Sains Malaysiana 50(2)(2021): 339-349

http://dx.doi.org/10.17576/jsm-2021-5002-06

Study of Thermostable Chitinase Isolated and Purified From Oryctes rhinoceros Larvae Gut

(Kajian Termostat Enzim Kitinolitik Diasingkan dan Ditulenkan daripada Perut Larva Kumbang Tanduk Oryctes rhinoceros)

AISYAH NABILA IDRIS¹, TANG SWEE SEONG¹ & AHMAD FARIS MOHD ADNAN^1,2*

¹Institute of Biological Sciences, Faculty of Sciences, University of Malaya, 50603 Kuala Lumpur, Federal Territory, Malaysia

²Centre of Ionics, University of Malaya, 50603 Kuala Lumpur, Federal Territory, Malaysia

Received: 16 June 2019/Accepted: 17 July 2020

ABSTRACT

A chitinase-producing bacteria was locally isolated from rhinoceros beetle (Oryctes rhinoceros) larvae gut discovered in mushroom compost. A strain was selected from the source and labeled as RBLG1. It was identified using 16s rDNA sequencing and has strong homology to Bacillus cereus. Chitinase produced from RBLG1 was purified using ammonium sulfate precipitation, ion exchange chromatography, and gel filtration. The purified chitinase from RBLG1 shows the homogeneity 1.9 fold after the gel filtration process. The purified chitinase was identified and analyzed using SDS-PAGE and the molecular mass from the strain was approximately 40 kDa. Further assays were performed to study the effect of temperature, pH, and metal ion on the purified enzyme. The purified enzyme shows highest chitinase activity at pH 4 and 60 °C. For metal ion test, chitinase from RBLG1 was unaffected when treated by various ions. Kinetic performance of chitinase using Michaelis-Menten equation shows K_m and V_max values were 2.3 mM and 0.0294 [P] mM/min, respectively.

Keywords: Chitinase; Michaelis-Menten; purification; rhinoceros beetle larvae

ABSTRAK

Bakteria yang dapat menghasilkan kitinase ini telah diasingkan daripada perut larva kumbang tanduk (Oryctes rhinoceros) yang ditemui di dalam kompos cendawan. Satu strain bakteria dipilih daripada sumber tersebut dan dilabel sebagai RBLG1. Ia telah dikenal pasti menggunakan urutan 16s rDNA dan mempunyai homologi kuat kepada Bacillus cereus. Kitinase yang dihasilkan daripada RBLG1 ini kemudian menjalani proses penulenan menggunakan pemendakan amonium sulfat, kromatografi pertukaran ion dan penapisan gel. Kitinase yang dibersihkan daripada RBLG1 menunjukkan homogeniti 1.9 kali ganda selepas proses penapisan gel. Kitinase yang telah dibersihkan telah dikenal pasti dan dianalisis dengan menggunakan SDS-PAGE dan jisim molekul adalah pada saiz 40 kDa. Kajian lanjut dijalankan untuk mengkaji kesan enzim pada suhu, pH, kepekatan substrat dan juga ion logam pada enzim yang telah dibersihkan. Enzim yang ditulenkan menunjukkan aktiviti tertinggi pada pH 4 dan 60 °C. Bagi ujian ion logam, kitinase daripada RBLG1 tidak menunjukkan perubahan apabila ditindakkan dengan pelbagai ion. Kajian prestasi kinetik bagi kitinase dilakukan menggunakan prinsip Michaelis-Menten menunjukkan nilai Km dan Vmax masing-masing adalah 0.3 mM dan 0.0294 [P] mM/min.

Kata kunci: Kitinase; larva kumbang tanduk; Michaelis-Menten; penulenan

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*Corresponding author; email: ahmad_farisz@um.edu.my