黑颈鹤粪便分离菌Arthrobacter sp. GN14的α-半乳糖苷酶基因克隆、表达与酶学特性

A novel α-galactosidase from Arthrobacter sp. GN14 isolated from Grus nigricollis feces: gene cloning,heterologous expression and characterization

【目的】克隆高原唯一珍惜鹤类——黑颈鹤粪便分离菌Arthrobacter sp.GN14的α-半乳糖苷酶基因agaAGN14,并对该酶进行序列分析、系统发育分析和重组酶的酶学特性分析。【方法】利用简并PCR和GCTAIL-PCR方法获得agaAGN14全长,并对其氨基酸序列(AgaAGN14)进行比对和neighbor-joining系统发育树的构建。将agaAGN14重组到载体pET-28a(+)中并转化到Escherichia coli BL21(DE3)中异源表达。利用组氨酸标签纯化重组α-半乳糖苷酶rAgaAGN14并进行酶学性质分析。【结果】agaAGN14全长2109 bp,GC含量66.8%,编码702个氨基酸(77.5 kDa)。AgaAGN14与数据库中序列的最高一致性为53.7%,与其余胃肠道环境α-半乳糖苷酶的一致性<43%。系统发育分析将AgaAGN14聚于具有催化域KWD和SDXXDXXXR的α-半乳糖苷酶分支,与土壤微生物来源α-半乳糖苷酶距离相对较近,而与其余胃肠道环境α-半乳糖苷酶距离相对较远。rAgaAGN14可水解pNPG、棉籽糖、密二糖、水苏糖、菜粕和棉籽粕,表观最适pH为6.5,在pH 6.0-pH 9.0的范围内稳定并维持50%以上的酶活性。rAgaAGN14的表观最适温度为45℃,在10℃、20℃和37℃内稳定并分别具有约28%、30%和80%的酶活。在45℃pH 6.5条件下,rAgaAGN14对pNPG的Km、Vmax和kcat分别为0.41 mmol/L、18.28μmol/min/mg和25.36 s-1。rAgaAGN14受Ag+、Hg2+及SDS抑制,受K+、Ca2+、Mn2+、Fe3+、Ni2+、Cu2+和β-mercaptoethanol部分抑制,受Co2+、Pb2+、Zn2+、Mg2+、Na+和EDTA的影响较小。【结论】首次报道从黑颈鹤粪便中分离到Arthrobacter菌,并对该属细菌α-半乳糖苷酶进行序列分析、系统发育分析、异源表达和重组酶的酶学特性分析。rAgaAGN14序列较新颖,其酶学特性可能是同时适应黑颈鹤肠道环境和高原淡水湿地环境的结果。

[Objective] Cloning and heterologously expressing the α-galactosidase gene（agaAGN14） from Arthrobacter sp.GN14 isolated from feces of black-neck crane（Grus nigricollis）.[Methods] The full-length agaAGN14 was cloned based on degenerate PCR and GC TAIL-PCR（thermal asymmetric interlaced PCR）,ligated into pET-28a（＋） vector and expressed in Escherichia coli BL21（DE3） cells.The recombinant α-galactosidase（rAgaAGN14） was purified to electrophoretic homogeneity by Ni2＋-NTA metal chelating affinity chromatography,and then the enzyme characterizations were determined.Amino acids sequences of agaAGN14（AgaAGN14） and α-galactosidases from Actinobacteria and gastrointestinal microorganisms were aligned and used for constructing a neighbor-joining phylogenetic tree.[Results] The 2109-bp full-length agaAGN14（66.8% GC content） encodes a 702-residue polypeptide（AgaAGN14;77.5 kDa）.AgaAGN14 showed the highest identity of 53.7% with α-galactosidases in public databases,and 43% identities with α-galactosidases from gastrointestinal microorganisms.AgaAGN14 was put in a phylogenetic branch sharing the catalytic motifs KWD and SDXXDXXXR,and close to α-galactosidases from soil microorganisms and far from α-galactosidases from gastrointestinal microorganisms.The purified rAgaAGN14 efficiently hydrolyzed pNPG,raffinose,melibiose,stachyose,rapeseed meal and cottonseed meal;showed apparent optimal at pH 6.0 and 45℃,stability and activity（50%） at pH 6.0-9.0,and activities of 28%,30% and 80% at 10℃,20℃ and 37℃,respectively;exhibited Km,Vmax and kcat values of 0.41 mmol/L,18.28 μmol/min/mg and 25.36 s-1,respectively,using pNPG as the substrate at 45℃ and pH 6.5;strongly inhibited by Ag＋,Hg2＋ and SDS,partial inhibited by K＋,Ca2＋,Mn2＋,Fe3＋,Ni2＋,Cu2＋ and β-mercaptoethanol,and little influenced by Co2＋,Pb2＋,Zn2＋,Mg2＋,Na＋ and EDTA.[Conclusion] The Arthrobacter strain isolated from feces of Grus nigricollis,and the sequence analysis,phylogenetic analysis,heterologous expression and recombinant enzyme＇s biochemical characterizations of an α-galactosidase from Arthrobacter strain were first reported.rAgaAGN14 was a novel α-galactosidase.