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Objective To screen out a strain with the ability to degrade penicillin G (PENG) and identify the key enzymes involved in PENG catabolism, providing strain and gene resources for the biological treatment of penicillin waste. Methods Bacterial strains capable of utilizing penicillin G potassium (PGK) as the sole carbon source were screened by enrichment culture. Key enzymes involved in the catabolism of PGK were identified by genome and transcriptome analyses, and their evolutionary origins were examined. The key enzymes were expressed and purified, and their kinetics were analyzed. The physiological roles of the key genes in bacterial growth on PGK were revealed by gene knockout and complementation. Results The obtained strain Delftia sp. PG-8 can degrade PGK and utilize it as the sole carbon source for growth. The strain showed the best performance in PENG degradation and growth at pH 7.0, 35 ℃, and 10.00 mmol/L PGK. PgkA catalyzed the rapid degradation of PGK, with Km=(99.19±19.45) μmol/L and kcat/Km=(1.96±0.55)×105 L/(mol·s). Compared with the functionally characterized β-lactamases, PgkA had a unique evolutionary origin. PgkB also had the ability to catalyze the transformation of PGK, while its substrate affinity was only 1/5 that of PgkA, in addition to the lower catalytic efficiency. The degradation and utilization of PGK for growth by strains PG-8-ΔpgkA and PG-8-ΔpgkB were significantly reduced, with PG-8-ΔpgkA showing a more pronounced decline. Although PG-8-ΔpgkAB, in which both pgkA and pgkB were knocked out, still degraded a certain amount of substrate, it was almost unable to use PGK as the sole carbon source for growth. Conclusion PG-8 is the first strain of Delftia capable of using PGK as the sole carbon source for growth. Both pgkA and pgkB play important physiological roles during PG-8 growth on PGK, with pgkA playing a dominant role.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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