Sains Malaysiana 52(12)(2023):3407-3419
http://doi.org/10.17576/jsm-2023-5212-05
Rand
Protease: The Role of Calcium-Binding Site on Stability with Mutagenesis and
the Effectiveness on Leather Dehairing
(Rand
Protease: Peranan Tapak Pengikat Kalsium terhadap Kestabilan dengan Mutagenesis dan Keberkesanan terhadap Penyahbuluan Kulit)
PHANG ZI WEI1,
NUR ALIYAH MOHD AZRIN2, MOHD SHUKURI MOHAMAD ALI1,2 &
NOOR DINA MUHD NOOR1,2,*
1Department of
Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
2Enzyme and
Microbial Research Center, Faculty of Biotechnology
& Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
Diserahkan: 7 Mei 2023/Diterima: 21 Disember 2023
ABSTRACT
Bacillus subtilis produces a number of proteases, which are highly demanded in various
industries, especially the thermostable one. Rand protease, originally isolated
from B. subtilis, has thermostability and
other remarkable properties such as organic solvent tolerance and pH stability.
However, its vulnerability to instability-induced degradation has limited its
applications. Because Rand protease contains two calcium ions for folding,
activation, and, above all, stability, previous studies have shown that
boosting the calcium-binding affinity enhances stability. Therefore, Rand
protease’s susceptibility to degradation could be remedied by discovering the
calcium-binding site having the greatest impact on stability for further
calcium-binding affinity improvement. This was done with an in silico mutagenesis approach whereby one
calcium-binding site was mutated to alanine and evaluated either the RMSD, the
deviation of the mutated configuration from the original configuration using
YASARA, or stability in terms of kcal/mol using HotSpot Wizard. The result found that
calcium-binding sites Leu75 from YASARA and Tyr171 from HotSpot Wizard have higher influences on stability (our target). This result was also
validated using Pymol, ExPASy ProtParam, and Molprobity.
Additionally, Rand protease-chemical formulation dehairs leather best without additional metal ions at pH 7.0 and for 18 h. It also produced higher-quality
leather with smaller pores and softer leather than chemical
formulations. In contrast, hair breakage was observed in calcium treatment,
which is compatible with the low dehairing activity
achieved. In conclusion, Leu75 and Tyr171 are vital for calcium stabilisation
and this enzyme has demonstrated its crucial efficacy in the leather dehairing industry.
Keywords: Calcium-binding site; Leather dehairing industry; Rand protease; stability
ABSTRAK
Bacillus subtilis menghasilkan sejumlah protease yang amat diperlukan
dalam pelbagai industri, terutamanya yang berkaitan dengan kestabilan terma.
Protein Rand yang dipencilkan daripada B. subtilis mempunyai kestabilan
terma dan ciri luar biasa lain seperti toleransi terhadap pelarut organik dan
kestabilan pH. Walau bagaimanapun, kelemahannya terhadap degradasi akibat
ketidakstabilan telah mengehadkan penggunaannya. Oleh kerana protein Rand
mengandungi dua ion kalsium untuk lipatan, pengaktifan dan yang paling penting
sekali kestabilan, kajian terdahulu telah menunjukkan bahawa peningkatan
keafinan pengikat kalsium mampu meningkatkan kestabilan. Oleh itu, kerentanan
protein Rand terhadap degradasi boleh diperbaiki dengan meneliti tapak pengikat
kalsium yang mempunyai kesan paling besar terhadap kestabilan untuk
menambahbaik keafinan pengikat kalsium. Ini boleh dilakukan dengan pendekatan
mutagenesis secara in silico dengan satu tapak pengikat kalsium dimutasi kepada
alanina dan penilaiannya dilakukan sama ada daripada segiRMSD, sisihan
konfigurasi bermutasi daripada konfigurasi asal menggunakan YASARA, atau daripada
segi kestabilan, kcal/mol menggunakan HotSpot Wizard. Hasilnya, didapati bahawa
tapak pengikat kalsium Leu75 daripada YASARA dan Tyr171
daripada HotSpot Wizard, mempunyai pengaruh yang lebih tinggi terhadap
kestabilan (sasaran) berbanding tapak pengikat
kalsium yang lain. Keputusan ini juga telah disahkan menggunakan Pymol,
ExPASy ProtParam dan Molprobity. Tambahan pula, formulasi kimia protein Rand
mengenyah kulit dengan baik tanpa ion logam tambahan pada pH 7.0, selama 18 jam. Ia juga menghasilkan kulit
berkualiti tinggi seperti liang pori yang lebih kecil dan kulit yang lebih
lembut berbanding formulasi kimia. Selain itu, tahap kepatahan rambut yang
turut diperhatikan dalam rawatan kalsium, menunjukkan keserasian apabila
aktiviti penyahbuluan kulit mencapai tahap yang rendah. Kesimpulannya, Leu75
dan Tyr171 adalah penting untuk penstabilan kalsium dan enzim ini telah
menunjukkan keberkesanannya yang penting dalam industri penyahbuluan kulit.
Kata kunci: Industri penyahbuluan
kulit; kestabilan; protein Rand; tapak pengikat kalsium
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*Pengarang untuk surat-menyurat; email: dina@upm.edu.my
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