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 ALI^1,2 & NOOR DINA MUHD NOOR^1,2,*

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

Received: 7 May 2023/Accepted: 21 December 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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*Corresponding author; email: dina@upm.edu.my