Purification, physico-chemical and kinetic properties of the deglycosylated Talaromyces thermophilus lipase

• Published in: International journal of biological macromolecules 2012-12, Vol.51 (5), p.892-900
• Main Authors: Romdhan, Ines belhaj-ben, Fendri, Ahmed, Frikha, Fakher, Gargouri, Ali, Belghith, Hafedh
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acids; Binding Sites; Biocatalysis; Carbohydrates - chemistry; Catalysis; catalytic activity; Chemical Phenomena; Covalent immobilization; culture media; enzyme stability; enzymes; gel chromatography; Glycosylation; Kinetics; Lipase - chemistry; Lipase - isolation & purification; Lipase - metabolism; Models, Molecular; Molecular Sequence Data; molecular weight; Protein Conformation; Purification; Substrate Specificity; Talaromyces; Talaromyces - enzymology; Talaromyces thermophilus lipase; temperature; thermal stability
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2012.06.034

► An appearance of a second shorter form of Talaromyces thermophilus lipase. ► This form was purified to homogeneity and its N-terminal sequence was identified. ► The glycan chain content of this form confirmed its deglycosylation at Asn 33. ► N-glycans are determinant for its optimal catalytic activity and thermal stability. The Talaromyces thermophilus strain produces only one form of lipase called TTLI. When the culture medium was concentrated and stored at 4°C during a few days, we noticed the appearance of a second short form of lipase named TTLII. This second form was purified to homogeneity using gel filtration and FPLC-Anion exchange chromatography. The NH2-terminal 24 amino acid residues were found to be identical to those of TTLI. The treatment of the TTLI with endoglycosidase H decreased its apparent molecular weight from 39 to 30kDa which corresponds to the molecular weight of TTLII. This difference was mostly attributed to the N-glycosylation of the enzyme. In fact, the glycan chain content and concavaline A–Sepharose affinity column confirmed that the TTLII was completely deglycosylated. Compared to TTLI, the TTLII activity was completely decreased over a broad range of temperature and pH. Furthermore, the deglycosylation of the enzyme reduced its specific activity by 50% toward different substrates; strongly suggest that the N-glycans are determinants for optimal catalytic activity and thermal stability of this enzyme. Covalent immobilization of the enzymes on supports suggests the involvement of the glycan moiety in enzyme–polymer interactions. In the case of TTLI the glycan moiety can constitute an extra site for the covalent linkage of the enzyme on the carrier.