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Xylanases are key enzymes for the degradation of arabinoxylans, the major non-starch polysaccharides of cereal cell walls. Due to the heterogeneity of arabinoxylans, xylanases with different properties are required for industrial applications. In this work, xylanase was produced by cultivation of Thermomyces lanuginosus on barley husk in solid-state fermentation conditions (ms = 30 g, nmycelial discs = 5, ϕmycelial discs = 1 cm, wH2O = 70 %, t = 8 days, T = 45 °C). 80 g of the fermented sample was extracted in 650 mL of the phosphate buffer (pH 6.0, 0.1 M) during 60 min at 450 rpm to obtain xylanase-rich extract. To find optimal pH and T, activity of xylanase was measured at a pH range from pH 3.0 to pH 8.0 in two different buffers (citrate and phosphate buffer) and at temperature range from 30 - 90 ºC in phosphate buffer (pH 6.0, 0.1 M) using arabinoxylan as a substrate. The highest enzyme activity at pH 6.0 and T = 65 ºC was found. The hydrolysis of arabinoxylan by a xylanase-rich crude extract was studied in different types of reactors. First, it was performed in a 12 mL batch reactor (Vreaction mixture = 3 mL, t = 30 min, T = 65 ºC, n = 250 rpm, c (arabinoxylan) = 10 mg mL-1 ; V.A. (xylanase) = 8500 U mL-1). Samples were taken at regular intervals and the concentration of reducing sugars was measured spectrophotometrically using the DNS method. The reaction was then studied in two types of microreactors: a) Y-shaped microreactor (V = 88 μL) and b) microreactor with micromixers (V = 20.85 μL). Xylanase was fed through one inlet, whereas arabinoxylan was fed through the other inlet by syringes placed on pumps and connected to the microreactors with PTFE tubing. All experiments were performed at 65 °C by immersing the microreactor in a thermostatic water bath. Different residence times (constant flow rates from 1.6 to 19.2 µL min-1) were tested. It was found that the maximum concentration of reducing sugars released in the batch reactor was 2.07 mg mL-1 after 30 minutes. A slightly higher concentration of reducing sugars (2.55 mg mL-1) was obtained in a microreactor at a residence time of 27.50 minutes. However, when the reaction took place in a microreactor with micromixers, only 13.03 minutes were required to achieve reducing sugars concentration of 3.37 mg mL-1. The results are presented at Figure 2. At the end, 4 reactions on a single chip comprised of 4 microreactors with micromixers were performed simultaneously. HPLC analysis of simple sugars was done for all performed experiments. It was shown that arabinose was the main liberated sugar after arabinoxylan hydrolysis with xylanase. The maximum concentration of arabinose liberated in the batch reactor after 30 min was 0.304 mg mL-1. After intensification of the substrate hydrolysis by microreactor with micromixer, arabinose concentration was increased for 1.8-fold after 13.03 minutes reaching the value of 0.548 mg mL-1. |
