The massive accumulation of polyethylene (PE) in the natural environment has caused persecution to the ecological environment. At present, the mechanism of microbial degradation of PE remains unclear, and the related enzymes for degrading PE need to be further explored. In this study, a strain of Klebsiella pneumoniae Mk-1 which can effectively degrade PE was obtained from the soil. The degradation performance of the strains was evaluated by weight loss rate, SEM, ATR/FTIR, WCA, and GPC. The key gene of PE degradation in the strain was further searched, which may be the laccase-like multi-copper oxidase gene. Then, the laccase-like multi-copper oxidase gene (KpMco) was successfully expressed in E.coli and its laccase activity was verified, which reached 85.19 U/L. The optimum temperature and pH of the enzyme are 45 °C and 4.0, respectively; it shows good stability at 30-40 °C and pH 4.5-5.5; Mn²⁺ and Cu²⁺ can activate the enzyme effect. After the enzyme was applied to the degradation of PE film, it was found that the laccase-like multi-copper oxidase did have a certain degradation effect on PE film. This study provides new strain and enzyme gene resources for the biodegradation of PE, thereby promoting the process of PE biodegradation.

Urethane hydrolase can degrade the carcinogen ethyl carbamate (EC) in fermented food, but its stability and activity limit its application. In this study, a mutant G246A and a double mutant N194V/G246A with improved cpUH activity and stability of Candida parapsilosis were obtained by site-directed mutagenesis. The catalytic efficiency (Kcat/Km) of mutant G246A and double mutant N194V/G246A are 1.95 times and 1.88 times higher than that of WT, respectively. In addition, compared with WT, the thermal stability and pH stability of mutant G246A and double mutant N194V/G246A were enhanced. The ability of mutant G246A and double mutant N194V/G246A to degrade EC in rice wine was also stronger than that of WT. The mutation increased the stability of the enzyme, as evidenced by decreased root mean square deviation (RMSD) and increased hydrogen bonds between the enzyme and substrate by molecular dynamics simulation and molecular docking analysis. The molecule modification of new cpUH promotes the industrial process of EC degradation.

Ethyl carbamate (EC) is classified as a Class 2A carcinogen, and is present in various fermented foods, posing a threat to human health. Urethanase (EC 3.5.1.75) can catalyze EC to produce ethanol, CO₂ and NH₃. The urethanase (cpUH) from Candida parapsilosis can hydrolyze EC, but its low affinity and poor stability hinder its application. Here, the structure of cpUH from Candida parapsilosis was determined with a resolution of 2.66 Å. Through sequence alignment and site-directed mutagenesis, it was confirmed that cpUH contained the catalytic triad Ser-cisSer-Lys of the amidase family. Then, the structure-oriented engineering mutant N194V of urethanase was obtained. Its urethanase activity increased by 6.12 %, the catalytic efficiency (k_cat/Km) increased by 21.04 %, and the enzyme stability was also enhanced. Modeling and molecular docking analysis showed that the variant N194V changed the number of hydrogen bonds between the substrate and the catalytic residue, resulting in enhanced catalytic ability. MD simulation also demonstrated that the introduction of hydrophobic amino acid Val reduced the RMSD value and increased protein stability. The findings of this study suggest that the N194V variant exhibits significant potential for industrial applications due to its enhanced affinity for substrate binding, improved catalytic efficiency, and increased enzyme stability.

A large amount of highly acidic wastewater, known as Huangshui, is produced during liquor brewing. To improve its utilization rate and achieve resource recovery of liquor by-products. A strain with high esterifying ability was screened from Daqu and identified as Acinetobacter baumannii . The strain exhibited an esterification enzyme activity of (16.79 ± 0.03) U/mL and an esterification capacity of (45.37 ± 0.33) mg/g. It was able to grow normally under conditions of 7% ethanol and pH 4.0, demonstrating strong acid and ethanol tolerance, indicating its potential for preparing a Huangshui esterification solution. Subsequently, Huangshui was esterified using ethanol, caproic acid and Daqu powder prepared with this strain. Analysis of trace components in Huangshui before and after esterification showed that the ethyl caproate content in the esterified Huangshui reached (649.26 ± 2.39) mg/L, which was 13.19 times higher than that before esterification, while ethyl lactate decreased by 15.35%. These results indicate a notable ability to enhance ethyl caproate and reduce ethyl lactate. It is expected that the application of this Huangshui esterification solution could improve the flavor and quality of Non-gxiangxing Baijiu by increasing ethyl caproate and decreasing ethyl lactate, thus providing a reliable experimental basis for the resource utilization of Huangshui.