The Preparation Of A Multi-site Enzyme Microreactor And Its Application In The Screening Of Active Ingredients Of Drunk Horse Grass

Aims:On the basis of single enzyme micro-reactor,a multi-enzyme micro-reactor was prepared using trypsin and ?-mannosidase as the targets for screening.The micro-reactor was combined with CE to recognize the active components in the extract of Achnatherum inebrians.In addition,separation conditions of Achnatherum inebrians was explored,and the electrophoretogram of Achnatherum inebrians was gained.Methods:1.Physical adsorption and chemical covalent binding were used to prepare CE enzyme micro-reactor.Advantages and disadvantages of them were compared.2.To confirm the effect of the micro-reactor for screening inhibitors from Chinese medicinal herb,BH and SW were respectively selected as the known trypsin and ?-mannosidase inhibitors.3.Trypsin and ?-mannosidase were separately immobilized on the inner of the same capillary through regulating the pH of buffer and lengths of different kinds of enzyme solution.4.Combined to CE,multi-enzyme micro-reactor was used to screen the effective components from the extract of Achnatherum inebrians.5.The extract of Achnatherum inebrians was prepared by alcohol quenching method.The separation conditions of Achnatherum inebrians were identified by CE.Results:1.Trypsin and a-mannosidase was easily coated on the inner of capillary wall by physical adsorption.On the contrary,it was difficult for a-mannosidase immobilized by covalent reaction.The reason maybe the active site of a-mannosidase was covered,and its activity damaged.2.BH was used in the experiment for the inhibition of immobilized trypsin.The mixture of BAEE and BH were injected into the enzyme micro-reactor.Without BH,the peak area of BA was significantly decreased,indicating that BH interacted with trypsin.3.SW was used in the experiment for the inhibition of immobilized a-mannosidase.The mixture of PNP-a-Man and SW were injected into the enzyme micro-reactor.Without SW,the peak of PNP disappeared,indicating that SW interacted with a-mannosidase.4.5 mM BAEE and 5 mM PNP-a-Man were injected into the multi-enzyme micro-reactor.The concentration of 0.1 M,pH=6.0 acetate buffer solution was used as running buffer.According to the addition method,the first peak was the substrate BAEE absorption peak,and the third peak was the product PNP absorption peak.The addition of product BA and substrate PNP-a-Man in solution,and the second peak obviously increased,the second peak was the product PNP and the substrate PNP-a-Man absorption peak under this condition.5.The extract of Achnatherum inebrians was found to inhibit the activity of a-mannosidase.6.The optimizing separation system of the extract of Achnatherum inebrians was constructed with the detection wavelength of 200 nm,sample-injected time for 0.5 psi×5 s,and separation voltage for 25 kV.The concentration of 0.2 M,pH=8.5 boric acid buffer solution was used as running buffer.The separation conditions of Achnatherum inebrians were established.Conclusions:1.It is demonstrated that trypsin micro-reactor and a-mannosidase micro-reactor based CE is available for specifically identifying inhibitors.2.Various types of enzymes are immobilized on the inner of the same capillary by regulating the pH of buffer and different lengths of enzyme solution.The method could structure multi-enzyme micro-reactor,which offers a new way to immobilize kinds of enzymes in the same micro-reactor.3.The multi-enzyme micro-reactor uniting with CE could be used to screen active constituents from the extract of Achnatherum inebrians.The results show that the prepared micro-reactor could be as potential inhibitor screening tool.4.The CE conditions of Achnatherum inebrians are established,and its provid the scientific basis for quality control of Achnatherum inebrians.