Preparation And Application Of Immobilized Angiotensin-converting Enzyme On Chitosan

Angiotensin converting enzyme (ACE) plays an important role in the Renin-angiotensin system and Kallilrein-kinin system. ACE raises blood pressure by converting the inactive decapeptide angiotensin I to the potent vasoconstrictor octapeptide angiotensin Ⅱ, as well as inactivating the vasodilating nonapeptide bradykinin. ACE inhibition has been considered to be an effective therapeutic approach in the treatment of high blood pressure, and ACE inhibitors (ACEI) have become important drugs in the management of blood pressure levels. However, the synthetic ACEIs are believed to cause certain side effects, therefore, recent studies are dedicated to the finding of novel, no side effect, safe and natural ACEIs for the prevention of bioactive of hypertension. And many researchs have been focused on screening ACEI from foods, animals, plants and traditional Chinese medicines (TCMs). Hitherto, about70kinds of TCMs have been used in the traditional Chinese prescription for treatment of hypertensive and have been proved effective. Thus, the TCMs could be a important compounds library for screening of nature ACEI. However, the complex compositions make it difficult to develop a simple, rapid and reliable method for screening of ACEIs.A large quantity of free ACE are needed for the realization of the properties of ACE, and for the screening ACEIs, but the purification of ACE is difficult, the commercial ACE preparations are expensive, and its activity loses fast. The immobilization of enzyme on suitable carriers have been proved effective in improve ACE stability and reusability, while the catalysis properties are maintained. Furthermore, the immobilized enzyme can be subjected to a variety of physical and chemical treatment, with the aim of further improving its activity and stability. The immobilized enzyme with good performance can be used as the affinity absorbent for the high throughput screening of ACEI.In this dissertation, a series of studies were conducted, about the methods for ACE immobilization, the preparation of carriers, regulation of the activity and stability of immobilized ACE, the application of the immobilized ACE in screening of ACEIs, the approach for assay of ACEIs from complex extracts and the evaluation of the bioactivity contribution of each TCM in the traditional Chinese prescription:(1) ACE was immobilized on crosslinked chitosan beads (CCB-ACE) by chemical binding and on modified crosslinked chitosan beads (MCB-ACE) by physical adsorption, respectively. The influence of immobilization strategy on the activity and the effectiveness factor of the immobilized ACE was investigated. The characteristics of CCB-ACE and MCB-ACE including optimal pH and temperature, Km, Vmax, stability and reusability were studied. The results showed that, the activity, effectiveness factor, pH stability, thermal stability, storage stability and reusability of MCB-ACE were superior to that of CCB-ACE. However, for the enzyme immobilized by physical adsorption, the binding force between the enzyme and the carriers was weak, thus the adsorbed enzyme on MCB was more leakiness than the enzyme chemical bonded on CCB. But the leakage rate was low. It could be concluded that the immobilization of ACE on MCB by physical adsorption was preferable and reliable.(2) Although the activity and stability of CCB-ACE was lower than MCB-ACE, the binding between ACE and CCB was more tighter than that of MCB-ACE. The excess aldehyde groups in the carriers of CCB-ACE contributed so largely to its lower activity and stability. Therefore, alkylamines (methylamine, ethylamine, n-butylamine, diethylamine and triethylamine) were used as the quenching agents for deactivation of the excess aldehyde groups in the carriers to regulating the activity and stability of the CCB-ACE. The modification conditions were optimized. The characteristics of CCB-ACE before and after modification including activity, kinetic parameters, optimal pH and temperature, stability and reusability were investigated. The results showed that, under optimal conditions, after modification with2240mM methylamine, the acivity of CCB-ACE was increased2-4times, the surface of the carriers became inert and the the microenvironment around the immobilized ACE was altered.Km decreased from4.33mM to2.61mM, which indicated an improvement of the affinity between immobilized ACE and substrate.(3) Alkylamines had been proved effective in the improvment of the activity and stability of CCB-ACE by deactivating the excess aldehyde groups in the carriers of CCB-ACE. When CCB-ACE was use as affinity absorbent for the screening of ACEIs, the excess aldehyde groups in the surface of the carries will react with some solutes to cause non-specific adsorption (NSA) and give a false binding activity, which is adverse for the specific affinity adsorption of inhibitor screening. To overcome the drawbacks, alkylamines were used as the quenching agents to minimize NSA. Combined with HPLC method, five ACEIs (captopril, lisinopril, perindopril, benazepril and fosinopril) were chose as models to evaluate the changes of the specific adsorption and NSA. The nucleosides in Dracocephalum Tanguticum Maxim were used as mode for validation the shielding effect of alkylamines on NSA. Furthermore, the extract of Hawthorn was screened and identified by affinity adsorption with methylamine-modified CCB-ACE and HPLC analysis. Two main ingredients were identified, they were chlorgenic acid and epicatechin, and the IC50of them were2.98mM and7.65mM, respectively. The method of affinity selection of ACEI from natural drug using immobilized ACE has proved to be simple, rapid, effective and low-consumption. And it was shown that the quenching treatment with alkylamines did minimized NSA that ensured the accuracy and efficiency of the affinity screening method.(4) To develop an online affinity chromatography method for rapid screening ACEI, the core is the preparation of high performance immobilized ACE enzyme column. Compared to the column which packed with chitosan beads, the monolithic carrier was more uniform, and has more surface areas for ACE immobilization. Based on the property of chitosan that it is soluble in acid, but do not dissolve in alkali, a monolithic chitosan column was prepared by coagulating with coagulation olution; Monolithic chitosan column and monolithic albumin/chitosan column were prepared via cryogelation. The preparation conditions of the there column were optimized, and their structures and performances were characterized. Furthermore, the monolithic albumin/chitosan column was used for the ACE immobilization via crosslinking by glutaraldehyde, and the conditions for immobilization were investigated. The characteristics of the immobilized ACE including activity, Km,Vmax,optimal pH and temperature, stability and reusability were studied. The results showed that, the monolithic albumin/chitosan column was a good carrier for ACE immobilization, which was easy to prepared and had good mechanical stability, good mass transport, good convective flow properties and high capacity. The activity and stability of ACE were improved by immobilization. Which laid a foundation for the establishment of an online affinity chromatography method for rapid screening of ACEI.(5) On the basis of the classical method of the determination of ACE activity, the extract from pig lung was used as the enzyme source for ACE, HHL was used as substrate, the solvent induced phase separation extraction (SIPSE) method and UV method were employed for the extraction and determination of hippuric acid (HA) formed during enzymatic reaction. The conditions of SIPSE method were optimized, and the developed SIPSE method had been compared with common LLE approach and HPLC method. Captopril was used as the model for the verification of the developed method. Based on the method, several vegetable juices and extracts of TCMs were measured for ACE inhibition. The results showed that the crude extract of ACE had meet the requirements of the determination of ACE inhibitory activity; SIPSE method was highly selective and efficient in extracting of HA, and the recovery of HA was97.47±0.55%(n=3). By necessary correction, the SIPSE-UV method was accurate and sufficient for the direct evaluation of the ACE inhibitory activity of high-matrix samples.(6) Based on the SIPSE-UV method for the determination of the ACE inhibitory activity in vitro, the inhibitory rate of a traditional Chinese prescription (Gastrodia and Uncaria Decoction, GOD) and the11 kinds of TCMs in the prescription were measured. The HPLC-UV fingerprintings of GOD and the11kinds of single TCM were determined and the differences were analysed. The bioactivity contributions of each TCM for the global bioactivity of the prescription were evaluated. Some TCMs which have high ACE inhibition were combined to form some groups, the inhibition of those groups to ACE and the bioactivity contributions of each groups were measured. The determination of the inhibition and the bioactivity contributions could provide information for the investigation of the antihypertensive mechanism of TCMs from the perspective of the ACE inhibition, for the screening new natural ACEI drugs, and for the quality control of TCMs.