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Study on Enzymatic Properties of Feeding Xylanase
Xylanase is a group of enzymes capable of breaking down xylan into oligosaccharides and xylose. It includes exo-β-1,4-xylanase, endo-β-1,4-xylanase, and β-xylosidase [1]. These enzymes have broad applications in various industries such as feed, food, paper, textiles, medicine, and energy. In the feed industry, xylanase helps eliminate the anti-nutritional effects of xylan, improving the digestion and absorption of roughage by livestock and poultry. As a pulp bleaching agent, it reduces the need for chlorine, thus addressing environmental pollution issues in the paper industry. Additionally, xylanase breaks down hemicellulose in plants, producing pentose sugars that are used in the production of xylitol, alcohol, and organic acids, playing a key role in the biotransformation of renewable resources [2].
In the early stages of laboratory research, an efficient xylan-degrading strain was isolated [3]. This experiment focused on studying the enzymatic properties of xylanase produced through solid-state fermentation, including optimal pH and pH stability, optimal reaction temperature, thermal stability, and resistance to pepsin and trypsin. The goal was to gain a deeper understanding of the enzyme's characteristics and provide guidance for its practical application.
**1 Materials and Methods**
1.1 Xylanase was obtained through solid-state fermentation in our laboratory.
1.2 Reagents
Oat xylan (X0627), pepsin (P7000), and trypsin (T1005) were purchased from Sigma. The DNS reagent was prepared according to the method described in GB/T 23874-2009. All other reagents were of analytical grade.
1.3 Enzyme Activity Determination
Enzyme activity was measured using the DNS spectrophotometric method, following the procedure outlined in GB/T 23874-2009. One unit of enzyme activity is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute from a 5 mg/mL xylan solution at 37°C and pH 5.5.
**2 Experimental Design**
2.1 Optimal pH for Xylanase Reaction
Buffers with different pH values (2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0) were prepared using 0.1 mol/L sodium dihydrogen phosphate-citrate buffer for pH ≤ 8.0, and glycine-sodium hydroxide buffer for pH > 8.0. The xylan substrate was prepared accordingly, and enzyme activity was measured according to national standards. The highest activity was set as 100%, and relative activities were calculated based on this value.
2.2 pH Stability of Xylanase
Equal volumes of buffers with varying pH (2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0) were mixed and incubated at 37°C for 4 hours. After cooling in an ice bath, enzyme activity was measured. The highest activity was considered 100%.
2.3 Optimal Reaction Temperature
Using the optimal pH buffer, the reaction was carried out at temperatures of 30, 40, 45, 50, 55, 60, 65, and 70°C. Enzyme activity was measured according to the standard method, and the temperature yielding the highest activity was identified as the optimal reaction temperature.
2.4 Thermal Stability of Xylanase
The enzyme was treated at 50, 55, and 60°C for 10, 20, 40, 60, 80, 100, and 120 minutes. After treatment, enzyme activity was determined using the standard method. The untreated enzyme served as the reference, with its activity set at 100%.
2.5 Effect of Pepsin and Trypsin on Xylanase Activity
A 0.5 mL xylanase solution was incubated with 0.5 mL of 0.01 mg/mL pepsin (prepared in 0.01 mol/L HCl at pH 2.0) or 0.5 mL of 0.2 mg/mL trypsin (prepared in potassium phosphate monobasic buffer at pH 7.0). The mixtures were incubated at 37°C for 30, 60, and 90 minutes. Enzyme activity was then measured according to the standard method, with the untreated sample serving as the control (100% activity).