Three Phase Partitioning and Immobilization of Bacillus methylotrophicus Y37cellulase into organo-bentonite and its Kinetic and Thermodynamic Properties

Yonca Duman, A. Uğur Kaya, Çiğdem Yağci
2020 Clay minerals  
In this study Bacillus methylotrophicus Y37 cellulase was first time purified and recovered in single step by three phase partitioning (TPP). Optimal purification parameters for TPP were 40% ammonium sulfate saturation (m/v) with 1.0:1.0 (v/v) ratio of crude extract:t -butanol which gave 5.8 purification fold with 155% recovery of cellulase. Non-covalent immobilization of the partitioned cellulase was performed using bentonite as support material. The activity observed in the 20 th experiment
more » ... 20 th experiment was 100%. Optimum pH and temperatures determined for the free enzyme and the immobilized enzyme were 5.0 -6.0 and 45 °C − 50 °C, respectively. The Arrhenius activation energy (E a ) was decreased of the immobilized enzyme. The Michaelis-Menten constant (K m ) and maximum 2 velocity (V m ) obtained for the immobilized enzyme were increased. The turnover number (k cat ) and the catalytic performance (k cat /K m ) were showed catalytic properties of immobilized enzyme higher than free enzyme. It has been observed that immobilization of cellulase is thermodynamically preferred. INTRODUCTION Enzymes are naturally existing biocatalysts. As enzymes offer environmentally friendly processes, they are advantageous in industrial applications. The advantages of using enzymes are substrate specificity, absence of undesirable side reactions, mild conditions required for reactions, and noncontaminated product generation. Enzyme-based reactions tend to have lower waste treatment costs, which renders it possible to construct and control the facility using enzymatic reactions at much lower capital and energy cost (Patel et al., 2019; Hasan et al., 2006) . Cellulase is a multicomponent enzyme consisting of three different enzymes, namely, endocellulase, cellobiohydrolase, and β-glucosidase, which together catalyze the hydrolysis of cellulose, generating soluble sugars. Cellulase has become one of the most important catalysts due to its wide range of industrial applications in the fields of food bioconversion, agriculture, pulp and paper, and textiles. Cellulase is particularly crucial in the bio-refinery industry, where it is utilized for catalyzing cellulose into sugars. Purification of this enzyme by operating various routine purification methods such as salting out and various chromatographic techniques is a costly and time-consuming procedure (Duman & Kaya, 2013a). Among the bioseparation techniques; three-phase partitioning (TPP) is an alternative method due to it can be directly used for crude suspension and includes principles of salting out, isoionic precipitation, and cosolvent precipitation. As its rather simple and low-cost purification technique, TPP has been used to purify proteins, enzymes and inhibitors have use at last decades (Duman & Kaya, 2013b). It uses ammonium sulfate with certain saturation to precipitate the protein and t-3 butanol is added to make three phase layers and to remove lipids, phenolic compounds and some detergents (Rao et al., 1998) . Most biological processes occur in the presence of biocatalysts. The acquisition of biocatalysts is expensive, thus the overall economy of their use depends on their recovery and stability. The production costs of the enzyme constitute 50% of the total cost for hydrolysis; industrial applications of hydrolysis may become expensive. In order to achieve efficient recovery and stability of biocatalysts, immobilization has recently emerged as a system that provides suitable results in terms of high product yield in favorable conditions and allowing the reuse of the biocatalysts (Rao et al., 1998) . The speed and efficiency of the immobilization process depend on the type of carrier (support material), the method of immobilization, concentration, pH, temperature, and reaction time. Strong ionic, hydrophilic or hydrophobic, and/or hydrogen bond interactions between the enzyme and the carrier affect the stability of an enzyme since these strong interactions lead to irreversible adsorption of the enzyme on the carrier, resulting in the loss of enzyme activity. Such strong interactions may also cause conformational changes in the tertiary structure of the enzyme, which again leads to the loss in enzyme activity. These effects may be particularly observed in the case of multiple interactions on the surface of solid carriers (Worsfold, 1995) . A variety of support materials especially porous materials are available for industrial applications, for example, alginate, collagen, chitosan, agar-agarose, polyacrylamide, polyanhydrides, and clays. On the basis of the production type, cost, product specificity, biocompatibility, and usability, the support material must be considered (Naik & Goss, 2016; Bilal et al., 2018) . General expectations from an immobilization process are as follows: providing a balanced bio-hybrid; providing necessary conditions in which the biomolecules do not get denatured during the process; allowing sufficient contact between the enzyme and the substrate; and allowing estimating the quantity of biomolecules required for the process (Vitola et al., 2017) . Several immobilization techniques are available for the immobilization of enzymes. However, the most appropriate one should be selected by considering the result that is required to be achieved.
doi:10.1180/clm.2020.18 fatcat:tixgdbkvojbkvcrg2smj4i3tiu