评论：塑料结合模块促进聚对苯二甲酸乙二醇酯的酶法降解

编者按 从本期开始，《生物工程学报》新增设了“评论”栏目，邀请领域内的专家，就近期发表在著名权威期刊中的重要突破性成果或进展进行评论，希望为读者带来启发和参考。 本期邀请德国亚琛工业大学生物技术研究所季宇博士和Ulrich Schwaneberg教授团队，针对近期发表在期刊Chem Catalysis的一项关于塑料结合模块促进酶法降解塑料的研究成果进行了评论，以飨读者。 对LCCYCCG-CBMs的性能进行表征后，在PET薄膜的低负荷(2 wt%)、40–90 ℃的温度下检测LCCYCCG和LCCYCCG-CBMs的无定形PET水解活性。结果表明，在40−70 ℃的温度范围内，酶的活性随着温度的增加而增强，随后在70−90 ℃的温度范围内酶的活性随着温度的增加而下降。LCCYCCG-TrCBM1和TtCBM10比LCCYCCG和其他LCCYCCG-CBM表现出较高的活性。然而，LCCYCCG与TrCBM1的等摩尔混合物不能提高PET的降解活性，表明只有通过共价连接LCCYCCG和CBM才能提高PET的降解活性。 此外，在1 wt%−20 wt%的PET负载条件下，检测了LCCYCCG、LCCYCCG-TrCBM1、LCCYCCG-TtCBM10和LCCYCCG-StCBM64的PET降解活性。结果表明，在PET负载低于5 wt%时，与LCCYCCG相比，LCCYCCG-TrCBM1、LCCYCCG-TtCBM10和LCCYCCG-StCBM64从PET降解中产生了更多的单体。然而，对于10 wt%− 20 wt%的PET负载，即使酶浓度提高至1 mmol/L，LCCYCCG-CBMs的活性也并未提高。为了评估塑料结合模块在工业相关条件下的作用，将反应体系扩大至1 L，并将PET负载设定为20 wt%。在此反应条件下，LCCYCCG-TrCBM1并没有显示出比LCCYCCG更好的活性。 综上所述，Graham等[13]的报道表明，当PET负载较低(15 wt%)时，CBMs可以发挥促进PET降解的作用，而当PET负载较高(10 wt%− 20 wt%)时，CBMs的作用变得很弱。因此，本团队提出了一些策略来发挥塑料结合模块在促进PET降解方面的作用。首先，塑料结合模块对高负载PET的降解作用降低的原因尚不清楚。由于塑料结合模块加速PET降解与PET结合强度有关，了解塑料结合模块的PET结合机制将有助于解决这一问题。其次，不同的PET应在降解过程中进行测试，如不同的类型、结晶度、化学纯度和商业的PET塑料瓶。第三，许多研究人员已经研究了塑料结合模块的改造以提高塑料结合强度[14-15]，应用具有更高的塑料结合强度的塑料结合模块突变体将会在更高的PET负载下促进塑料的降解。相信经过深入地研究，可以实现PET或其他种类的聚酯在较高塑料负载下的加速降解。 The indiscriminate use of plastics leads to serious environmental hazards including increased land and water pollution, which has largely affected the natural ecosystem[1]. Therefore, there are significant environmental, ecological, economic, and social necessity for the development of novel environmentally friendly enzymatic technologies to degrade plastic wastes and generate added-value chemicals that will mitigate the present detrimental effects of plastics pollution and at the same time establish a new source of raw materials, in conformity to the notion of a sustainable and circular economy. To improve the degradation efficiency of plastic degradation enzymes, protein engineering strategies were utilized to modify enzymes and ultimately improve their catalytic performance. Protein engineering can be used to tailor different enzyme properties, including improving enzyme thermostability, enhancing the binding of enzyme to substrate, and reducing product inhibition effects. Fusing polymer degradation enzymes with polymer binding modules is an alternative approach to advance polymer enzymatic degradation. In the past decades, polymer binding modules have been utilized to bind with a variety of solid materials including natural and synthetic polymers[2]. With unparalleled binding affinity, polymer binding modules have been widely applied in biological surface functionalization (e.g., antifouling and antimicrobial properties)[3-4], enzyme immobilization[5], material assembling[6], plant protection[7], and plastic degradation[8]. Especially, polymer binding modules display significant roles in the acceleration of polymer enzymatic degradation[9]. Nature generates carbohydrate binding modules (CBM) to accelerate cellulose degradation by cellulases. In general, cellulases, the carbohydrate-active enzymes used to degrade natural polymers like cellulose into sugar monomers, contain a single domain or multiple domains and the catalytic domain is connected with CBM through a linker[10]. Inspired by nature, polymer binding modules have enormous potential in accelerating the activity of polymer degrading enzymes. For instance, fusing anchor peptide Tachystatin A2 with cutinase Tcur1278 enhanced the degradation of polyester-polyurethane nanoparticles by a factor of 6.6 compared with the Tcur1278 wild-type[8]. In the last decades, extensive work in poly(ethylene terephthalate) (PET) enzymatic degradation has revealed that the polymer binding modules could enhance enzymatic PET hydrolysis[11-12]. Here, we introduced a recent work, which studied the role of binding modules in enzymatic PET hydrolysis at high-solids loadings[13]. Taking industrial application into consideration, Graham et al.[13] investigated the catalytic efficiency of PET-hydrolyzing enzymes at low and high-solids loading after fusing with binding modules, which facilitates the rapid deployment of feasible approaches to realistically address plastics pollution. Rosie Graham and coworkers[13] first generated a new thermostable variant (LCCYCCG) of leaf compost cutinase (LCC) and fused it with five type A CBMs (LCCYCCG-CBM) (Figure 1). Several properties of LCCYCCG-CBM fusion proteins, including esterase activity, PET binding activity, and thermal stability, were compared with that of LCCYCCG. The esterase activity was measured from the hydrolysis of the diester bis(2-hydroxyethyl) terephthalate (BHET), and the LCCYCCG esterase activity remained unchanged after fusing with CBMs. In addition, on top of LCCYCCG-BsCBM3, other four LCCYCCG- CBMs showed similar PET binding and all of them displayed higher PET binding than LCCYCCG. Among them, LCCYCCG-TrCBM1 displayed the highest PET binding affinity. The temperatures of thermal unfolding transition for LCCYCCG and LCCYCCG-CBMs were measured by differential scanning calorimetry (DSC). Results showed that LCCYCCG and LCCYCCG-CBMs had similar temperatures of thermal unfolding transition, indicating that CBMs posed limited effects on the properties of LCCYCCG. Thus, fusing LCCYCCG with CBMs could improve the PET binding while CBMs did not affect the esterase activity and thermal stability of LCCYCCG. After characterization of LCCYCCG-CBMs properties, amorphous PET hydrolysis activity of LCCYCCG and LCCYCCG-CBMs was detected from 40 ℃ to 90 ℃ at low loadings of PET films (2 wt%). Generally, the enzyme activity was increased from 40 ℃ to 70 ℃ and later decreased until the temperature reached 90 ℃. LCCYCCG- TrCBM1 and TtCBM10 exhibited the highest activity than LCCYCCG and other LCCYCCG-CBMs. However, the activity cannot be improved for the PET degradation by equimolar mixtures of LCCYCCG with TrCBM1, indicating the enhanced PET degradation activity can only be achieved by covalently linked LCCYCCG and CBMs. Moreover, the PET degradation activity of LCCYCCG, LCCYCCG-TrCBM1, LCCYCCG-TtCBM10, and LCCYCCG-StCBM64 were measured at 1 wt%– 20 wt% PET loading. In comparison to LCCYCCG, LCCYCCG-TrCBM1, LCCYCCG-TtCBM10, and LCCYCCG-StCBM64 generated a higher number of monomers from PET degradation when the PET loading is lower than 5 wt%. However, no improved activity of LCCYCCG-CBMs can be observed for 10 to 20 wt% PET loading even when the enzyme concentration was elevated to 1 mmol/L. In order to evaluate the role of polymer binding modules in industrially relevant conditions, the reaction system was enlarged into 1 L and the PET loading was set as 20 wt%. Under these reaction conditions, LCCYCCG-TrCBM1 did not show improved activity than LCCYCCG. In summary, Graham et al.[13] reported that CBMs could exert the role of facilitating PET degradation when the PET loading was low (1 wt%−5 wt%) and the effects of CBMs became weak when the PET loading was high (10 wt%− 20 wt%). To exert the role of polymer binding modules in advancing PET degradation, we put forward some approaches. First, the reasons underlying the reduced effects of polymer binding modules on the degradation of high loading PET are not clear. As the accelerated PET degradation of polymer binding modules is associated with the PET binding strength, understanding the binding mechanism behind will be helpful for solving this problem. Second, different PET substrates such as different types, crystallinity, chemical purity, and commercial bottles, should be tested during degradation. Third, the engineering of polymer binding modules to improve polymer binding strength has been studied by many researchers[14-15]. Applying polymer binding module variants with higher polymer binding strength would be a strategy to improve polymer degradation with higher PET loading. We believed that the accelerated degradation of PET or other kinds of polyesters with higher polymer loading can be realized after extensive investigation. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]