Process Integration of Production, Purification, and Immobilization of β-Glucosidase by Constructing Glu-linker-ELP-GB System

Abstract

In enzymatic conversion of biomass, how to degrade cellulose into fermentable glucose in an economic, efficient, and clean way has become an important subject. As for the application of cellulase in cellulose degradation, the process optimization in enzyme engineering is urgently desired. The traditional multistep purification processes lead to rising production costs and reduced activity of cellulase; meanwhile, the difficulty in reusability of cellulase has also become a big baffle in the cost-effective application of cellulase in biomass degradation. In this paper, the biocatalyst Glu-linker-ELP-GB (GLEGB) containing binary tags, elastin-like polypeptide (ELP), and graphene-binding (GB), was constructed to simplify the purification and immobilization of βglucosidase (Glu) from Coptotermes formosanus. A high recovery rate (97.2%) and purification fold (18.7) of GLEGB was obtained by only one round of inverse transition cycling (ITC) with 0.5 M (NH 4 ) 2 SO 4 at 25 °C in a short incubating time of 10 min. The purification performance of the one-round ITC method is superior to the commonly used Ni-NTA resin affinity method. Furthermore, the high loading amounts of GLEGB immobilized on GO (698.2 mg g −1 ) and C 3 N 4 (527.3 mg g −1 ) were achieved by the synergistic effects of ELP and GB tags. The storage stability and thermal stability of GLEGB was significantly enhanced after immobilization. The recombinant GLEGB immobilized on GO, MGO, graphite, C 3 N 4 , C200, and C400 retained 71.4%, 69.5%, 75.1%, 61.2%, 73.5%, and 80.2% of their initial activities respectively after eight cycles. It is worth mentioning that the K m values of GLEGB immobilized on lamellar carbon materials including GO, MGO, and C 3 N 4 are very close to free GLEGB, showing a high affinity of recombinant GLEGB to substrate. To our knowledge, this is the first report on enzyme-linker-ELP-GB system with wide application prospect in the efficient purification and immobilization of enzyme, which can achieve the goal of reducing cost and improving efficiency of biocatalyst in enzymatic conversion of biomass.

Recyclable β-Glucosidase by One-Pot Encapsulation with Cu-MOFs for Enhanced Hydrolysis of Cellulose to Glucose

“…Biofuel ethanol, the ethanol produced from renewable cellulose (an almost inexhaustible polymeric raw material), has attracted wide attention as an alternative and green liquid energy in recent years. , However, conversion of cellulose into glucose is an important step of biofuel ethanol production, which mainly depends on the degradation capacity of a complex cellulase system including endo-β-1,4-glucanase (EG), cellobiohydrolase (CBH), and β-glucosidase (β-G). The EG and CBH act synergistically to degrade cellulose to cellobiose, which is further hydrolyzed into glucose by β-G .…”

Immobilization of Alcohol Dehydrogenase on Titania Nanoparticles To Enhance Enzyme Stability and Remove Substrate Inhibition in the Reaction of Formaldehyde to Methanol

“…Therefore, surface charge in enzyme immobilization is a key factor in the immobilization yield and determining the density of immobilized enzyme. − Actually, with no conformational changes observed after immobilization the enzyme retains the most initial activity, whereas adsorption of the enzyme onto a carrier is relatively weak and the enzyme easily detaches from the support under gentle conditions . However, covalent linkage between enzyme and support ensures the highest strength of the bonding and improves the stability of enzyme in different conditions. − One of the most widely used reagents in covalent enzyme immobilization is glutaraldehyde, which can be used as a linker between support and enzyme. Glutaraldehyde can react with different portions of the enzyme, mainly involving the primary amino groups of proteins, and forms imine bonds between the biomolecule and the support …”

Construction of Multienzyme Co-immobilized Hybrid Nanoflowers for an Efficient Conversion of Cellulose into Glucose in a Cascade Reaction

“…The recombinant plasmid of pET-Glu-linker-ELP-6His (pET-GLEH) in this work was constructed in our previous report. 36 And the recombinant plasmids of pET-EG-linker-ELP-6His (pET-EGLEH) and pET-CBH-linker-ELP-6His (pET-CBHLEH) were constructed by Synbio Tech (Suzhou, China). The detailed process of expression and purification of recombinant proteins is shown in the Supporting Information.…”