Development of enzymatically-active bacterial cellulose membranes through stable immobilization of an engineered β-galactosidase

Abstract

Enzymatically-active bacterial cellulose (BC) was prepared by non-covalent immobilization of a hybrid enzyme composed by a β-galactosidase from Thermotoga maritima (TmLac) and a carbohydrate binding module (CBM2) from Pyrococcus furiosus. TmLac-CBM2 protein was bound to BC, with higher affinity at pH 6.5 than at pH 8.5 and with high specificity compared to the non-engineered enzyme. Both hydrated (HBC) and freeze-dried (DBC) bacterial cellulose showed equivalent enzyme binding efficiencies. Initial reaction rate of HBC-bound enzyme was higher than DBC-bound and both of them were lower than the free enzyme. However, enzyme performance was similar in all three cases for the hydrolysis of 5% lactose to a high extent. Reuse of the immobilized enzyme was limited by the stability of the β-galactosidase module, whereas the CBM2 module provided stable attachment of the hybrid enzyme to the BC support, after long incubation periods (3 h) at 75 °C.

Revalorization of cellulosic wastes from Posidonia oceanica and Arundo donax as catalytic materials based on affinity immobilization of an engineered β-galactosidase

“…The binding yield of the cellulose cryogel from P. oceanica (7.5 mg enzyme/g of support) obtained after incubation of 30 mg of support with 0.25 mg of enzyme was within the range of the values reported for the freeze-dried bacterial cellulose (3.6 -9.2 mg enzyme/g of support), assayed under similar conditions (incubation of 30 mg of support with 0.15 -0.35 mg of enzyme) (Estevinho et al, 2018). This result suggests that the available number of binding sites in both supports is comparable.…”

“…The hybrid enzyme used in this work (TmLac-CBM2) was constructed by fusing a galactosidase from Thermotoga maritima (TmLac) (Marín-Navarro et al, 2014) with a carbohydrate binding module (CBM2) from Pyrococcus furiosus chitinase (Nakamura et al, 2008). The genetic construct encoding the hybrid protein, as well as the production of both TmLac-CBM2 and the wild type TmLac by Escherichia coli, have been described in a previous work (Estevinho et al, 2018). Because both TmLac and TmLac-CBM2 are thermoresistant proteins, purification was carried out by heat shock treatment of crude extracts at 85 0 C during 10 min.…”

“…The soluble fraction was dialyzed against phosphate buffer 50 mM pH 6.5 and concentrated by ultrafiltration using a membrane with a 20 KDa cutoff (Thermo Fisher Scientific). Enzyme concentration was determined as the ratio of -galactosidase activity (expressed in mol galactose • min -1 • ml -1 sample) divided by the intrinsic activity of TmLac-CBM2 ( 28μmol galactose•min −1 •mg enzyme −1 ), as previously described (Estevinho et al, 2018).…”

“…Both the chemical characteristics of the solid support and the restrictions imposed to diffusion of substrates and products contribute to the change in the microenvironment of the immobilized enzyme, compared to the free form (Bolivar & Nidetzky, 2019). A particular case of physical adsorption, referred as bioadsorption or affinity immobilization, is based on the affinity of a protein or protein module to a specific substrate (Estevinho et al, 2018;Mislovičová, Masárová, Vikartovská, Gemeiner, & Michalková, 2004). In this context, molecular engineering techniques can be used to create hybrid proteins by fusion of the enzymes to a module with affinity for a specific biopolymer.…”

“…They are classified into different families by sequence homology, which is related to their substrate specificity (Boraston, Bolam, Gilbert, & Davies, 2004). In a recent work, Estevinho et al (2018) showed the suitability of hydrated and freeze-dried bacterial cellulose (BC) to immobilize a hybrid enzyme composed by a -galactosidase from Thermotoga maritima (TmLac) and a CBM2 from Pyrococcus furiosus, with high specificity compared to the non-engineered enzyme. The CBM2 provided a stable attachment of the hybrid enzyme to the BC support at high temperatures.…”