Chitin Binding Proteins Act Synergistically with Chitinases in Serratia proteamaculans 568

Genome sequence of Serratia proteamaculans 568 revealed the presence of three family 33 chitin binding proteins (CBPs). The three Sp CBPs (Sp CBP21, Sp CBP28 and Sp CBP50) were heterologously expressed and purified. Sp CBP21 and Sp CBP50 showed binding preference to β-chitin, while Sp CBP28 did not bind to chitin and cellulose substrates. Both Sp CBP21 and Sp CBP50 were synergistic with four chitinases from S. proteamaculans 568 (Sp ChiA, Sp ChiB, Sp ChiC and Sp ChiD) in degradation of α- and β-chitin, especially in the presence of external electron donor (reduced glutathione). Sp ChiD benefited most from Sp CBP21 or Sp CBP50 on α-chitin, while Sp ChiB and Sp ChiD had major advantage with these Sp CBPs on β-chitin. Dose responsive studies indicated that both the Sp CBPs exhibit synergism ≥0.2 µM. The addition of both Sp CBP21 and Sp CBP50 in different ratios to a synergistic mixture did not significantly increase the activity. Highly conserved polar residues, important in binding and activity of CBP21 from S. marcescens (Sm CBP21), were present in Sp CBP21 and Sp CBP50, while Sp CBP28 had only one such polar residue. The inability of Sp CBP28 to bind to the test substrates could be attributed to the absence of important polar residues.解黏沙雷菌568的基因组序列分析显示，该菌株编码3种33家族几丁质结合蛋白（chitin binding proteins, CBPs）。本研究对这3种解黏沙雷菌CBPs（Sp CBP21、Sp CBP28及Sp CBP50）进行了异源表达与纯化。其中Sp CBP21与Sp CBP50对β-几丁质（β-chitin）具有结合偏好性，而Sp CBP28无法结合几丁质与纤维素底物。Sp CBP21与Sp CBP50均可与解黏沙雷菌568的4种几丁质酶（chitinases，Sp ChiA、Sp ChiB、Sp ChiC及Sp ChiD）协同降解α-几丁质与β-几丁质，该协同效应在外源电子供体还原型谷胱甘肽（reduced glutathione）存在时更为显著。在α-几丁质降解体系中，Sp ChiD从Sp CBP21或Sp CBP50的协同作用中获益最为明显；而在β-几丁质降解时，Sp ChiB与Sp ChiD可借助这两种CBPs获得最大活性提升。剂量响应实验结果显示，这两种具有底物结合活性的CBPs（Sp CBP21与Sp CBP50）的协同作用浓度阈值均不低于0.2 μM。将Sp CBP21与Sp CBP50以不同比例添加至协同反应体系中，并未显著提升整体酶活。粘质沙雷菌CBP21（Sm CBP21）的结合功能与酶活依赖高度保守的极性残基，Sp CBP21与Sp CBP50中均存在这类保守极性残基，而Sp CBP28仅含有1个此类残基。Sp CBP28无法结合实验所用底物的原因，可归结为其缺乏关键的功能性极性残基。