Characterization of a poly(butylene adipate-co-terephthalate)-hydrolyzing lipase from Pelosinus fermentans

• Published in: Applied microbiology and biotechnology 2016-02, Vol.100 (4), p.1753-1764
• Main Authors: Biundo, Antonino, Hromic, Altijana, Pavkov-Keller, Tea, Gruber, Karl, Quartinello, Felice, Haernvall, Karolina, Perz, Veronika, Arrell, Miriam S, Zinn, Manfred, Ribitsch, Doris, Guebitz, Georg M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Anaerobic bacteria; Anaerobiosis; Biodegradation; Biomedical and Life Sciences; Biosynthesis; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Chemical properties; Chemicals; Cloning, Molecular; Composition; Crystallization; Crystallography, X-Ray; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; E coli; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Extraction (Chemistry); Fatty acids; Firmicutes - enzymology; Firmicutes - isolation & purification; Food packaging; Gene Expression; groundwater; Groundwater - microbiology; Hydrogen-Ion Concentration; hydrolases; Hydrolysis; Identification and classification; Life Sciences; Lipase; Lipase - chemistry; Lipase - genetics; Lipase - isolation & purification; Lipase - metabolism; Liquid chromatography; Mass spectrometry; Methods; Microbial Genetics and Genomics; Microbiology; Plasmids; Polyesters; Polyesters - metabolism; Polymers; Protein Conformation; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; recycling; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Studies; Substrate Specificity; Temperature; zinc
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-015-7031-1

Certain α/β hydrolases have the ability to hydrolyze synthetic polyesters. While their partial hydrolysis has a potential for surface functionalization, complete hydrolysis allows recycling of valuable building blocks. Although knowledge about biodegradation of these materials is important regarding their fate in the environment, it is currently limited to aerobic organisms. A lipase from the anaerobic groundwater organism Pelosinus fermentans DSM 17108 (PfL1) was cloned and expressed in Escherichia coli BL21-Gold(DE3) and purified from the cell extract. Biochemical characterization with small substrates showed thermoalkalophilic properties (T ₒₚₜ = 50 °C, pHₒₚₜ = 7.5) and higher activity towards para-nitrophenyl octanoate (12.7 U mg⁻¹) compared to longer and shorter chain lengths (C14 0.7 U mg⁻¹ and C2 4.3 U mg⁻¹, respectively). Crystallization and determination of the 3-D structure displayed the presence of a lid structure and a zinc ion surrounded by an extra domain. These properties classify the enzyme into the I.5 lipase family. PfL1 is able to hydrolyze poly(1,4-butylene adipate-co-terephthalate) (PBAT) polymeric substrates. The hydrolysis of PBAT showed the release of small building blocks as detected by liquid chromatography-mass spectrometry (LC-MS). Protein dynamics seem to be involved with lid opening for the hydrolysis of PBAT by PfL1.