一株产卡拉胶酶细菌的分离鉴定及其酶学性质

Isolation, identification of a κ-carrageenase-producing bacterium and κ-Carrageenase characterization

【目的】从红树林土壤腐叶中分离出能够产生卡拉胶酶的菌株,对其进行鉴定,并研究其酶学性质。【方法】利用以卡拉胶为唯一碳源的培养基,分离出产卡拉胶酶的菌株;通过形态学观察、16S r DNA序列分析对其进行种属鉴定;对该菌株所产卡拉胶酶进行纯化并采用DNS测酶活的方法测定酶学性质。【结果】从红树林土壤腐叶中分离出1株高产κ-卡拉胶酶的菌株ASY5,经鉴定该菌株为假交替单胞菌属(Pseudoalteromonas sp.ASY5)。纯化得到的κ-卡拉胶酶的分子量约为30 k Da;酶学性质试验表明,其最适反应温度和p H分别为60℃和7.5,在50℃以下酶的稳定性较好,在p H 7.0-9.0范围内酶活力较稳定,对κ-卡拉胶具有良好的底物特异性,以κ-卡拉胶为底物时Km值和Vmax值分别为2.28 mg/m L和147.06μmol/(min·mg),Na+、K+、Ca2+、Mg2+、Al3+等对酶活有显著的促进作用,而Ag+、Zn2+、Cd2+及SDS对酶活有强烈的抑制作用。【结论】分离到的细菌假交替单胞菌Pseudoalteromonas sp.ASY5产生的κ-卡拉胶酶在较高的温度和碱性条件下均具有较高的酶活性,为利用卡拉胶水解酶产生卡拉寡糖的研究和应用奠定了基础。

[Objective] The aim of this study was to screen and identify carrageenase-producing strain from mangrove soil leaf and to characterize produced carrageenase. [Methods] The culture medium withκ-carrageenan as sole carbon source was used to isolate the strain exhibiting carrageenase activity. The isolated strain was identified by morphology observation and 16 S r DNA sequencing. κ-carrageenase produced by Pseudoalteromonas sp. ASY5 was purified and characterized by DNS method. [Results] A bacterial strain ASY5 with high carrageenase activity was isolated from mangrove soil humus,and was identified as Pseudoalteromonas sp. The molecular mass of the purifiedenzyme was estimated to be 30 k Da. The optimal temperature and p H of the enzyme were 60℃ and 7. 5,respectively. The enzyme was stabileat 50℃,and more stable between p H 7. 0 and 9. 0. The enzyme could convert κ-carrageenan. The Kmand Vmaxvalues of the enzyme for κ-carrageenan was 2. 28 mg / m L and 147. 06μmol /（ min·mg）,respectively. The enzyme was significantly stimulated by Na＋,K＋,Ca2 ＋,Mg2 ＋and Al3 ＋. The enzyme was inhibited strongly by Ag＋、Zn2 ＋、Cd2 ＋and SDS. [Conclusion] κ-carrageenase produced by Pseudoalteromonas sp. ASY5 was stable at high temperature and alkaline p H,with potential applicationin carrageenan oligosaccharides production.