Protein surface engineering and interaction studies of maltogenic amylase towards improved enzyme immobilisation
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, cilt.213, ss.70-82, 2022 (SCI-Expanded, Scopus)
- Yayın Türü: Makale / Tam Makale
- Cilt numarası: 213
- Basım Tarihi: 2022
- Doi Numarası: 10.1016/j.ijbiomac.2022.05.169
- Dergi Adı: INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
- Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, BIOSIS, Biotechnology Research Abstracts, CAB Abstracts, Chemical Abstracts Core, EMBASE, Food Science & Technology Abstracts, INSPEC, MEDLINE, Veterinary Science Database
- Sayfa Sayıları: ss.70-82
- Anahtar Kelimeler: Computational analysis, Protein engineering, Enzyme immobilisation, Protein orientation, Protein secondary structure, HETEROFUNCTIONAL CARRIER, COVALENT IMMOBILIZATION, CANDIDA-RUGOSA, STABILITY, LIPASE, ORIENTATION, STRATEGIES, SUPPORTS, SITE, BENTONITE
- Marmara Üniversitesi Adresli: Evet
Özet
A combined strategy of computational, protein engineering and cross-linked enzyme aggregates (CLEAs) approaches was performed on Bacillus lehensis G1 maltogenic amylase (Mag1) to investigate the preferred amino acids and orientation of the cross-linker in constructing stable and efficient biocatalyst. From the computational analysis, Mag1 exhibited the highest binding affinity towards chitosan (-7.5 kcal/mol) and favours having interactions with aspartic acid whereas glutaraldehyde was the least favoured (-3.4 kcal/mol) and has preferences for lysine. A total of eight Mag1 variants were constructed with either Asp or Lys substitutions on different secondary structures surface. Mutant Mag1-mDh exhibited the highest recovery activity (82.3%) in comparison to other Mag1 variants. Mutants-CLEAs exhibited higher thermal stability (20-30% activity) at 80 C whilst Mag1-CLEAs could only retain 9% of activity at the same temperature. Reusability analysis revealed that mutants-CLEAs can be recovered up to 8 cycles whereas Mag1-CLEAs activity could only be retained for up to 6 cycles. Thus, it is evident that amino acids on the enzyme's surface play a crucial role in the construction of highly stable, efficient and recyclable CLEAs. This demonstrates the necessity to determine the preferential amino acid by the cross-linkers in advance to facilitate CLEAs immobilisation for designing efficient biocatalysts.