engleski

Intensification of protein extraction in a microextractor - purification of lignocellulosedegrading enzymes produced during fungal-based solid-state fermentation on food industry by-products

In solid-state fermentation, microorganisms, mainly filamentous fungi, are cultivated on lignocellulosic substrates and synthesize a multienzyme complex that causes the degradation and modification of lignocellulosic structural units1. A large amount of lignocellulosic materials is generated in the food industry. For example, sugar beet pulp, a by-product from sugar industry, was used as substrate for cultivation of Trametes versicolor to produce endo-1, 4-xylanase, while brewer's spent grain, a by-product of brewing industry, was used as substrate to produce laccase. Both enzymes have been attracting increasing attention in recent decades due to their potential application in lignocellulosic biorefineries and circular bioeconomy. The enzyme endo-1, 4-xylanase, or xylanase in general, is used in feed production, food industry (bakery, fruit juice and beer clarification), leather processing, pulp and paper bleaching, and biofuel production from lignocellulosic biomass. The enzyme laccase is widely used in the textile, paper and pulp, and petroleum industries, as well as in bioremediation. After the enzyme is produced, and before it is used for industrial application, the enzyme must be purified. The purification process, which usually involves precipitation and separation on columns, is an expensive and time-consuming process. Nowadays, there is a growing interest in environmentally friendly extraction, and great efforts have been made to develop clean, sustainable, and efficient extraction processes. Aqueous two-phase systems (ATPSs) are considered as an effective, versatile and important green-technology for downstream processing of biomolecules. ATPSs are liquid- liquid systems formed when two incompatible polymers such as polyethylene glycol (PEG) and dextran are mixed. The ATPS can also be obtained by mixing certain polymers (e.g. PEG) with salts (e.g. Na2HPO4) or detergents, ionic liquids and short chain alcohols2. In this work, enzymes laccase and xylanase derived from Trametes versicolor were produced by solid-state fermentation. After solid-state fermentation was performed, the material was mixed and homogenized to obtain the crude enzyme extract. To optimize the extraction process, the extraction was firstly carried out with commercial enzymes. In the first phase, a suitable ATPS had to be selected. Thus, ATPSs based on polyethylene glycol (PEG1540) and various salts: sodium sulfate, sodium citrate dihydrate, sodium formate, potassium sodium tartrate, and ammonium sulfate were tested. Based on the experimental results obtained, the best ATPSs for the extraction were selected, namely, PEG1540–H2O–(NH4)2SO4 for laccase extraction, and PEG1540–H2O–C6H5Na3O7·2H2O for the extraction of xylanase. For both extraction processes an extraction efficiency of about 73% was obtained. To achieve higher extraction efficiency, optimization of xylanase and laccase extraction processes were performed using the experimental Box-Behnken design at three levels with three factors analyzing the influence of extraction time (t), protein concentration (γ) and mass content of polymers in the ATPS (wPEG) on the extraction efficiency (E) and purification factor (PF).The obtained results shows that the highest purification factor for extraction of laccase, PF = 2.52 (E = 59.76%), was achieved for following process conditions ; t = 15 min, γ = 1.6 mg/mL and wPEG = 0.179. On the other hand, the highest PF = 6.50 (E = 100%) for extraction of xylanase was obtained at following process conditions: t = 15 min, γ = 0.3 mg/mL, and wPEG = 0.210. After optimization, the process was performed at optimal process conditions in a microextractor where the influence of retention time, inlet strategy and channel diameter on extraction efficiency were additionally investigated. The highest PF = 6.61 (E = 100%) for xylanase extraction performed in a microextractor was achieved for residence time of τ = 1 min. When laccase extraction was performed in a microextractor, a PF = 6.42 (E = 86.02%) was achieved for τ = 8.61 min. In addition, a 2D mathematical model describing the extraction process in a microextractor was developed and successfully validated on independent set of experimental results. Furthermore, the developed batch and continuous extraction was applied on crude xylanase derived from Trametes versicolor produced by solid-state cultivation with the extraction efficiency of 73%.

aqueous two-phase system ; laccase ; xylanase ; microextractor ; process optimization ; solidstate fermentation

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