Earticle

This study focuses on isolating and characterizing novel polyhydroxybutyrate (PHB) depolymerase-producing bacteria to enable efficient PHB biodegradation. PHB is a promising biodegradable alternative to petroleum-based plastics, requires PHB depolymerase to facilitate its effective depolymerization and mineralization in the environment. We identified Janibacter terrae COS4-44 from oil contaminated tidal flat in Taean, Republic of Korea, demonstrating high PHB degradation efficiency via clear zone formation and enzymatic activity. Scanning electron microscopy (SEM) revealed increased surface roughness and porosity in degraded PHB films, while Fourier-transform infrared spectroscopy (FTIR) confirmed the cleavage of ester bonds, indicating enzymatic breakdown. The PHB depolymerase gene was successfully cloned and expressed in E. coli, yielding a functional 58 kDa enzyme. Optimal degradation conditions were 40°C, pH 8.0, and 96 h incubation, with 0.15% PHB concentration. Glucose and yeast extract enhanced enzyme activity, whereas heavy metals like Zn²⁺ and Co²⁺ slightly stimulated depolymerase production. These findings highlight Janibacter terrae COS4-44 as a potent PHB degrader, offering potential for sustainable plastic waste management. Further research on degradation metabolites and enzyme engineering could enhance industrial applications.

두 개의 프로모터 (trc와 Pp)를 Alcaligenes eutrophus에서 유래된 PHA 오페론에 삽입하여 재조합 대장균에서 분자량이 큰 polyhydroxybutyrate (PHB)를 얻고자 하였다. 두 개의 프로모터는 hydroxybutyric CoA와 PHA 중합반응의 유전자 발현을 각각 독립적으로 제어하기 위해 설계된 것이다 새로운 합성오페론을 포함한 플라즈미드는 E. coli DH5a 에 transformation 되어 PHB 생산에 이용되었다. 본 실험의 가설로서 PHA 합성오페론의 IPTG에 의한 유도가 없을 경우 낮은 pHA synthase의 활성이 고분자 중합반응의 개시제 농도를 줄여주어 결과적으로 높은 연결수의 고분자를 생성할 것이라는 모델을 세웠다. 실제로 IPTG의 공급이 없는 발효실험을 통해 평균분자량이2.5x107 인 거대 고분자를 얻을 수 있었다. PHA 생합성에 관여는 효소의 활성 분석으로 3-hydroxybutyric CoA의 중합을 촉매하는 효소인 PHA synthase의 활성을 가지고 In vivo에서 분자량이 제어됨을 확인하였다

Two promoters (trc and Pp) were inserted in PHA operon derived from Alcaligenes eutrophus to obtain high chain molecules of polyhydroxybutyrate (PHB) in recombinant Escherichia coli. Newly designed PHA operon was used to control the gene expressions of hydroxybutyric CoA and PHA polymerization, separately. Plasmids containing new synthetic operon was transformed into E. coli DH5a and analyzed for PHB production. Without induction of the PHA biosynthetic operon, PHA synthase which has low activity might supply low concentration of initiator during the polymerization reaction, resulting very high molecular weight of polymer. An increase of PHB average molecular weight was observed with decreased IPTG (isopropyl B-Dithiogalactosidase) concentration. When no IPTG was added to the culture of E. coli DH5a /p SJS1 which contained two promoters in PHA operon, high chain polymer having an average molecular weight of 2.5x107 was achieved. Analysis of the enzyme activities of PHA biosynthetic enzymes suggests that PHA synthase, the enzyme responsible for polymerizing 3-hydroxybutyric CoA, controls the molecular weight of PHB produced in vivo.