Genetic Engineering Laboratory, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia; Plant Molecular Biotechnology Laboratory, Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi 6205, Bangladesh.
Genetic Engineering Laboratory, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia.
Int J Biol Macromol. 2024 Nov;279(Pt 4):135533. doi: 10.1016/j.ijbiomac.2024.135533. Epub 2024 Sep 13.
Although plants don't have chitins, they produce chitinases to protect themselves from biotic and abiotic stressors. There are two forms of chitinases found in organisms: glycosyl hydrolase 18 (GH18) and 19 (GH19) families. Plant GH19 chitinases are well known for their role in protecting against pathogens, but the roles of GH18 chitinases have not been fully elucidated. This study aimed to produce and characterise two recombinant GH18 chitinases from Metroxylon sagu. Two GH18 chitinase genes, MsChi1 and MsChi2, were identified, with nucleotide sequences of 1009 and 1308 bp, respectively. The proteins encoded by MsChi1 and MsChi2 genes were single polypeptide chains of 310 and 300 amino acids with predicted molecular masses of 31.21 and 30.15 kDa, respectively. Both cDNAs were cloned and expressed in the GS115 strain of Pichia pastoris. Recombinant MsChi1 and MsChi2 exhibited optimal activity at 60 °C with acidic pH 4.0 and 5.0, respectively. Both recombinant enzymes could hydrolyze synthetic and natural substrates (colloidal chitin). rMsChi1 preferred 4-nitrophenol N,N'-diacetyl-β-D chitobioside, while rMsChi2 preferred 4-nitrophenol N,N',N″-triacetyl-β-D chitotriose, suggesting they might function as exochitinase and endochitinase, respectively. They also demonstrated antifungal activities against tested fungi. Homology modeling indicated ASP and GLU as essential residues for proton donation and acceptance.
虽然植物没有几丁质，但它们会产生几丁质酶来保护自己免受生物和非生物胁迫。在生物体中发现有两种形式的几丁质酶：糖苷水解酶 18（GH18）和 19（GH19）家族。植物 GH19 几丁质酶以其在抵御病原体方面的作用而闻名，但 GH18 几丁质酶的作用尚未完全阐明。本研究旨在从西谷椰子中生产和表征两种重组 GH18 几丁质酶。鉴定出两个 GH18 几丁质酶基因，MsChi1 和 MsChi2，它们的核苷酸序列分别为 1009 和 1308bp。MsChi1 和 MsChi2 基因编码的蛋白质分别为 310 和 300 个氨基酸的单多肽链，预测分子量分别为 31.21 和 30.15kDa。这两个 cDNA 都被克隆并在毕赤酵母 GS115 菌株中表达。重组 MsChi1 和 MsChi2 在 60°C 时表现出最佳活性，最佳 pH 值分别为酸性 pH4.0 和 5.0。两种重组酶都可以水解合成和天然底物（胶体几丁质）。rMsChi1 优先水解 4-硝基苯酚 N,N'-二乙酰基-β-D 壳二糖，而 rMsChi2 优先水解 4-硝基苯酚 N,N',N″-三乙酰基-β-D 壳三糖，这表明它们可能分别作为外切几丁质酶和内切几丁质酶发挥作用。它们还表现出对测试真菌的抗真菌活性。同源建模表明 ASP 和 GLU 是质子供体和受体的必需残基。
