Nano-immobilization of PETase enzyme for enhanced polyethylene terephthalate biodegradation

https://doi.org/10.1016/j.bej.2021.108205Get rights and content

Highlights

  • Immobilized PETase was successfully performed via biomimetic mineralization.

  • The catalytic performance of PETase were enhanced owing to the nano-effect.

  • The immobilized PETase degraded the surface of PET effectively within two days.

Abstract

PET hydrolase (PETase), discovered in Ideonella sakaiensis, is a promising agent for the biodegradation of polyethylene terephthalate (PET) capable of PET decomposition under mild reaction conditions with limited stability and productivity. Here, the immobilization of His-tagged PETase was achieved by synthesizing enzyme-inorganic nanoflowers, PETase@Co3(PO4)2, which was designed based on the principle of biomimetic mineralization. Immobilization of PETase onto nanostructured Co3(PO4)2 enjoys high enzyme loading and low mass transfer inhibition due to large specific surface area, high movement speed, and large surface curvature caused by small particle size. The nano-effect of inorganic carriers materialize the 10 °C optimum temperature swelling of the immobilized PETase with enhanced pH tolerance (6.0–10.0) than the free counterpart. The long-duration reaction showed that the productivity of terephthalic acid (TPA) was 3.5 times higher than that of the free enzyme. PETase@Co3(PO4)2 still retained 75% of the initial activity after 12 days compared with the free enzymes, which showed almost no activity. The excellent and stable catalytic performance of PETase@Co3(PO4)2 with low cost demonstrates the synthetical usefulness of immobilization via biomimetic mineralization in the enzyme utilization in industrial PET depolymerization.

Graphical Abstract

Schematic illustration of PETase@Co3(PO4)2 nanoflowers synthesis (a), immobilization of PETase enzyme by biomimetic mineralization, and PET film degradation using the immobilized enzyme(b).

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Introduction

Plastics have been massively produced over the past few decades and become essential to modern society, driven by their durability, plasticity, and chemical stability. However, poor management and disposal methods engendered explosive accumulation worldwide [1]. The environmental threat of plastic pollution, especially in marine ecosystems, is further recognized for the ultra-long lifetimes of plastics [2], [3], [4]. Especially for PET, with only two simple monomers, TPA and ethylene glycol (EG), connected by an ester bond, is relatively solid and difficult to degrade naturally. The current treatment methods of PET waste like recycling, landfill treatment, and incineration treatment, have apparent limitations, for example, the considerable investment in PET recycling, land pollution causing by landfill treatment, and air pollution resulting from incineration.

Biodegradation has been considered an effective method to control PET pollution profit for its eco-friendly and cost-effective characteristics [4], [5], [6]. The biodegradation of PET could be performed by the whole-cell organism or by the produced enzymes. Several enzymes that catalyze PET hydrolysis have been discovered in the past few years and identified as promising and environmental biocatalysts to alternate chemical or physical recycling [7], [8], [9], [10], [11], [12]. For example, after extracting the cutinase named LCC from a fosmid library of a leaf-branch compost metagenome [12], Tournier et al. improved its hydrolysis capacity that ultimately achieved a minimum of 90% PET depolymerization into monomers over 10 h [10]. Polyester hydrolase TfCut2 from Thermobifida fusca KW3 is another candidate with a high-PET hydrolytic activity [11]. Roth and his co-workers [13] structurally characterized TfCut2 and proposed a model for the binding of the enzyme towards its polymeric substrate. In 2016, a PET-specific hydrolase named PETase (EC: 3.1.1.101, PDBID:5XJH), identified from bacterium Ideonella sakaiensis strain 201-F6, exhibits high depolymerization activity against PET films under mild reaction conditions [14]. However, its instability during the long-term storage and plastic depolymerization process hinders its application in industrial large-scale PET biodegradation.

Immobilization of PETase using the facile methods is a suitable solution to the abovementioned problems. In the past few decades, immobilized enzymes influenced by carrier choices have demonstrated superior performance due to their higher stability, better reusability, and easier separation from the reaction mixture than their free forms [15]. Traditional immobilization approaches mainly include three groups, binding to supports, carrier-free insolubilized enzyme aggregates, and entrapment in polymer materials [31]. Compared with traditional materials, nanostructures can stabilize enzymes for a long time in different systems with nano-effects such as the small size and the large surface curvature of the nanoparticles [16].

Recently, the biomimetic mineralization process has caught many eyeballs with its ease operation, ultrahigh enzyme activity recovery rate, mild reaction conditions, and the specially enhanced stabilization effect of enzyme−inorganic hybrid nanoflowers on enzymes component [17]. Unlike any traditional immobilization method, the biomimetic mineralization process is a new immobilized enzyme technology accompanied by inorganic salt precipitation. A low-concentration buffer is used to slow down the rate of inorganic salt formation, with enzyme molecules introduced in the reaction to allow inorganic salt precipitation to adhere to the enzyme slowly [24]. Then, embedding was carried out to immobilize of the enzyme. The method utilizes the specific chelation of the imidazole group in his-tag exposed on the protein surface and metal ions to immobilize the protein, whose affinity then be purified. The inorganic nanoparticles as carriers reduced the steric hindrance and substrate diffusion limiting. Following the protein-inorganic (Cu3(PO4)2) hybrid system by Ge et al. [18], and subsequently, some scientists synthesized several other hybrid nanoflowers using different inorganic or organic components for the efficient immobilization of hydratase, lipase and transaminase [19], [20], [21]. Encouraged by these early achievements, a biomimetic mineralization strategy that introduces an affinity tag at the N-terminal of the PETase to realize the purification and immobilization of the enzyme was adopted. PETase flower-like cobalt phosphate nanoparticles were produced by the nucleation reaction of the cobalt ions and PETase enzyme that was assembled on the surface of particles. Catalytic activities and stabilities of the free and immobilized PETase toward PET films were studied. The reusability efficiency and storage performance of the immobilized PETase enzyme were also examined.

Section snippets

Materials

The complete gene sequence of PETase from Ideonella sakaiensis strain 201-F6 (Genbank GAP38373.1) was codon-optimized and synthesized, then inserted into the target vector pET-30a (+) for the expression in Escherichia coli BL21 (DE3). For immobilized PETase, a 6x His tag was added into the N-terminal of its sequence. The sequence of the resulting plasmids was confirmed using Sanger sequencing at GenScript China, Inc. Acetonitrile (HPLC grade) was purchased from Shanghai Macklin Biochemical Co.,

Specificity of cobalt phosphate to His-Tagged PETase from cell Lysates

Cell lysates containing PETase were mixed directly with Co(NO3)2 and PBS solution without any purification process to prepare PETase@Co3(PO4)2. Ni-NTA column witnessed the unsatisfactory effect in the enzyme purification, as shown in Fig. 1, lane 2. This may be attributed to Ni2+ can bind to some non-His-tagged proteins, resulting in poor selectivity and affecting the purification effect [33], [34]. In contrast, Co2+ enjoys a higher purity target protein with the weaker non-specific binding

Conclusions

In summary, the facile and efficient biomimetic mineralization process witnessed the effective immobilization of the PETase into the Co3(PO4)2 nanoflower at room temperature. Based on the strong affinity interaction between the His-tag located on PETase and cobaltous phosphate, the oriented purification and immobilization of PETase from crude enzyme without pre-purification has been realized. Owing to the unique structure, the hybrid nanoflowers biocatalyst (PETase@Co3(PO4)2) showed

CRediT authorship contribution statement

Yunpu Jia: Conceptualization, Methodology, Validation, Data curation, Writing – original draft, Writing – review & editing. Nadia A. Samak: Methodology, Writing – review & editing. Xuemi Hao: Writing – review & editing, Supervision. Zheng Chen: Validation. Gama Yang: Validation. Xuhao Zhao: Validation. Tingzhen Mu: Validation. Maohua Yang: Validation. Jianmin Xing: Conceptualization, Writing – review & editing, Supervision, Project administration.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

The authors gratefully acknowledge the financial support provided by the National Natural Science Foundation of China (grant numbers 31961133017, 31961133018, 31961133019). These grants are part of “MIXed plastics biodegradation and UPcycling using microbial communities” MIX-UP research project, which is a joint NSFC and EU H2020 collaboration. In Europe, MIX-UP has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 870294.

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