【For INTERNATIONAL COLLABORATIVE RESEARCH FY2023】

DATE: Day 1 Month May Year 2024

SUMMARY ofFY2023 RESEARCH RESULTS REPORTFor International Collaborative Research with IPR, Osaka University
Research Title

Crystallization and X-ray diffraction analysis of halophilic cellulase(CelGH5) from Indonesia local isolate

ApplicantName

Prof. Dr. Ni Nyoman Tri Puspaningsih, M.Si.

Affiliation(1) Department of Chemistry, Faculty of Science and Technology,
Universitas Airlangga, INDONESIA(2) University-CoE-ResearchCenter

for

Bio-MoleculeEngineering,

Universitas Airlangga, INDONESIA
Present Title
Research Collaborator (Host PI)

ProfessorProf. Genji KURISU

SummaryCelGH5 is a lignocellulose-degrading enzyme that has a high novelty in enzyme structure. Understanding thestructural interactions between an enzyme and its substrate can be used to engineer the properties of enzymes toimprove the ability and capability of enzyme catalytic processes. The three-dimensional structure of the enzyme
can elucidate the catalytic mechanism of the enzyme against the substrate, and then how the enzyme interacts withthe substrate and the residues involved can be explained. Therefore, this study was conducted to determine thethree-dimensional structure of CelGH5 and its interaction with the substrate/ligand using an X-ray crystallographicapproach. The specific aims of this research are (1) crystallization of CelGH5 using the optimum condition and (2)exploration of x-ray crystallographic data. The method used in this study consists of enzyme production, enzymepurification, crystallization and X-ray diffraction of enzyme crystals. According to the optimization ofcrystallization, CelGH5 can be crystallized using 18 mg/mL of protein concentration and crystallization conditionof 0.28 M LSM, 23% of PEG 4000, and 0.1 M Tris-HCl pH 8.5 after 7 days of incubation. Some good shapes ofsingle crystals indicated that CelGH5 has the potential to be processed to obtain crystal diffraction data. Thecrystals were picked and cryoprotected with the solution containing 0.30 M LSM, 25% PEG 4000, 0.1 M Tris-HCl
pH 8.5, and 15% glycerol and flash-cooled in a cold nitrogen stream for Spring-8 X-ray diffraction analysis. The
resolution of the diffracted CelGH5 crystal was in the range of 48.08–2.13 (2.17–2.13) Å. The molecularreplacement analysis demonstrated the space group to be P 21 21 2 and cell constants (a, b, c, α, β, γ) to be 166.85
Å, 286.88 Å, 50.11 Å, 90°, 90°, 90°. The CelGH5 enzyme derived by the metagenome approach from palm oil
waste compost soil has enormous potential to be crystallized and determine its three-dimensional protein structure.
Then, the research related to determining the structure of CelGH5 needs to be continued to obtain the final
structure of CelGH5 from this research collaboration. This research is still ongoing to obtain the final structure ofCelGH5 and can be published scientifically. Thus, the achievement of this research in terms of research
papers/articles has not been achieved. The next researcher, who is also a member of our research group, is expected
to get the desired results so that the expected joint scientific publications can be achieved.
*Deadline: May 10, 2024
*Please submit it to E-mail: tanpakuken-kyoten@office.osaka-u.ac.jp.
*Please describe this summary within 1 sheet. Please DON’T add some sheets.
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