Immobjllization Of Amaranthus Tircolor Hydroperoxide Lyase And Its Application In C6Aldehydes Production

Hydroperoxide lyase is a key enzyme in the lipoxygenase (LOX) enzymatic pathway toproduce green Leaf Volatiles (GLVs). However, its instability is an important limitation in itsindustrial application. In this study, the stabilization, immobilization and biocatalysis ofimmobilized hydroperoxide lyase (HPL) from amaranthus tricolor leaves were investigated.The key problem for enzyme immobilization is the rational design and preparation ofcarrier. For HPL immobilization, the comparative study of membrane-bound HPL (M-HPL) toCHT-immobilized HPL (CHT-HPL) was firstly conducted. A maximum activity and proteinloading of M-HPL was determined to be64.67±0.4mg/g and45.27±0.3U/g. However, themaximum activity of CHT-HPL was only2.85±0.1U/g under the optimized couplingconditions. The optimal reaction pH was6.0,6.0and7.5for free HPL, M-HPL and CHT-HPLrespectively, while their respective optimal reaction temperature was30,35and35oC.However, the thermostablity and operational stability of M-HPL were nearly equal to that offree HPL and much lower than that of CHT-HPL. Thus, the more suitable support carrier forHPL immobilization need to be explored to obtain an immobilized enzyme with high activityand improved stability.1,6-hexamethylenediamine attached chitosan-γ-carrageenan (CCH-HMDA-HPL) withchloroplast membrane biomimetic hydrophobic surface was proved to be the more suitablecarrier support for HPL immobilization. A maximum activity of7.49±0.19U/g and a yield of90.3±1.04%were obtained under optimized coupling condition. Meanwhile, the affinitybetween HPL and substrates was not reduced after immobilization. Furthermore, the thermal,operational and storage stabilities of HPL were significantly improved after immobilizationonto CCH-HMDA. Using CCH-HMDA-HPL as the catalyst in the batched reaction, the yieldof2(E)-hexenal and hexanal reached1374.8±51.8mg/L and1987.9±67.9mg/L, respectively.The catalytic productivity of hexanal and2(E)-hexenal were calculated to be1.92±0.07mg/Uand0.344±0.01mg/U.Followed by, the nanosized magnetic particles (NMP) with larger surface area andsurface hydrophobicity were tried to be used for HPL immobilization. HNMP-HPL exhibitedthe higher catalytic activity and protein loading (27.45±1.56mg/g,35.16±1.87U/g) thanCHT-HPL and CCH-HMDA-HPL. Furthermore, when the immobilization of HPL wasconducted in1M PBS, the HNMP-HPL obtained presented a significantly improved stability,including the thermal and operational stabilities of HPL. Meanwhile, the affinity betweenenzyme and substrates was not reduced after immobilization, as evidenced by the fact that theKmvalue of hydroperoxide lyase almost unchanged after immobilization. Using HNMP-HPLas the catalyst in the batched reaction, the yield of2(E)-hexenal and hexanal reached1576±87.9mg/L and3668±121.8mg/L, respectively. The catalytic productivity of hexanaland2(E)-hexenal were calculated to be0.394±0.02mg/U and2.45±0.08mg/U.The cheap and easy prepared CCH-HMDA-HPL was used in the continuous synthesis ofC6aldehydes. In the beginning of the catalytic reaction, the largest volumetric productivity ofhexanal reached3560±102.1mg/L when16U of immobilized HPL was used to catalyze43.5 mM13-HPOD at the residence time of61min. For the continuous reaction, the stability ofimmobilized HPL was significantly improved in the packed-bed reactor. The catalyticproductivity of hexanal was determined to be5.35±0.21mg/U, much higher than that of otherplant original nature HPLs in batch stirred reaction. The reason can be attributed to the higherratio between the immobilized enzyme amount and the substrate in packed-bed reactor.Finally, the purity of hexanal synthesized in this study was determined to be same with thatsynthesized by free HPL. However, this reaction manner was not suitable for2(E)-hexenalsynthesis.Finally, the catalytic activity and kinetic behavior of immobilized HPLs werecomparative studied when it was respective combined with three different support carriers,including porous CHT, spacer arm attached CCH-HMDA and nanosized HNMP. Themaximum catalytic activity of them was achieved when the protein loading (YP/B) was2.45,6.95and16.5mg/g respectively. Kinetic assay showed that KMaof CHT-HPL was not onlygreater than KMbut also increased with YP/B, whereas that of CCH-HMDA-HPL andHNMP-HPL was nearly equal to KM. Additionally, Vmaxof all immobilized HPLs tended todecrease with YP/B. The diffusion-reaction model and multi-layer immobilization model canbe well used to predict catalytic activity of CHT-HPL and CCH-HMDA-HPL, respectively,suggesting that the diffusion limitation of the low-soluble substrate and the multi-layerimmobilization of zymoprotein was the main contributor to the change in their kineticparameters, respectively. Thus, NMP was demonstrated to be the most suitable carrier, wherediffusion resistances of substrate in the reaction process are completely overcome and thelargest surface area allowed more zymoprotein placed as a monolayer.