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.

In this study, a novel type of multi-armed polymer (poyltehylene glycol, PEG) magnetic graphene oxide (GO) composite (GO@Fe3O4@6arm-PEG-NH2) has been synthesized as a support for immobilization of horseradish peroxidase (HRP) for the first time. The loading amount of HRP was relatively high (186.34 mg/g) due to the surface of carrier material containing a large amount of amino groups from 6arm-PEG-NH2, but degradation rate of phenols was also much higher (95.4 %), which is attributed to the synergistic effect between the free HRP (45.4 %) and the support material of GO@Fe3O4@6arm-PEG-NH2 (13.6 %). Compared with the free enzyme, thermal, storage and operational stability of the immobilized HRP improved. The immobilized HRP still retained over 68.1 % activity after being reused 8 times. These results suggest that the multi-armed magnetic composite has good application prospect for enzyme immobilization.

Inspired by natural biomineralization process, inorganic phosphates system has been selected as a candidate for the encapsulation of enzyme; however, during the long-term fabrication process, the loss of enzyme activity is unavoidable, and the biomimetic mineralization mechanism is still poorly understood. Meanwhile, the purification process plays a key role in the preparation of immobilized enzyme with high enzyme loading and activity, while the rapid, low-cost, and eco-friendly purification of biocatalyst from crude fermentation broth remains a critical challenge in biochemical engineering. Here, a binary tag composed of elastin-like polypeptide (ELP) and His-tag was presented for the first time to be fused with β-glucosidase (Glu) to construct a recombinant Glu-linker-ELP-His (GLEH) with the aim of developing a fast synthesis strategy combining purification and immobilization processes for a biocatalyst with better stability and recyclability. The purification fold and activity recovery of GLEH reached 18.1 and 95.2%, respectively, once a single inverse transition cycling was conducted at 25 °C for 10 min. Then, efficient biomineralization of hybrid enzyme-Cu3(PO4)2 nanoflowers was realized in 15 min by the action of His-tag and ultrasonic-assisted reaction method. The activity recovery and relative activity reached the maximum at 90.3 and 111.0%, respectively. We demonstrate that the crystal growth process of a hybrid nanoflower involves obvious nucleation, self-assembly, and the Ostwald ripening process, and the enzyme GLEH acts as a “binder” to assemble Cu3(PO4)2 nanoflakes. The immobilized GLEH nanoflowers show outstanding operation stability and recyclability, and their catalytic efficiency is close to that of free Glu.

Magnetic double-shell hybrid microspheres (FeO@SiO@p(NIPAM-co-GMA)) have been developed as a promising supported substrate for the immobilization of cellulase. Since the surface of the magnetic microspheres not only contains an epoxy group from GMA (glycidyl methacrylate) that can covalently bind to the enzyme, but also has an intelligent temperature response property from NIPAM (N-isopropylacrylamide), the cellulase can be covalently bonded to the magnetic microspheres and have a temperature-sensitive capability. The immobilized cellulase has the recovery ability of cellulase activity after a high-temperature inactivation. The average amount and activity of immobilized enzymes, respectively, was 233 mg g, 57.4 U mg under the optimized conditions. The experimental results show that the immobilized cellulase has a wider catalytic temperature range, better temperature and storage stability. The residual activity still remained about 65.6% of the initial activity after the sixth catalysis run, which indicated that the immobilized enzyme had high reproducibility.