Engineering a Disulfide Bond in the Lid Hinge Region of <em>Rhizopus chinensis</em> Lipase: Increased Thermostability and Altered Acyl Chain Length Specificity

<div><p>The key to enzyme function is the maintenance of an appropriate balance between molecular stability and structural flexibility. The lid domain which is very important for “interfacial activation” is the most flexible part in the lipase structure. In this work, rational design was applied to explore the relationship between lid rigidity and lipase activity by introducing a disulfide bond in the hinge region of the lid, in the hope of improving the thermostability of <em>R. chinensis</em> lipase through stabilization of the lid domain without interfering with its catalytic performance. A disulfide bridge between F95C and F214C was introduced into the lipase from <em>R. chinensis</em> in the hinge region of the lid according to the prediction of the “Disulfide by Design” algorithm. The disulfide variant showed substantially improved thermostability with an eleven-fold increase in the <em>t</em>_1/2 value at 60°C and a 7°C increase of <em>T</em>_m compared with the parent enzyme, probably contributed by the stabilization of the geometric structure of the lid region. The additional disulfide bond did not interfere with the catalytic rate (<em>k</em>_cat) and the catalytic efficiency towards the short-chain fatty acid substrate, however, the catalytic efficiency of the disulfide variant towards pNPP decreased by 1.5-fold probably due to the block of the hydrophobic substrate channel by the disulfide bond. Furthermore, in the synthesis of fatty acid methyl esters, the maximum conversion rate by RCLCYS reached 95% which was 9% higher than that by RCL. This is the first report on improving the thermostability of the lipase from <em>R. chinensis</em> by introduction of a disulfide bond in the lid hinge region without compromising the catalytic rate.</p> </div

Enzyme properties of RCLCYS and RCL.

<p>Enzyme properties of RCLCYS and RCL.</p

Proposed substrate binding region of RCLCYS in an open form.

<p>Red, the introduced disulfide bond; Blue, the catalytic triad of S145-H257-D204; Yellow, the substrate binding region.</p

Solvent-free conversion of soy bean oil to fatty acid methyl esters by RCLCYS and RCL at different temperatures.

<p>The various curves refer to the conversion of soy bean oil by lipases as a function of temperature at different intervals of reaction time.</p

Time course of solvent-free conversion of soy bean oil to fatty acid methyl esters by RCLCYS and RCL at 40°C.

<p>Time course of solvent-free conversion of soy bean oil to fatty acid methyl esters by RCLCYS and RCL at 40°C.</p

Thermal inactivation of RCLCYS and RCL at 60°C.

<p>Thermal inactivation of RCLCYS and RCL at 60°C.</p

Models of the simulated structure of RCL in a close form.

<p>Red, the lid hinge regions (⁸²GTNS⁸⁵ and ⁹²DMVFT⁹⁶), F214/F95 and N84/G266 as ball and stick; Blue, the catalytic triad of S145-H257-D204; Green, three original disulfide bonds; Pink, the free cysteine C177; Purple, the lid region (⁸⁶FRSAIT⁹¹).</p

Primers for plasmid pPIC9K-proRCLCYS construction.

<p>Primers for plasmid pPIC9K-proRCLCYS construction.</p

Heat-induced denaturation of RCLCYS and RCL.

<p>CD spectra of RCLCYS (•) and RCL (▪) were recorded at 220 nm at the temperatures indicated.</p