Introduction

With the topic of biochemical beneficiation of biomass, the main focus is on studies of metalloenzymes from soil bacteria. Lignin is a polyphenolic complex polymer whose amount in plant biomass varies from 10-50%. In various woody and herbaceous plants, lignin is much less abundant than cellulose, but due to its phenolic properties, lignin is seen as having great potential as an alternative to petrochemicals. Because chemically, lignin has a relatively stubborn structure, this also means that its natural decomposition is not easy at all. In nature, there are mainly four types of organisms that cope with this task - white and brown rot fungi and certain gram-positive and gram-negative bacteria. The laboratory of structural biology and lignin biochemistry focuses on the model system of gram-positive soil bacteria, one of the advantages of which compared to the model system based on fungi is the good temperature stability of enzymes derived from them and the ability to work in a wide pH range. In addition, enzymes from various microbes living in extreme environmental conditions have recently been investigated with high industrial potential. In addition to enzymes that degrade/modify lignin, enzymes that degrade carbohydrate components from extremophilic organisms were also examined.

Uniquely in Estonia, the laboratory of structural biology and lignin biochemistry of KBI applies X-ray crystallography of proteins as a method that allows studying the possibilities of rational improvement of enzymes. At the same time, this laboratory is the only one in Estonia that deals with enzyme structure-function studies in the same laboratory and cooperates with synchrotron beam channels both in the USA (Cornell High Energy Synchrotron Source) and in Spain (ALBA Synchrotron). The laboratory has active cooperation with the cellulose research group of the Institute of Molecular and Cell Biology of the University of Tartu.

Membership

Dr. Tiit Lukk Group leader
Dr. Eve-Ly Ojangu
Dr. Kairit Zovo
Hegne Pupart Doctoral student
I carry Thirumalmuthu  Doctoral student
Marcel Mager Master's student
Marja Lipp Doctoral student
Epp Väli Laboratory manager
Elina Ovsijenko Undergraduate student
Johanna Maria Jõgisu Undergraduate student

Alumni

  • Dr. Zeba Usmani - Postdoctoral Fellow
  • Elodie Mammar - Exchange student
  • Alexis Gallien - Exchange student
  • Melissa Ingela Bramanis - Master's student
  • Merli Jõemaa – master's student
  • Matthew Vince - Exchange Student
  • Ander Erik - Bachelor's student

Selected publications

Pupart, H.; Vaschjonok, D.; Lukk, T.; Väljämäe, P. (2024). Dye-Decolorizing Peroxidase of Streptomyces coelicolor (ScDyPB) Exists as a Dynamic Mixture of Kinetically Different Oligomers. ACS Omega. 9, 3866-3876 DOI: 10.1021/acsomega.3c07963.

Pupart, Hegne; Lukk, Tit; Väljämäe, Priit. (2024). Dye-decolorizing peroxidase of Thermobifida halotolerance displays complex kinetics with both substrate inhibition and apparent positive cooperativity. Archives of Biochemistry and Biophysics, #109931. DOI: 10.1016/j.abb.2024.109931.

Pupart, H.; Christmas, P.; in Braman, MI; Lukk, T. (2023). Characterization of the Ensemble of Lignin-Remodeling DyP-Type Peroxidases from Streptomyces coelicolor A3(2). Energies, 16(3). DOI: 10.3390/en16031557.

Zovo, K.; Pupart, H.; Wieren, VW; Gillilan, RE; Huang, Q.; Majumdar, S.; Lukk, T. (2022). Substitution of the Methionine Axial Ligand of the T1 Copper for the Fungal-like Phenylalanine Ligand (M298F) Causes Local Structural Perturbations that Lead to Thermal Instability and Reduced Catalytic Efficiency of the Small Laccase from Streptomyces coelicolor A3(2). ACS Omega. DOI: 10.1021/acsomega.1c06668

Usmani, Z.; Kulp, M.; Lukk, T. (2021). Bioremediation of lindane contaminated soil: Exploring the potential of Actinobacterial strains. Chemosphere, 278, #130468. DOI: 10.1016/j.chemosphere.2021.130468.

Usmani, Z.; Sharma, M.; Awasthi, AK; Lukk, T.; Tuohy, MG; Gong, L.; Nguyen-Tri, P.; Goddard, AD; Bill, RM; Nayak, SC; Gupta, VK (2021). Lignocellulosic biorefineries: The current state of challenges and strategies for efficient commercialization. Renewable and Sustainable Energy Reviews, 148, #111258. DOI: 10.1016/j.rser.2021.111258.

Technical ability

The group's activities are supported by the presence of modern infrastructure. In addition to the use of standard microbiology and molecular biology methods (PCR machines, incubators, electrophoresis equipment), the laboratory is also equipped with chromatography systems for the purification of heterologously expressed proteins.

Protein purification:

  • GE Healthcare AKTA Pure 25 system
  • Cytiva AKTA Start
  • Avestin Emulsiflex C5

Protein liquid chromatography columns:

  • Superdex 200 Increase 5/150 GL - Analytics
  • HiLoad 16/600 Superdex 200 pg
  • HisTrap FF (15x)
  • Hitrap Phenyl FF (5x)
  • HiPrep 26/10 Desalting (2x)
  • HiPrep DEAE FF 16/10

Enzyme kinetics:

  • Shimadzu UV-2700, with thermostatic cuvette holder
  • Shimadzu UV-1900i

Crystallography of proteins:

  • Art Robbins Instruments Crystal Gryphon
  • Rigaku Complact HomeLab diffractometer
  • Molecular Dimensions vibration free crystal incubators (2x)

Current projects

DIGIBIO Center for Digitization of Biology - Next Generation Sustainable Products (European Commission, ETIS)

Completed projects

MOBTT60 – The role of actinomycete metalloproteins in lignin depolymerization and soil chemistry (ETIS e-profile)

RESTA11 – Development of technologies for chemical and biochemical beneficiation of bleached chemical-thermal wood pulp (BCTMP) and untreated secondary wood pulp streams (ETIS e-profile)

RESTA22 – Technologies of chemical and enzymatic beneficiation of lignin: products developed from lignin and phenolic fragments separated from it – basic compounds used for the synthesis of materials, resins, adhesives, oils, plastics, building materials (ETIS e-profile)