Although the efficiency of poly(ethylene terephthalate)(PET)degradation has been successfully improved by depolymerase engineering,mostly by using Goodfellow-PET(gf-PET)as a substrate,efforts to degrade unpretreated PET materials with high crystallinity remain insufficient.Here,we endeavored to improve the degradation capability of a WCCG mutant of leaf-branch compost cutinase(LCC)on a unpretreated PET substrate(crystallinity>40%)by employing iterative saturation mutagenesis.Using this method,we developed a high-throughput screening strategy appropriate for unpretreated substrates.Through extensive screening of residues around the substrate-binding groove,two variants,WCCG-sup1 and WCCG-sup2,showed good depolymerization capabilities with both high-(42%)and low-crystallinity(9%)substrates.The WCCG-sup1 variant completely depolymerized a commercial unpretreated PET product in 36 h at 72℃.In addition to enzyme thermostability and catalytic efficiency,the adsorption of enzymes onto substrates plays an important role in PET degradation.This study provides valuable insights into the structure-function relationship of LCC.
我国是塑料产品生产大国,塑料产品的使用产生了大量难以降解的塑料垃圾。为了能获得可高效降解塑料的菌株,本研究以聚乙烯塑料为唯一碳源,筛选出塑料降解菌,通过失重法测定其降解效率,采用16S rDNA序列测定对其进行核酸分类学鉴定。结果:从半降解带土塑料中筛选出9株塑料降解菌,塑料降解菌的降解率为0.33%~4.24%。9株塑料降解菌中,6株为恶臭假单胞菌(Pseudomonas putida),3株为产吲哚金黄杆菌(Chryseobacterium indologenes),恶臭假单胞菌的塑料降解率高于产吲哚金黄杆菌,有进一步开发应用的潜力。China is a major producer of plastic in the world, while the use of plastic products creates a large amount of plastic waste which is difficult to be degraded. In order to obtain strains that can efficiently degrade plastics, polyethylene plastic was used as the sole carbon source to screen plastic degrading bacteria. The degradation efficiency was determined by weight-loss method, and the strains were identified by nucleic acid taxonomy based on 16S rDNA sequencing. Results: 9 plastic degrading bacteria were screened from semi degraded soil plastics, with the degradation rate of 0.33% to 4.24%. Among the 9 strains of plastic degrading bacteria, two-thirds are Pseudomonas putida and one-thirds are Chryseobacterium indologenes. The plastic degradation rate of P. putida is higher than that of C. indologenes. That indicates P. putida has the potential for further development and application.
