Extraction purification and characterisation of a milk-clotting protease from kesinai (Streblus asper Lour.) leaves

M. Pagthinathan, . and H. M. Ghazali, . and A. M. Yazid, . and Foo H. L., . Extraction purification and characterisation of a milk-clotting protease from kesinai (Streblus asper Lour.) leaves. pp. 913-922. ISSN 2231-7546

Extracts from kesinai (Streblus asper) leaves were investigated as a potential source of enzymes that can serve as an alternative to calf rennet in cheese making. Different types of extraction buffers were investigated namely sodium acetate buffer (pH 4.2-5.0) phosphate buffer (pH 6.0-7.0) and Tris-HCl buffer (pH 7.0-9.0). Finally the milk-clotting enzyme was extracted using 100 mM Tris-HCl buffer (pH 7.4) with and without 5.0 mg/mL polyvinylpyrrolidone 0.015 mL/mL Triton X-100 and 2 mM sodium metabisulphite. Purification was carried out using acetone precipitation and ion-exchange and size-exclusion chromatographic techniques. Results showed that 100 mM Tris-HCl buffer (pH 7.4) was the most efficient extraction buffer among the buffers used in the extraction study. After the final purification step of size-exclusion chromatography the enzyme was purified 3.3-fold with 42.3 of recovery. The enzyme showed an optimum temperature and pH at 60C and pH 7.4 respectively. The enzyme was stable up to 70C for one hour and the partially purified enzyme retained 83 and 96 of its original activity at pH 6.0 and 8.0 respectively. The molecular weight of the partially enzyme was estimated to be 75.8 kDa on SDS-PAGE. The milk-clotting activity of kesinai enzyme was found to be lower than that of commercial Mucor rennet.

Full text available from: Official URL: http://www.ifrj.upm.edu.my/26%20(03)%202019/21%20-...

Abstract

Extracts from kesinai (Streblus asper) leaves were investigated as a potential source of enzymes that can serve as an alternative to calf rennet in cheese making. Different types of extraction buffers were investigated namely sodium acetate buffer (pH 4.2-5.0) phosphate buffer (pH 6.0-7.0) and Tris-HCl buffer (pH 7.0-9.0). Finally the milk-clotting enzyme was extracted using 100 mM Tris-HCl buffer (pH 7.4) with and without 5.0 mg/mL polyvinylpyrrolidone 0.015 mL/mL Triton X-100 and 2 mM sodium metabisulphite. Purification was carried out using acetone precipitation and ion-exchange and size-exclusion chromatographic techniques. Results showed that 100 mM Tris-HCl buffer (pH 7.4) was the most efficient extraction buffer among the buffers used in the extraction study. After the final purification step of size-exclusion chromatography the enzyme was purified 3.3-fold with 42.3 of recovery. The enzyme showed an optimum temperature and pH at 60C and pH 7.4 respectively. The enzyme was stable up to 70C for one hour and the partially purified enzyme retained 83 and 96 of its original activity at pH 6.0 and 8.0 respectively. The molecular weight of the partially enzyme was estimated to be 75.8 kDa on SDS-PAGE. The milk-clotting activity of kesinai enzyme was found to be lower than that of commercial Mucor rennet.

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Item Type:	Article
AGROVOC Term:	Streblus asper
AGROVOC Term:	Leaves
AGROVOC Term:	Leaf extracts
AGROVOC Term:	Milk clotting enzyme
AGROVOC Term:	Purification
AGROVOC Term:	Proteases
AGROVOC Term:	Acetone
AGROVOC Term:	Ion exchange chromatography
AGROVOC Term:	Gel filtration chromatography
AGROVOC Term:	Protein content
Depositing User:	Mr. AFANDI ABDUL MALEK
Last Modified:	24 Apr 2025 00:54
URI:	http://webagris.upm.edu.my/id/eprint/8246

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