; temps exceeding 55 C resulted in a loss of activity [17]. The stability Verbenalinp of the recombinant (crude, dialyzed) enzyme at different pH and temps was tested. The enzyme loses virtually all activity in ca 2 h at 50 C. (Number S3a, Supplementary Data). Consequently a Rabbit Polyclonal to GR lower temp (40 C), where the enzyme has an activity of ca 60% compared to the optimum temp but is quite stable, was selected. Nevertheless, actually at 50 C the overall performance of rutinosidase during rutin bioconversion was very satisfactory. The complete conversion of rutin at a concentration of 200 g/L was accomplished within 5C6 h (Number S3b, Supplementary Material). It is obvious the enzyme in the presence of substrate is much more stable than in a mere buffer. The pH optimum of recombinant rutinosidase is at pH 3.0. At pH 5.0 the enzyme still maintains 50% of its maximum activity and at pH 2.0 and pH 7. 0 it is virtually inactive [17]. To support the above hypothesis the overall performance of the enzyme in a real reaction Verbenalinp system with a high rutin concentration (200 g/L) was.