Summary of biology knowledge points from Xuzhou No. 2 Middle School in 2011 (optional module 1)\x0d\\x0d\Test point 1. Application of enzymes: application of enzymes in washing and other aspects; preparation and application of solid-phase enzymes\x0d \1. Application of enzymes in washing and other aspects\x0d\1. Enzyme-added laundry detergent refers to laundry detergent containing enzyme preparations. Currently, there are four commonly used enzyme preparations: protease, lipase, amylase, and cellulase. Among them, the most widely used and most effective ones are alkaline protease and alkaline lipase. Alkaline protease can hydrolyze macromolecular proteins contained in blood stains, milk stains, etc. into soluble amino acids or small molecule peptides, making the stains fall off the clothes. Lipase, amylase and cellulase can also hydrolyze large molecules of fat, starch and cellulose into small molecular substances respectively, giving the laundry detergent a stronger decontamination ability. \x0d\Characteristics of enzyme preparations: It is acid-resistant, alkali-resistant, can withstand surfactants and higher temperatures, and the enzymes are wrapped layer by layer with special chemicals to isolate them from other components of the laundry detergent. \x0d\2. Factors that affect enzyme activity include temperature, pH and surfactant. Enzymes cannot be added directly to laundry detergent because the surfactants in the laundry detergent will reduce the activity of the enzyme. The enzyme produced by genetic engineering is wrapped in a special water-soluble substance and isolated from other ingredients of the washing powder. \x0d\3. Enzyme-added laundry detergent can reduce environmental pollution, because enzyme-added laundry detergent can reduce the amount of surfactant and sodium tripolyphosphate, making detergents develop in the direction of low-phosphorus and phosphorus-free. (The chemical ingredients of ordinary laundry detergents include: surfactants, water softeners, alkali agents, bleaching powder and other ingredients. Some laundry detergents also contain whitening agents, flavors and pigments, and fillers.) \x0d\? Ordinary Washing powder plus enzyme washing powder\x0d\The same point is that surfactant can produce foam and can disperse oil molecules, and the water softener can disperse dirt\x0d\The different point is that enzymes can decompose large molecular organic matter into small molecular organic matter. Small molecules Organic matter is easily dissolved in water and thus separated from the fiber\x0d\4. The residual condition of the dirt can be compared after washing, such as it has disappeared, the color has become lighter, the area has shrunk, etc. to judge the washing effect. \x0d\ 2. Preparation and application of immobilized enzymes \x0d\ 1. Immobilized enzymes refer to enzymes that play a catalytic role within a certain spatial range and can be used repeatedly and continuously. \x0d\Principle: The enzyme is fixed on a water-insoluble carrier, so that the enzyme can easily catalyze the reaction, be easily recovered, and can be reused. \x0d\2. Using immobilized enzyme technology, the enzyme is fixed on a granular carrier, and then these enzyme granules are loaded into a reaction column. The bottom of the column is equipped with a sieve plate with many small holes. The enzyme particles cannot pass through the pores of the sieve plate, but the reaction solution can enter and exit freely. During the production process, the glucose solution is injected from the upper end of the reaction column, causing the glucose solution to flow through the reaction column, contact with the immobilized glucose isomerase, convert it into fructose, and flow out from the lower end of the reaction column. The reaction column can be used continuously for half a year, which greatly reduces production costs and improves fructose yield and quality. \x0d\3. Immobilized enzymes and immobilized cells are technologies that use physical or chemical methods to fix enzymes or cells in a certain space, including embedding, chemical binding, and physical adsorption. Generally speaking, enzymes are more suitable for immobilization using chemical binding and physical adsorption methods, while cells are mostly immobilized using embedding methods. This is because cells are large and enzyme molecules are small; large cells are difficult to adsorb or bind, while small enzymes easily leak out of the embedding material. \x0d\4． The immobilized cell method is to embed microbial cells in a water-insoluble carrier. Commonly used carrier materials include gelatin, agarose, sodium alginate, cellulose acetate, and polyacrylamide.

\x0d\Immobilized cells are developed on the basis of immobilized enzymes. Its advantages are as follows: (1) It eliminates the separation procedures of enzymes. It is a multi-enzyme system and does not require regeneration of cofactors; (2) Cells grow quickly , and there are many, and the reaction is fast; (3) Continuous fermentation is possible, which saves costs, and there is no need to separate and remove cells before distillation and extraction. The fermentation liquid can be drained out while cultivating, eliminating product inhibition and consumption; (4) Maintaining the original state of the enzyme in the cell increases the stability of the enzyme, especially the resistance to contaminating factors. \x0d\ Disadvantages: (1) The integrity of the bacterial cell must be maintained to prevent autolysis of the bacterial cell, otherwise, it will affect Product purity; (2) It is necessary to prevent intracellular proteases from decomposing the required enzymes, and at the same time, it is necessary to inhibit the activity of other intracellular enzymes to prevent the formation of by-products; (3) Cell membranes and walls will hinder substrate penetration and diffusion. \x0d\Type advantages are insufficient\x0d\Direct use of enzymes has high catalytic efficiency, low energy consumption, low pollution, etc. It is very sensitive to environmental conditions and is easily inactivated; the enzyme in the solution is difficult to recover and cannot be reused, which increases production costs; the enzyme will be mixed in the product after the reaction, which may affect product quality. \x0d\Immobilized enzymes can both be in contact with the reactants and separated from the products. The enzymes immobilized on the carrier can also be used repeatedly. An enzyme can only catalyze one chemical reaction, and in production practice, the formation of many products can only be obtained through a series of enzymatic reactions. \x0d\Immobilized cells are low cost and easier to operate. The fixed enzymes or cells are not easily accessible to the reactants, which may lead to a decrease in the reaction effect. \x0d\Application: 1. Students in a certain experimental group want to conduct a glucose solution fermentation experiment by preparing immobilized yeast cells. The experimental materials and equipment are complete. (1) The method used to fix yeast cells is the embedding method. \x0d\ (2) Please improve the steps of the experimental group's preparation of immobilized yeast cells \x0d\ ① Use distilled water when preparing calcium chloride solution. ② To dissolve sodium alginate, use low heat and intermittently heat while stirring. ③The sodium alginate solution must be cooled to room temperature before adding yeast cells. ④The mixture of sodium alginate and yeast cells in the syringe should be dropped into the calcium chloride solution to form gel beads. \x0d\(3) The experimental team used the device as shown in the picture to conduct glucose fermentation\x0d\① In order to enable the immobilized yeast cells used in the experiment to be used repeatedly, they must be kept under sterile conditions during the experiment. proceed below. \x0d\②The operation after adding the reaction solution is to close piston 1 and piston 2. \x0d\③What is the function of the long catheter of the device? Release CO2 and reduce the pressure in the reaction column; prevent air from entering the reaction column. \x0d\ (4) During the experiment, phenomena that may be observed: bubbles are produced, and the smell of wine is emitted\x0d\ During the experiment, the reaction formula that may occur in the device is: (aerobic respiration, anaerobic respiration Produce alcohol and carbon dioxide)\x0d\Application: 2. Wort can penetrate into gel beads made of sodium alginate and brewer's yeast. Brewer's yeast can use a series of enzymes in its own cells to convert fermentable sugars into ethanol. The following is the experimental process of fermenting beer using immobilized yeast cells:\x0d\Step 1: Activation of yeast cells. Weigh 1g of dry yeast and place it in a 50mL beaker, add 10mL of distilled water, stir and let stand for 1 hour. \x0d\Step 2: Prepare a calcium chloride (CaCl2) solution with a substance concentration of 0.05mol/L. \x0d\Step 3: Prepare sodium alginate solution. Weigh 0.7g sodium alginate and place it in a 50ml beaker, add 10mL distilled water, place the beaker on an alcohol lamp and heat it over low heat or intermittently. \x0d\Step 4: Add activated yeast cells to the sodium alginate solution cooled to room temperature, mix thoroughly and transfer to syringe\x0d\Step 5: Immobilize yeast cells. Slowly drop the solution in the syringe into the prepared calcium chloride (CaCl2) solution at a constant speed to form gel beads, and let the gel beads soak in the calcium chloride (CaCl2) solution for 30 minutes. \x0d\Step 6: Rinse the immobilized yeast cells (gel beads) with distilled water 2 to 3 times. \x0d\Step 7: Put an appropriate amount of gel beads into a 500mL Erlenmeyer flask, add 300mL of sterilized wort, seal and ferment at 25°C. \x0d\ Read the above process carefully and answer the following questions: \x0d\ (1) The purpose of step 6 of rinsing with distilled water 2 to 3 times is to wash away impurities (calcium chloride) and bacteria to prevent contamination.

\x0d\(2) The fermentation product alcohol can be tested with potassium dichromate, but it needs to be under acidic conditions to appear gray-green. \x0d\(3) How to check whether the quality of gel beads is qualified? (Method one is to use tweezers to pick up a gel bead and put it on the experimental table and squeeze it with your hands. If the gel bead is not easy to break and no liquid flows out, it means that the gel bead is successfully made. Method two is to put it on the experimental table Smash the gel beads hard. If the gel beads bounce easily, it also indicates that the prepared gel beads are successful)\x0d\Test point 2. Application of biotechnology in food processing: Basic methods of fermented food processing\ x0d\ 1. Fermentation: Broadly speaking: it is a process of mass production of various metabolites through the cultivation of microorganisms. Including aerobic fermentation (such as acetic fermentation, glutamic acid fermentation) and anaerobic fermentation (such as alcoholic fermentation). Narrow sense: refers to the anaerobic respiration of microorganisms (including alcohol fermentation, lactic acid fermentation, etc.). So: fermentation ≠ anaerobic respiration. \x0d\Applications: wine making, steamed bread making, bread making, alcohol manufacturing, production of medicinal yeast tablets, production of vitamins, production of antibiotics, etc. \x0d\ 2. Principle of making fruit wine: strain: yeast, unicellular eukaryotic organism, heterotrophic facultative anaerobic type, budding and reproduction under suitable conditions 1. Facultative anaerobic lifestyle of yeast: in the presence of oxygen Under conditions, aerobic respiration and mass reproduction (asexual budding reproduction). Alcohol ferments under anaerobic conditions. The optimal temperature for reproduction: 20℃; the optimal temperature for alcohol fermentation: 18~25℃. \x0d\ In anoxic, 20°C (usually the temperature is controlled at 18-25°C, optimally 20°C), acidic fermentation liquid, yeast can reproduce and perform alcoholic fermentation. The vast majority of other microorganisms are suppressed because they cannot adapt to this environment. 2. The effect of temperature on fermentation: Yeast can only live at a certain temperature. When the temperature is below 10°C, yeast development is very slow. As the temperature increases, the reproduction rate accelerates, and the optimal reproduction temperature is 20°C. At this time, the yeast reproduces quickly and has strong vitality. Above 35°C, the growth of yeast is inhibited and the reproduction rate decreases rapidly. At 40°C, yeast stops budding and begins to die. If you want to obtain fermentation liquid with high alcohol concentration and reduce the final loss, you must control the fermentation temperature. \x0d\3. Measures to prevent the fermentation liquid from being contaminated: the juicer should be washed and dried, the fermentation bottle should be washed and disinfected with 70% alcohol, and the grape juice should be sealed after filling it\x0d\4. The wine will be red. Reason: During the fermentation process, as the alcohol content increases, the pigment from the red grape skin also enters the fermentation liquid, making the wine red. \x0d\ 3. Principle of making fruit vinegar: Bacteria: acetic acid bacteria, prokaryotes, heterotrophic aerobic type, binary fission reproduction 1. The principle of turning wine into vinegar: When oxygen and sugar sources are sufficient, acetic acid bacteria convert the grapes into vinegar. The sugar in the juice is decomposed into acetic acid; when there is a lack of sugar sources, acetic acid bacteria change ethanol into acetaldehyde, and then acetaldehyde into acetic acid. C2H5OH+O2→CH3COOH+H2O (Fermentation conditions: suitable temperature, timely ventilation, controlled sugar source supply) 2. The role of controlling fermentation conditions: ① Acetic acid bacteria are particularly sensitive to the content of oxygen. When deep fermentation is carried out, even if the oxygen is only interrupted for a short time , can also cause the death of acetic acid bacteria. ②The optimal growth temperature of acetic acid bacteria is 30~35℃. Control the fermentation temperature to shorten the fermentation time and reduce the chance of contamination by miscellaneous bacteria. 3. Source of acetic acid bacteria: Bacteria can be purchased from local vinegar-producing factories or strain preservation centers. Acetic acid bacteria can also be isolated from vinegar. The production process of fruit wine and vinegar: select grapes→rinse→juice→alcoholic fermentation→fruit wine (→acetic acid fermentation→fruit vinegar) 4. Principle of fermented bean curd production: strains: Mucor, eukaryotic, heterotrophic aerobic, spore Reproduction\x0d\1. A variety of microorganisms participate in the fermentation of tofu, such as Penicillium, yeast, Aspergillus, Mucor, etc. Among them, Mucor plays a major role. Mucor is a filamentous fungus. The protease produced by microorganisms such as Mucor can break down the protein in tofu into small molecule peptides and amino acids; the lipase can hydrolyze fat into glycerol and fatty acids. 2. Experimental process for making fermented bean curd: let mucor mold grow on the tofu → add salt and marinate → add brine and bottle → seal and marinate (1) Growth of mucor mold: Place the tofu pieces flat in the cage, and place the tofu in the cage The temperature is controlled at 15~18℃ and maintained at a certain temperature. After about 48 hours, Mucor began to grow. After 3 days, mycelium grew vigorously. After 5 days, the surface of the tofu block was covered with mycelium.

The Mucor growing on tofu blocks comes from Mucor spores in the air. Modern fermented bean curd production involves inoculating fine Mucor species directly onto tofu under strict sterile conditions. This can avoid contamination from other bacterial species and ensure Product quality. (2) Salt pickling: Arrange the tofu blocks covered with mucor mold neatly in layers in the bottle, and add salt layer by layer at the same time. As the number of layers increases, the amount of salt increases, and the salt close to the surface of the bottle mouth Be thicker. The salting time is about 8 days. Adding salt can precipitate the moisture in the tofu, making the tofu blocks hard and preventing them from becoming crispy prematurely during the later production process. At the same time, salt can inhibit the growth of microorganisms and prevent the tofu pieces from spoiling. (3) Preparing the stewed soup: The stewed soup is directly related to the color, aroma and taste of fermented bean curd. Braised soup is made from wine and various spices. The wine content in braised soup is generally controlled at about 12%. Adding wine can inhibit the growth of microorganisms and give fermented bean curd a unique aroma. Spices can modulate the flavor of fermented bean curd and also have antiseptic and sterilizing effects. 3. Experimental precautions (1) Control the amount of materials used: ① When marinating with salt, pay attention to controlling the amount of salt used. The salt concentration is too low, which is not enough to inhibit the growth of microorganisms and may cause the tofu to become spoiled; the salt concentration is too high. High will affect the taste of fermented bean curd. ②The alcohol content in the stewed soup should be controlled at about 12%. The higher the alcohol content, the greater the inhibitory effect on protease, which will extend the maturity period of fermented bean curd; the alcohol content is too low to inhibit the growth of microorganisms and the activity of protease is high. , speed up the hydrolysis of protein, bacteria breed quickly, tofu is easy to spoil and difficult to form into blocks. ③The water content of the tofu used is about 70%. If the water content is too high, it will not be easy to shape. \x0d\ (2) Prevent bacterial contamination: ① Glass bottles used for pickling fermented bean curd should be disinfected with boiling water after being cleaned. ②When bottling, operate quickly and carefully. After placing the tofu neatly and adding the stew, seal the mouth of the bottle with a tape. When sealing the bottle, it is best to pass the bottle mouth through the flame of an alcohol lamp to prevent the bottle mouth from being contaminated. ③The closer it is to the mouth of the bottle, the greater the possibility of bacterial contamination. Therefore, the amount of salt added should increase as the number of tofu layers increases, and the salt close to the surface of the bottle should be spread thicker. \x0d\Test point 3. Application of biotechnology in other aspects: protein extraction and separation\x0d\1. Basic principles and methods of protein extraction and separation\x0d\1. Experimental principle: physical and chemical properties of protein: shape, Various proteins are extracted and separated by their wide variation in size, charge nature and magnitude, solubility, adsorption properties, affinity, etc. 2. Gel chromatography (distribution chromatography): (1) Principle: Molecules with large molecular weight pass through the gaps of porous gel particles with a short distance and flow fast; molecules with small molecular weight pass through the interior of porous gel particles with a long distance. Flows slowly. (2) Gel material: porous, polysaccharide compounds, such as dextran and agarose. (3) Separation process: The mixture is put on the column → elute → large molecules flow quickly and small molecules flow slowly → collect large molecules → collect small molecules (elution: continuously inject buffer from the upper end of the chromatographic column to promote the differential flow of protein molecules . ) (4) Function: Separating proteins, determining the molecular weight of biological macromolecules, desalting proteins, etc. 3. Buffer solution: (1) Principle: It is composed of weak acid and corresponding strong base and weak acid salt (such as H2CO3-NaHCO3, HC-NaC, NaH2PO4/Na2HPO4, etc.). By adjusting the dosage of acid and salt, buffer solutions with different pH can be prepared. (2) Function: Resist the interference of external acids and bases on the pH of the solution and maintain pH stability. 4. Gel electrophoresis method: (1) Principle: Different proteins have different charged properties, charges, shapes and sizes, and the force size, direction and resistance they receive in the electric field are different, resulting in different movement directions and speeds of different proteins in the electric field. . (2) Separation methods: agarose gel electrophoresis, polyacrylamide gel electrophoresis, etc. (3) Separation process: At a certain pH, make the protein groups positively or negatively charged; add SDS with more negative charges to form a "protein-SDS complex", so that the protein migration rate only depends on the size of the molecule. \x0d\ 2. Experimental steps for protein extraction and separation: \x0d\ 1. Sample processing ① Washing of red blood cells: The purpose of washing red blood cells is to remove impurity proteins. The collected blood samples should be centrifuged at low speed and for a short time to separate the red blood cells in time, and then use Use a plastic tip straw to suck out the transparent yellow plasma in the upper layer, pour the dark red red blood cell liquid in the lower layer into the beaker, then add five times the volume of normal saline, stir slowly for 10 minutes, centrifuge at low speed for a short time, and repeat washing three times until there is no more liquid in the supernatant. Yellow indicates that the red blood cells have been washed.

②Release of hemoglobin?: Under the action of distilled water and toluene, red blood cells rupture and release hemoglobin. (Note: After adding distilled water, the volume of red blood cell solution should be the same as the original blood volume. The purpose of adding toluene is to dissolve the cell membrane, which is beneficial to the release and separation of hemoglobin.) 2. Rough separation ① Separation of hemoglobin solution: Centrifuge the stirred mixed solution , the solution in the test tube is divided into 4 layers. The first layer is a colorless and transparent toluene layer, the second layer is a thin white solid layer, which is the precipitation layer of fat-soluble substances, the third layer is a red transparent liquid, which is an aqueous solution of hemoglobin, and the fourth layer is a dark layer of other impurities. Red precipitate. Filter the liquid in the test tube with filter paper to remove the soluble precipitate layer. After letting it stand for a while in a separatory funnel, separate out the red transparent liquid in the lower layer. ② Dialysis: Put 1 mL of hemoglobin solution into a dialysis bag, put the dialysis bag into a phosphate buffer solution with a concentration of 20 mmol/L containing 300 mL of substance, and dialyze for 12 hours. Dialysis can remove smaller molecular weight impurities from a sample, or it can be used to replace the sample's buffer. 3. Purification: 4. Purity identification: SDS polyacrylamide gel electrophoresis\x0d\ 3. Notes 1. Electrophoresis technology: Electrophoresis technology uses the charged properties of various molecules in the sample to be separated and the molecules themselves under the action of an electric field. Differences in size, shape and other properties cause charged molecules to have different migration speeds, thereby achieving the purpose of separating, identifying or purifying samples. 2. Washing of red blood cells: If the stratification is not obvious, it may be due to the lack of washing times and failure to remove plasma proteins. In addition, if the centrifugation speed is too high and the time is too long, white blood cells and lymphocytes will precipitate together, and pure red blood cells will not be obtained, which will affect the subsequent purity of hemoglobin extraction. 3. How to check whether the gel chromatography column is successfully packed: Since gel is a translucent medium, you can place a fluorescent lamp perpendicular to the gel column next to the gel column to check whether the gel is evenly filled. In addition, macromolecules of colored substances can also be added to observe the movement of the ribbon. If the color bands are uniform, narrow, and flat, it indicates that the gel chromatography column is performing well. If lines or bubbles appear on the chromatographic column, gently tap the column to eliminate the bubbles. If the bubbles cannot be eliminated, repack the column. 4. Why does gel filling need to be tight and even? If the gel is not packed tightly and evenly enough, ineffective gaps will be formed in the chromatographic column, allowing sample molecules that should enter the gel to pass through these gaps, disrupting the flow order of the eluent, and affecting the separation effect. 5. The purpose of treating the wet gel with eluent added in a boiling water bath is not only to save time, but also to remove possible microorganisms in the gel and eliminate the air in the gel. 6. G-75: "G" represents the cross-linking degree, swelling degree and separation range of the gel. 75 represents the water value of the gel, that is, each gram of gel absorbs 7.5g of water when it swells. 7. After filling, immediately use eluent to elute the purpose: to make the gel packed tightly 8. What is the purpose of adding sodium citrate? Why centrifuge at low speed and for a short time? Why stir slowly? Prevent blood coagulation; prevent white blood cells from settling; prevent red blood cells from rupturing and releasing hemoglobin. 9. Compared with other eukaryotic cells, the characteristics of red blood cells and the significance of this feature for protein separation: Mature red blood cells of mammals and humans are double-concave round cake-shaped, without nuclei and organelles. The hemoglobin it contains is a colored protein, so the color can be observed during gel chromatography separation to determine when the dehydrated liquid should be collected. This makes the hemoglobin separation process very intuitive and greatly simplifies experimental operations. 10． How to test whether the separation of hemoglobin is successful: If the gel chromatography column is packed successfully and the separation operation is correct, you can clearly see that the red zone of hemoglobin is uniform, narrow, and flat, flowing out slowly with the eluent; if the red zone The zones are distorted, scattered, and broadened, indicating that the separation effect is not good, which is related to the packing of the gel chromatography column