Isolasi, Karakterisasi dan Ekspresi Heterolog Gen Penyandi Lakase Trametes hirsuta EDN0S82 dalam Sistem Pichia pastoris

Abstract

Lakase (EC 1.10.3.2) merupakan enzim multifungsi dari kelompok Multi-Copper Oxidase (MCO) yang mampu mengoksidasi berbagai jenis senyawa fenolik maupun non-fenolik. Enzim ini digunakan secara luas dalam berbagai bidang, termasuk penggunaanya sebagai "green catalysts" dalam bioremediasi limbah cair berbahaya. Lakase Native dari T. hirsuta EDN082 telah berhasil diisolasi dan diketahui aktivitas bioremediasinya pada penelitian terdahulu. Pada penelitian ini, salah satu gen penyandi lakase berhasil diskrining dari transkriptom T. hirsuta EDN082 menggunakan novel degenerate primer. Gen tersebut dinamai gen Thlacc1 yang kemudian dikarakterisasi secara bioinformatik dan menunjukan kekerabatan dengan kelompok minor lakase F, memiliki kemiripan tinggi terhadap lakase 3 (AOX15704.1) sebesar 99,20%, berukuran 1497 bp (GC content 62,66%) yang menyandi 499 asam amino mature protein. Gen tersebut membawa region pengikatan tembaga (Cu) yang umum dimiliki oleh gen-gen penyandi lakase berupa L1 (HWHGFFQ), L2 (HSHLSTQ), L3 (HPFHLHG) dan L4 (HCHIDFHL), serta termasuk dalam kelompok High-Redox Potential Laccase (HPRL) berdasarkan residu asam amino disekitar region L4. Gen Thlacc1 menyandi jenis lakase yang didominasi oleh region hidrofilik dengan titik isoelektrik pada pH 5,2 serta mengandung 4 situs potensial N-glikosilasi dan 11 situs potensial O-glikosilasi. Pemodelan struktur tiga dimensi (3D) protein mengungkap kemiripan tinggi dengan struktur lakase T. hirsuta (PDB ID: 3PXL, nilai RMSD 0.575 Å) dan memiliki kofaktor berupa 4 atom tembaga yang terdiri atas T1, T2 dan T3a/T3ß dengan rongga pengikatan substrat berpusat pada residu His458. Gen Thlacc1 berhasil dikonstruksi ke dalam pGEM-Teasy sebagai vektor kloning dan pPICZaB sebagai vektor ekspresi. Gen tersebut di-fusi pada N-terminal dengan sinyal sekresi a-mating factor lewat penambahan situs EcoRI pada ujung 5’ dan fusi C-terminal dengan 6xHis tag lewat penambahan situs NotI pada ujung 3’. Expression cassette pPICZaB_Thlacc1 berhasil diintegrasikan kedalam genom P. pastoris BG11 ?AOX1 pada lokus promoter AOX1 dan menghasilkan 64 strain, dimana 63 strain (98,4%) diantaranya mampu mensekresikan lakase rekombinan rThlacc1 kedalam medium yang diinduksi oleh methanol. Aktivitas oksidasi terhadap ABTS yang diamati mencapai rata-rata 0,0136 U/mL pada jam ke-18. Terdapat 2 strain terbaik yang menghasilkan rThlacc1 dengan aktivitas 0,434 Unit/mL (koloni 20) dan 0,411 Unit/mL (koloni 42) pada jam ke-72. Lakase rekombinan rThlacc1 memiliki ukuran 64 kDa atau 6,6 kDa lebih besar dibanding native lakase dari T. hirsuta EDN082.

Laccase (EC 1.10.3.2) is a multi-purpose enzyme belong to the Multi-Copper Oxidase (MCO) family, capable of oxidizing various phenolic and non-phenolic compounds. This enzyme is widely used in various fields, including its application as a "green catalyst" in the bioremediation of hazardous liquid waste. Native laccase from T. hirsuta EDN082 has been successfully isolated, demonstrating bioremediation activity in previous studies. In this study, a laccase-encoding gene was screened from the transcriptome of T. hirsuta EDN082 using novel degenerate primers. The gene was named Thlacc1 where the bioinformatic characterization showed a close relationship with the minor laccase group F, exhibiting a high similarity of 99,20% to laccase 3 (AOX15704.1), with a length of 1497 bp (GC content 62,66%) encoding a mature protein of 499 amino acids. The gene contains copper (Cu) binding regions commonly found in laccase-encoding genes, specifically L1 (HWHGFFQ), L2 (HSHLSTQ), L3 (HPFHLHG), and L4 (HCHIDFHL). It belongs to the High-Redox Potential Laccase (HPRL) group based on the amino acid residues surrounding the L4 region. The Thlacc1 gene encodes a laccase characterized by hydrophilic regions, with an isoelectric point at pH 5,2, four potential N-glycosylation sites and eleven potential O-glycosylation sites. Three-dimensional (3D) structural modeling of the protein revealed a high similarity to the laccase structure from T. hirsuta (PDB ID: 3PXL, RMSD value 0,575 Å), and it has four copper co-factors composed of T1, T2, and T3a/T3ß with substrate binding cavity centered at His458. The Thlacc1 gene was successfully constructed into pGEM-Teasy cloning vector and pPICZaB expression vector. The gene was fused at the N-terminus with the a-mating factor secretion signal by adding an EcoRI restriction site at the 5’ end and the C-terminus with a 6xHis tag by adding a NotI restriction site at the 3’ end. The expression cassette pPICZaB_Thlacc1 was successfully integrated into the genome of P. pastoris BG11 ?AOX1 at the locus of the AOX1 promoter, resulting in 64 strains. Among these, 63 strains (98.4%) could secrete the recombinant laccase rThlacc1 into the medium induced by methanol, exhibiting an average oxidation activity against ABTS of 0.0136 U/mL at 18 hours post-induction. The best two strains were observed to produce rThlacc1 with activities of 0.434 U/mL (colony 20) and 0.411 U/mL (colony 42) at 72 hours post-induction. The recombinant laccase rThlacc1 has a molecular weight of 64 kDa, which is 6.6 kDa larger than the native laccase from T. hirsuta EDN082.