海洋细菌Pseudoalteromonas sp.01中新型褐藻胶裂解酶AlyPC1的研究  

Study on the New Alginate Lyase AlyPC1 from Marine Bacterium Pseudoalteromonas sp.01

作　　者：张红秀 刘晓华 刘伟治[1] Zhang Hongxiu;Liu Xiaohua;Liu Weizhi(Key Laboratory of Marine Genetics and Breeding(Ministry of Education),College of Marine Life Sciences,Ocean University of China,Qingdao 266100,China)

机构地区：[1]中国海洋大学海洋生命学院海洋生物遗传学与育种教育部重点实验室,山东青岛266003

出　　处：《中国海洋大学学报（自然科学版）》2024年第2期84-88,105,共6页Periodical of Ocean University of China

基　　金：国家重点研究发展计划项目(2018YFC0311105)资助。

摘　　要：为挖掘新型褐藻胶裂解酶,使其开发成为制备褐藻寡糖的工具酶,本研究中表征了来源于海洋细菌Pseudoalteromonas sp.01的褐藻胶裂解酶AlyPC1。本文作者在生物信息学分析的基础上,对AlyPC1进行了重组表达及酶学性质的研究。序列比对结果显示,AlyPC1是PL7家族第五亚家族的一个褐藻胶裂解酶。酶学性质研究发现:AlyPC1最适反应温度为35℃,将AlyPC1置于5~30℃孵育1 h,仍保留80%以上的酶活;在Tris-HCl缓冲液中,AlyPC1最适反应pH为8.0,当pH在7.5~9.0之间时,酶活保留了90%以上,证明AlyPC1具有良好的温度稳定性以及宽泛的pH耐受性;反应体系中NaCl的含量为200 mmol/L时,AlyPC1的酶活最高;乙二胺四乙酸(EDTA)以及一些金属离子如Mn^(2+)、Zn^(2+)和Ni^(2+)等能够明显抑制AlyPC1的活性。AlyPC1能够降解褐藻胶、polyG以及polyM三种底物,因而说明AlyPC1是一种双功能褐藻胶裂解酶。同时,AlyPC1以内切的形式降解褐藻胶,产生二糖、三糖以及四糖。To explore new alginate lyases and develop them as the tool enzymes for the preparation of alginate oligosaccharides,an alginate lyase AlyPC1 from Pseudoalteromonas sp.01 was characterized.On the basis of bioinformatics analysis,the clone,recombinant expression,and enzymatic properties of AlyPC1 were studied.Sequence alignment showed that AlyPC1 belonged to the fifth subfamily of PL7.The optimal temperature of AlyPC1 was 35℃,and over 80%of the initial activity was retained after incubation at 5~30℃for 1 h.The optimal pH value of AlyPC1 was 8.0 in Tris-HCl buffer,and the enzyme activity of AlyPC1 was retained over 90%in the range of pH 7.5 to 9.0.It was proved that AlyPC1 showed good temperature stability and broad pH tolerance.AlyPC1 exhibited the maximum activity in the buffer with 200 mmol/L NaCl.EDTA and metal ions,such as Mn^(2+),Zn^(2+),and Ni^(2+),significantly inhibited the enzyme activity of AlyPC1.AlyPC1 was a bifunctional alginate lyase,which could cleave alginate,polyG,and polyM.Meanwhile,AlyPC1 degraded alginate into alginate oligosaccharides with the composition of disaccharides,trisaccharides,and tetrasaccharides.

关 键 词：褐藻胶裂解酶 褐藻寡糖 内切酶 双功能 稳定性 

分 类 号：Q557[生物学—生物化学]