D181A Site-Mutagenesis Enhances Both the Hydrolyzing and Transfructosylating Activities of BmSUC1, a Novel β-Fructofuranosidase in the Silkworm Bombyx mori.
D181A Site-Mutagenesis Enhances Both the Hydrolyzing and Transfructosylating Activities of BmSUC1, a Novel β-Fructofuranosidase in the Silkworm Bombyx mori.
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DOI：
10.3390/ijms19030683
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发表时间：
2018-02-28
影响因子：
5.6
通讯作者：
Meng Y
中科院分区：
文献类型：
--
作者：
Gan Q;Li X;Zhang X;Wu L;Ye C;Wang Y;Gao J;Meng Y
β-呋喃果糖苷酶（β-fructofuranosidase，β-FFase）属于糖基水解酶家族32（GH 32），可催化β-D-呋喃果糖苷水解蔗糖，并合成短链低聚果糖（FOS）。BmSuc 1是从家蚕中克隆并鉴定的第一个动物型β-FFase编码基因。推测BmSUC 1在家蚕-桑树适应系统中起重要作用。然而，关于BmSUC 1的酶核心位点的信息很少。在本研究中，我们通过定点突变将代表GH 32中β-FF酶活性的重要保守基序的三个氨基酸残基（D 63、D181和E234）分别突变为丙氨酸。通过Bac-to-Bac/BmNPV表达系统和BmN细胞获得了3个突变体和野生型BmSUC 1的重组蛋白。分析了这四种蛋白的酶活性、动力学性质和底物特异性。采用高效液相色谱法（HPLC）比较了D181 A和wtBmSUC 1的水解和转果糖基活性。我们的研究结果表明，D 63 A和E234 A突变失去了活性，这表明D 63和E234是BmSUC 1作为酶发挥功能的关键氨基酸残基。D181 A突变显著增强BmSUC 1的水解和转果糖基活性，表明D181可能不直接参与催化。该结果为深入了解BmSUC 1在B中的化学催化机制提供了线索。桑。BmSUC 1转果糖基活性的上调为B的利用提供了新的思路。mori β-FFase产生功能性FOS。
β-fructofuranosidase (β-FFase) belongs to the glycosyl-hydrolase family 32 (GH32), which can catalyze both the release of β-fructose from β-d-fructofuranoside substrates to hydrolyze sucrose and the synthesis of short-chain fructooligosaccharide (FOS). BmSuc1 has been cloned and identified from the silkworm Bombyx mori as a first animal type of β-FFase encoding gene. It was hypothesized that BmSUC1 plays an important role in the silkworm-mulberry adaptation system. However, there is little information about the enzymatic core sites of BmSUC1. In this study, we mutated three amino acid residues (D63, D181, and E234) that represent important conserved motifs for β-FFase activity in GH32 to alanine respectively by using site-directed mutagenesis. Recombinant proteins of three mutants and wild type BmSUC1 were obtained by using a Bac-to-Bac/BmNPV expression system and BmN cells. Enzymatic activity, kinetic properties, and substrate specificity of the four proteins were analyzed. High Performance Liquid Chromatography (HPLC) was used to compare the hydrolyzing and transfructosylating activities between D181A and wtBmSUC1. Our results revealed that the D63A and E234A mutations lost activity, suggesting that D63 and E234 are key amino acid residues for BmSUC1 to function as an enzyme. The D181A mutation significantly enhanced both hydrolyzing and transfructosylating activities of BmSUC1, indicating that D181 may not be directly involved in catalyzation. The results provide insight into the chemical catalyzation mechanism of BmSUC1 in B. mori. Up-regulated transfructosylating activity of BmSUC1 could provide new ideas for using B. mori β-FFase to produce functional FOS.
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影响因子：
12.3
作者：
Keeling CI;Yuen MM;Liao NY;Docking TR;Chan SK;Taylor GA;Palmquist DL;Jackman SD;Nguyen A;Li M;Henderson H;Janes JK;Zhao Y;Pandoh P;Moore R;Sperling FA;Huber DP;Birol I;Jones SJ;Bohlmann J
通讯作者：
Bohlmann J
影响因子：
5.1
作者：
Altenbach, Denise;Rudino-Pinera, Enrique;Ritsema, Tita
通讯作者：
Ritsema, Tita
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DOI：
10.1016/j.cbpb.2015.04.006
发表时间：
2015-08-01
影响因子：
2.2
作者：
Li, Wentian;Gong, Meixia;Meng, Yan
通讯作者：
Meng, Yan
影响因子：
--
作者：
Omori T;Ueno K;Muramatsu K;Kikuchi M;Onodera S;Shiomi N
通讯作者：
Shiomi N
影响因子：
2.8
作者：
Ozimek, LK;Kralj, S;Dijkhuizen, L
通讯作者：
Dijkhuizen, L