bioprocess practical 1.docx

1、bioprocess practical 1Title：ENZYME IMMOBILIZATION: Immobilization of Enzymes by Gel EntrapmentName：Zhang Xiaoyun X00097360Name of partner：Moji Olafisoye Zhang HaoranDate：24/09/2014Abstract:Enzyme immobilization has been developed for decades and become an important part of industry and lab. The expe

2、riment is about the immobilization by gel entrapment. The experiment determines a good working dilution of enzyme firstly，and then compare the activity and specific activity of enzyme(horseradish peroxidase) in Ca-alginate gel and Polyacrylamide acid and the soluble enzyme.Introduction Soluble enzym

3、e has been used for food industry for hundred years and has applications in the pharmaceutical and chemical industries in recent years. Their structure and mode of action are gradually being elucidated through a variety of well-known experimental means, such as X-ray and NMR. Modern genetic engineer

4、ing methods have been used to produce a large number of enzymes and modify their primary structure to change their physic-chemical and biological properties to let that some of the enzyme is more suitable for industrial production.The research of Immobilized enzyme began in 1910 and had been carried

5、 out around the world since 1970. Immobilization is the technology that let enzyme bound with solid materials or limited to a certain area and enzyme can still perform its catalytic reaction and it is recyclable and reusable. Compared with the free enzyme , immobilized enzyme can not only maintain i

6、ts high specificity and mild reaction, but also overcome the shortcomings of the free enzyme. It is more stable and can be separated and recovered easier than free enzyme. Immobilized enzyme has been applied popularly not only in the fields of chemistry , biology and biological engineering ,medical

7、and life sciences ,but also meet the requirements of sustainable development strategy because it saves resources and energy to reduce or prevent pollution.The main advantage of the immobilized biocatalyst is easy to control the reaction and the product easily recovered and purified and have a wide s

8、election of reactor. But it also has the following disadvantages: activity is reduced. Substrates of reaction and diffusion of products are limited and the cost increases.The technology of immobilized enzymes in the industry has developed rapidly and has increasingly become a matter of rational desi

9、gn. Numerous methods of immobilization on a variety of different materials have been developed. They can be classified into two main categories: Physical and Chemical methodsPhysical methods :1. Absorption 2. Entrapment Advantages of Physical methods :the enzyme does not participate in chemical reac

10、tions so the overall structure remains and the catalytic activity of the enzyme is well preserved. However ,because the embedded or semi-permeable membrane with a certain space and the hindrance effect , it can not be applied for some reaction.1. Absorption method : An enzyme or cell containing enzy

11、me are absorbed on the surface of the materials. 2. The carrier binding method: it is more common used than the Absorption method ,Chemical methods:1. Combining method (including ionic binding and covalent binding), crossing-link method.It connects the enzyme and the natural or synthetic polymer car

12、rier by a chemical bond.There are diverse forms of immobilized enzyme which can be made into particles with good mechanical properties for continuous production or have a batch stirred reaction in a reactor. It can also be made into enzyme membrane and enzyme tubes used in analytical chemistry. Micr

13、ocapsules with enzyme can be used as a treatment for clinical application. Nowadays, people can combine enzyme membrane(including membrane of cells, tissues, microorganism) and electrical sensitive components( light, heat) into a biosensor for the determination of organic compounds and harmful subst

14、ances in environment.Immobilized enzyme has more features than soluble enzyme solution, but it was prepared to go through a lot of complex separation and purification of the enzyme. One kind of immobilized enzyme can only be used for specific single-step reaction. So more and more industrial company

15、 started to use the immobilized cell technology. It obviates the time of separation and purification of the enzyme and simultaneous multi-enzyme reaction. Enzyme in the cell can keep the original form, increasing the stability of enzyme.Materials and MethodsReagents:1.Horseradish peroxidase stock en

16、zyme solution at 1.0mg.ml-1 in HEPES buffer2.Peroxidase substrate solution: dissolve 10mg phenol in 40ml dH2O; add 25mg of 4-amino-antipyrine ;dilute to a final volume of 50ml with dH2O3.H2O2 solution(prepare fresh: 1.7mM) used to start the reaction: mix 1ml of 30% with 99ml of dH2O; dilute 1 ml of

17、this solution to 50ml with 0.2M HEPES buffer4.2% w/v Sodium alginate(Alginic acid- sodium salt )5.Ammonium persulfate solution(fresh; 0.1g/ml in 0.2M phosphate buffer)6.KOH solution7.Stock acrylamide solution 40% w/v acrylamide 8.Stock bis-acrylamide solution 2%w/v of methylene bis-acrylamide9.HCL s

18、olution10.0.2M Phosphate buffer, PH7.211.0.03M CaCl212.TEMED13.HEPES buffer,0.2M PH7.2 containing 0.2M CaCl214.0.2M CaCl2Method3.1 Determination of a Working Dilution of Horseradish Peroxidase Preparation of blank and used blank to zero a spectrophotometer Blank contained: 0,5ml dH2O, 1.0ml of amino

19、antipyrine-phenol substrate reagent and 1.0ml H2O2 and waited for 2mins to make sure no reaction in it.Preparation of the different dilution of enzyme (1:25,1:50,1:100) Dilution Enzyme dH2O1:250.04ml(40ul)0.96ml(960ul)1:500.02ml(20ul)0.98ml(980ul)1:1000.01ml(10ul)0.99ml(990ul)In a clean test tube sh

20、ould contain:0.5ml diluted enzyme , 1.0ml aminoantipyrine-phenol substrate reagent and 1.0ml H2O2(finally added to start the reaction)Place those three dilution of enzyme into spectrophotometer record the extinction at 510nm at 30s intervals over 5 mins. Have a graph of each enzyme dilution.3.2Measu

21、rement of Horseradish Peroxidase Activity and Specific ActivityBlank: 0.5ml of dH2O ,1.0ml substrate reagent and 1.0ml H2O2Selected the best working dilution of enzyme （1：100 ） and do the enzyme assayIn a clean cuvette: Added the enzyme and 1.0ml aminoantipyrine-phenol substrate reagent Finally adde

22、d 1.0ml H2O2 to start the reaction.Difference with manual:Manual: In a clean cuvette: 1.0ml aminoantipyrine-phenol substrate reagent and 1.0ml H2O2Finally added the 0.5ml of the diluted enzyme to start the reaction.Allowed the reaction to proceed for 3 minutes exactly. Recorded the extinction at 510

23、nm(A510nm) on the 3min mark. The reaction should be performed in triplicate(1 blank 2 assay).Calculated the enzyme activity per ml of neat enzyme and the specific activity.3.3Immobilization of Horseradish Peroxidase in Polyacrylamide Gel.Didnt do the 3.33.4Preparation od Blank Ca-Alginate Gel BeadsM

24、ixed 10ml of 2%w/v Na-alginate and 5ml of 0.2M HEPES buffer well . Set up a beaker containing :40ml of 0.2M CaCl2 and Stir continuously. Put the burette over the beaker. Dropped the solution into the CaCl2 solution .Allowed the beads to cure for 15mins and then washed with 0.03MCaCl2 solution.3.5Imm

25、obilization of Horseradish Peroxidase in Ca-Alginate GelMixed 4ml of 2%w/v Na-alginate and 2ml of horseradish peroxidase（1mg.ml-1 in HEPES buffer）Transferred them into a burette on a retort stand. Dropped the solution into a stirred beaker contained of 20ml of 0.2MCaCl2 and stirred continuously. All

26、owed the beads to cure for 15mins and then washed with 0.03MCaCl2 solution.Weigh the beads with enzyme and with no enzyme.3.6Enzyme Progress Curves for Soluble and Immobilized Horseradish PeroxidaseSoluble enzyme： the enzyme activity curves developed under method 3.1Immobilized enzyme: Firstly, Set

27、up a blank reaction: To a universal tube, added 0.5g of gel without enzyme and 2.5ml of aminoantipyrine-phenol substrate reagent .Finally added 2.5mlH2O2(time 0)Mixed well, the tube should be mixed gently and continuously during the assay period.Checked the absorbance again at 3 minutes.Use the tube

28、 to zero your spectrophotometer.Secondly, Prepared 0.25g gelTo a universal tube,Added 0.25g of gel immobilized enzyme ,2.5ml of aminoantipyrine-phenol substrate reagent. Finally added 2.5ml H2O2(time 0) to start the reaction. Mixed well, the tube should be mixed gently and continuously during the as

29、say period.At exactly 45, 90,135 and 180 seconds, recorded the extinction at 510nm.Plotted a graph of A510nm versus time.Thirdly, Prepared 0.5g gelTo a universal tube,Added 0.25g of gel immobilized enzyme ，2.5ml of aminoantipyrine-phenol substrate reagent. Finally added 2.5ml H2O2(time 0) to start t

30、he reaction. Mixed well, the tube should be mixed gently and continuously during the assay period.At exactly 45, 90,135 and 180 seconds, recorded the extinction at 510nm.Plotted a graph of A510nm versus time.3.7Assay of Immobilized Peroxidase to Measure Immobilized Enzyme ActivityAs a blank reaction

31、: Added 0.5g of gel without enzyme ，2.0ml of aminoantipyrine-phenol substrate reagent and 2.0ml H2O2As an enzyme assay ：Added 0.5g of gel with enzyme， 2.0ml of aminoantipyrine-phenol substrate reagent and 2.0ml H2O2Allow the assay to proceed for a length of time equivalent to the lag phase. Take thi

32、s time as time 0，and recorded the extinction. Mix well and continue to mix gently and continuously during the assay period. At exactly 3 minutes after time 0 decanted the substrate solution minus the immobilized enzyme to a clean cuvette and recorded A510nm.Calculate the specific activity of the immobilized enzyme.Results3.1 Figure 1Time(S) Blank dilution1:25 dilution1:50 dilution1:100 dilution00.0000.1780.0680.029300.0000.6300.0920