A complex saccharifying enzyme and a saccharifying method thereof

Abstract

The application discloses a kind of compound saccharifying enzyme and saccharifying method thereof, belong to compound saccharifying enzyme technical field, the compound saccharifying enzyme is prepared by the following steps: removing transglycosidase, mixing;The removal transglycosidase, after saccharifying enzyme is mixed with purified water, stirring is carried out at room temperature, obtain the saccharifying enzyme after dilution, the pH value of saccharifying enzyme after dilution is adjusted to 3-3.2, precipitant is added, stirring, filtration, take filtrate, obtain the saccharifying enzyme after removing transglycosidase;The mixing, after pulanase is mixed with auxiliary, stirring is carried out at 25-30 DEG C, remove transglycosidase after saccharifying enzyme is added, stirring, obtain compound saccharifying enzyme;The application can improve saccharification efficiency and the purity of saccharification liquid, low in cost, when using high concentration starch slurry and ultra-high concentration starch slurry as substrate, still can realize high saccharification efficiency, and obtain high-purity saccharification liquid.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of compound saccharifying enzyme, and particularly relates to a compound saccharifying enzyme and a saccharifying method thereof. BACKGROUND

[0002] The preparation of glucose by amylase is an important part of the starch deep processing industry, and has formed a large-scale industry of millions of tons in China. The process route of preparing glucose by amylase is generally as follows: under the mediation of alpha-amylase, starch molecules are enzymatically degraded from the inside to low-molecular-mass dextrin and low-polymerization-degree malt oligosaccharide, i.e. starch liquefaction liquid, and then the starch liquefaction liquid is saccharified under the synergistic action of saccharifying enzyme and pullulanase to maximize the release of glucose.

[0003] However, when saccharifying the starch liquefaction liquid, the following problems exist:

[0004] The first problem is the low saccharification efficiency when using high-concentration starch slurry and ultra-high-concentration starch slurry as the substrate. According to the content disclosed in New-type saccharifying enzyme for improving glucose yield, Zhou Xiujin, Sichuan Food and Fermentation, May 1994, glucose amylase (saccharifying enzyme) can decompose the combined state of alpha-1,4 and alpha-1,6 glucoside of starch, and can almost completely decompose into glucose for a dilute starch solution, but there is a problem of difficult complete saccharification for a high-concentration starch solution with a mass concentration of 30% or more. In addition, according to the content disclosed in Application research on ultra-high-concentration liquid saccharification, Li Yi, Peng Hui, Bai Nixi, Tao Jin, Xu Hongxian, Tong Yi, Agricultural Products Processing, September 2019, in the traditional starch sugar-making process, a starch slurry substrate with a mass concentration of 25-35% is generally used for liquefaction. Since the concentration of the starch slurry substrate is relatively dilute, a large amount of water has to be evaporated in the downstream process, i.e. the sugar liquid concentration process, which consumes a large amount of energy and increases the production cost. At present, most enterprises are aware of the advantages of high-concentration starch slurry for sugar production, and gradually begin to optimize the saccharification process. However, the rate of enzyme-catalyzed reaction in the saccharification process is greatly affected by the diffusion of the substrate and the enzyme. With the increase of the concentration of the starch slurry substrate, the migration rate of water molecules decreases. When the mass concentration of the starch slurry substrate increases from 10% to 45%, the migration rate of water molecules decreases by more than 90%. The relatively low migration rate of water molecules will hinder the diffusion of the enzyme, and the increase of the starch concentration will reduce the effective combination of the enzyme and the substrate, thereby negatively affecting the saccharification of the saccharifying enzyme and the pullulanase.

[0005] The second problem is that the residual glycosidase in the saccharifying enzyme has a great influence on the saccharification efficiency. According to the new ideas for improving the purity of saccharifying enzyme in industrial production, the contents published in Gwanqing, Wang Juan, Mai Guoqin, Yu Shaowen. Food Science and Technology. April 2010, in recent years, great progress has been made in the activity and yield of saccharifying enzyme and the fineness of enzyme preparation, but there is a problem that the activity of glucosidase transferase in saccharifying enzyme is generally high. Glucosidase transferase can cut the alpha-1, 4 glycosidic bond of maltose and connect the free glucose residue to another glucose or maltose molecule in the form of alpha-1, 6 glycosidic bond to generate non-fermentable sugars such as isomaltose and panose, which has a great influence on the quality of saccharifying enzyme, especially in the glucose production industry, the conversion rate and purity of glucose will be reduced after using such saccharifying enzyme.

[0006] To solve the above problems, the following methods are used in the prior art:

[0007] To solve the above first problem, according to the application research of ultra-high concentration liquid saccharification, Li Yi, Peng Hui, Bai Nixi, Tao Jin, Xu Hongxian, Tong Yi. Agricultural Products Processing. September 2019, the current solution is mainly to improve the quality of saccharifying enzyme and introduce effective auxiliary enzyme preparation that can significantly improve the saccharification effect to improve the saccharification efficiency of ultra-high concentration starch slurry. However, in improving the quality of saccharifying enzyme, the method of genetic modification is mainly used, which has high research and development cost; in introducing effective auxiliary enzyme preparation that can significantly improve the saccharification effect, the current method is mainly to compound saccharifying enzyme and pullulanase and control the quality ratio of the two, but according to the research on saccharification of high-concentration malt dextrin by enzyme method, Qu Shiyang. Master's thesis of Jiangnan University. June 2014, malt dextrin occurs simultaneously during saccharification. The forward reaction and the complex reaction are particularly serious when the substrate concentration is high. Moreover, according to the new saccharifying enzyme for improving glucose yield, Zhou Xiujin. Sichuan Food and Fermentation. May 1994, for high-concentration starch solution, the transglycosidase in saccharifying enzyme has an inverse effect, which will polymerize the generated glucose, and when using pullulanase, it will also cause the content of trisaccharide in the saccharified liquid to increase. Further, the current solution has a small improvement in saccharification efficiency and also causes high trisaccharide content in the saccharified liquid.

[0008] For the above second problem, according to the new idea of improving the purity of glucoamylase in industrial production. Gan Wenqing, Wang Juan, Ma Guoqin, Yu Shaowen. Food Science and Technology. In April 2010, the contents of the public, at present, the method of removing alpha-glucosidase in glucoamylase mainly for precipitation method, diatomite method and ion exchange resin method. Among them, the precipitation method is mainly through the treatment of phosphotungstic acid, sodium dodecyl sulfate, tannic acid three reagents on the glycosylase, the precipitation method is based on the treatment of liquid glucoamylase fermentation broth with negatively charged reagent, because alpha-glucosidase is positively charged, glucoamylase is negatively charged, therefore, the negatively charged reagent can be targeted to neutralize the positively charged alpha-glucosidase, further, realize the purpose of precipitating and separating the glycosylase; In the precipitation method, according to the different reagents used, the removal efficiency of alpha-glucosidase is also different, and the activity of glucoamylase is also affected to different degrees, among them, the effect is better, tannic acid can remove glycosylase, increase the activity of glucoamylase by 3.8%, but the removal rate of tannic acid on glycosylase can only reach 70.3%. Diatomite method is to use the surface of diatomite with negative charge, when treating liquid glucoamylase fermentation broth, diatomite can adsorb positively charged alpha-glucosidase and other positively charged substances in the fermentation broth; But the removal rate of diatomite method on glycosylase is not high, the removal rate is only 41.6%, and it will reduce the activity of glucoamylase. Ion exchange column method is to pass the liquid glucoamylase fermentation broth through the cation exchange column, the cation exchange column can be targeted to retain alpha-glucosidase on the column, so as to achieve the effect of separation, the efficiency of removing alpha-glucosidase in glucoamylase by this method is as high as 90%, and it has no effect on the enzyme activity of glucoamylase, but this method also has the defect that the influence degree of various influencing factors on the exchange process is relatively fuzzy, and the cation exchange column also has the problems of easy failure and difficulty in regeneration, which further leads to the problem of high cost. Thus, the current solution has little effect on the conversion rate and purity of glucose, or has the problem of high cost.

[0009] In summary, when preparing glucose by amylase method, there are problems of low saccharification efficiency when using high concentration starch slurry and ultra-high concentration starch slurry as substrate and the influence of residual glycosidase in glucoamylase on saccharification efficiency, and the existing solutions have certain limitations in solving the above two problems respectively, further, leading to small improvement in saccharification efficiency, low purity of saccharification liquid and high cost when saccharifying, especially using high concentration starch slurry and ultra-high concentration starch slurry as substrate. SUMMARY

[0010] In view of the deficiencies of the prior art, the present application provides a compound glucoamylase and a saccharification method, which can improve the saccharification efficiency and the purity of the saccharification liquid, has low cost, and can still achieve high saccharification efficiency and obtain high-purity saccharification liquid when using high-concentration starch slurry and ultra-high-concentration starch slurry as substrate.

[0011] To solve the above technical problems, the technical solutions adopted by the present application are as follows:

[0012] A compound saccharifying enzyme is prepared by the following steps: removing transglycosidase, mixing;

[0013] After the transglycosidase is removed, the saccharifying enzyme is mixed with purified water, and then stirred at a stirring speed of 30-60 rpm at room temperature for 10-30 min to obtain diluted saccharifying enzyme. Hydrochloric acid is added to the diluted saccharifying enzyme to adjust the pH value to 3-3.2. A precipitant is added, and the stirring is continued for 30-40 min. Filtration is performed, and the filtrate is obtained to obtain the saccharifying enzyme after the transglycosidase is removed.

[0014] In the step of removing the transglycosidase, the mass ratio of the saccharifying enzyme to the purified water is 1:15-16.

[0015] The mass ratio of the precipitant to the purified water is 1.5-1.6:100.

[0016] The saccharifying enzyme is in a liquid dosage form, and the enzyme activity is 100,000 U / mL.

[0017] The molar concentration of the hydrochloric acid is 1 mol / L.

[0018] In the preparation method of the precipitant, corn starch is mixed with a first portion of buffer, and then stirred at a stirring speed of 30-60 rpm at 90-92°C for 60-70 min. Cooling is performed to room temperature. Tannic acid is added, and then stirred at a stirring speed of 30-60 rpm at room temperature for 60-90 min. Standing is performed at room temperature for 25-30 h. Centrifugation is performed at a centrifugal speed of 8000-9000 rpm for 15-20 min. The supernatant is removed, and the precipitate is obtained. The precipitate is washed with anhydrous ethanol for 2-3 times. The first freeze-drying is performed to obtain a complex. The complex is mixed with a second portion of buffer, and then stirred at a stirring speed of 30-60 rpm at room temperature for 10-30 min. Sodium dodecyl sulfate is added, and then stirred for 60-80 min. Sodium bicarbonate aqueous solution is added to adjust the pH value to 6-6.2. Centrifugation is performed at a centrifugal speed of 8000-9000 rpm for 15-20 min. The supernatant is removed, and the precipitate is obtained. The second freeze-drying is performed to obtain the precipitant.

[0019] In the preparation of the precipitant, the mass ratio of the corn starch to the first portion of buffer is 2:98-100.

[0020] The mass ratio of the corn starch to the tannic acid is 2:1.2-1.4.

[0021] The mass ratio of the complex to the second portion of buffer is 2:20-25.

[0022] The mass ratio of the complex to sodium dodecyl sulfate is 2:0.8-0.9;

[0023] Both the first and second buffer solutions were acetate-sodium acetate buffer solutions, both with a pH of 4 and a molar concentration of 0.2 mol / L.

[0024] The mass concentration of the sodium bicarbonate aqueous solution is 10%.

[0025] The temperature for the first and second freeze-drying processes is -40°C to -35°C, and the time is 34-36 hours.

[0026] The mixing process involves mixing pullulanase with the adjuvant, stirring at 30-60 rpm for 2-2.5 hours at 25-30°C, adding glucoamylase after removing transglycosylase, and continuing to stir for 60-80 minutes to obtain a compound glucoamylase.

[0027] In the mixture, the mass ratio of pullulanase to the adjuvant is 100:1-1.1;

[0028] The mass ratio of pullulanase to glucoamylase after the removal of transglycosidase is 100:2500-2600;

[0029] The pullulanase is a liquid preparation with an enzyme activity of 5000 U / mL;

[0030] The preparation method of the additive is as follows: corn starch is mixed with the first part of hydrochloric acid, and stirred at 30-60 rpm for 120-150 min at 60-65℃. Sodium hydroxide aqueous solution is added to adjust the pH value to 7, and stirred at 30-60 rpm for 50-60 min at 85-90℃. The temperature is then lowered to 60-65℃ while maintaining the stirring speed. Sodium dodecyl sulfate is added, and the second part of hydrochloric acid is added to adjust the pH value to 4-4.5. The stirring is continued for 30-40 min, sodium bicarbonate aqueous solution is added to adjust the pH value to 6-6.2, calcium chloride aqueous solution is added, and the stirring is continued for 60-70 min to obtain a liquid. 4-5 times the volume of ethanol is added to the liquid, and the stirring is continued for 30-40 min. The mixture is allowed to stand for 25-30 h, and then centrifuged at 8000-9000 rpm for 15-20 min. The supernatant is removed, the precipitate is collected, and freeze-dried to obtain the additive.

[0031] In the preparation of the auxiliary agent, the mass ratio of corn starch to the first part of hydrochloric acid is 10:28-30;

[0032] The mass ratio of corn starch to sodium dodecyl sulfate is 10:0.9-1;

[0033] The mass ratio of corn starch to calcium chloride aqueous solution is 10:0.9-1;

[0034] The molar concentration of the first and second hydrochloric acid is 1 mol / L;

[0035] The molar concentration of the sodium hydroxide aqueous solution is 10 mol / L;

[0036] The mass concentration of the sodium bicarbonate aqueous solution is 10%;

[0037] The mass concentration of the calcium chloride aqueous solution is 10%;

[0038] The volume concentration of the ethanol is 95%;

[0039] The temperature of the freeze-drying is -40℃ to -35℃, and the time is 34-36h.

[0040] A saccharification method of the foregoing complex saccharifying enzyme, comprising the following steps: liquefaction, saccharification;

[0041] In the liquefaction, the high-concentration corn starch material liquid is added with hydrochloric acid to adjust the pH value to 5.6-5.8, then medium-temperature α-amylase and anhydrous calcium chloride are added, and the stirring speed is 50-100 rpm at 70-75℃ for 25-30 min, the temperature is increased to 92-94℃, the stirring speed is unchanged, high-temperature α-amylase is added, and the stirring is continued for 18-19 min to obtain a liquefied liquid;

[0042] In the liquefaction, the mass concentration of the high-concentration corn starch material liquid is 40-45%;

[0043] The medium-temperature α-amylase is in a powder form, and the enzyme activity is 2000 U / g;

[0044] The high-temperature α-amylase is in a liquid form, and the enzyme activity is 20,000 U / mL;

[0045] The molar concentration of the hydrochloric acid is 1 mol / L;

[0046] The amount of the medium-temperature α-amylase used is 14-15 U / g of corn starch;

[0047] The mass ratio of corn starch to anhydrous calcium chloride is 40-45:0.2-0.21;

[0048] The amount of the high-temperature α-amylase used is 12-13 U / g of corn starch;

[0049] In the saccharification, the liquefied liquid is stirred at 62-65℃ with a stirring speed of 50-100 rpm, hydrochloric acid is added to the liquefied liquid to adjust the pH value to 4.3-4.4, then the complex saccharifying enzyme is added, and the stirring is continued for 40-42h, then filtration is performed, the filtrate is taken to obtain a saccharified liquid;

[0050] The molar concentration of the hydrochloric acid is 1 mol / L in the saccharification.

[0051] The amount of the saccharifying enzyme in the compound saccharifying enzyme is 80-82 U / g of corn starch.

[0052] Compared with the prior art, the present application has the following advantages:

[0053] (1) In the preparation of the compound saccharifying enzyme of the present application, the steps include removing transglycosidase and mixing; wherein, the removing transglycosidase is using a precipitant to precipitate the transglycosidase in the saccharifying enzyme, the preparation method of the precipitant is first mixing corn starch and tannic acid, there is hydrogen bond between the corn starch and the tannic acid, thereby reducing the solubility of the corn starch and the tannic acid, obtaining a precipitate containing corn starch and tannic acid, then mixing the precipitate with sodium dodecyl sulfate, the sodium dodecyl sulfate and the precipitate are combined by intermolecular forces, obtaining a complex of sodium dodecyl sulfate, tannic acid and corn starch, i.e. the precipitant; the outer layer of the precipitant is sodium dodecyl sulfate, the inside is a porous complex of corn starch and tannic acid, both the sodium dodecyl sulfate and the tannic acid have negative charges, which can selectively adsorb the transglycosidase, the complex formed by the corn starch and the tannic acid in the inside can further improve the adsorption capacity of the transglycosidase, and since the complex formed by the combination of the corn starch and the tannic acid has negative charges, it can also avoid the adsorption of the saccharifying enzyme; the mixing is mixing pullulanase, an additive and the saccharifying enzyme after removing the transglycosidase, the additive is mixing corn starch and sodium dodecyl sulfate, then adding calcium ions, the sodium dodecyl sulfate can improve the dispersibility of the corn starch, the calcium ions can combine part of the dodecyl sulfate, obtaining a mixture of calcium dodecyl sulfate and corn starch, at the same time, the remaining sodium dodecyl sulfate can play a dispersing role, thereby obtaining an additive with an outer layer of sodium dodecyl sulfate and an inner layer of a mixture of calcium dodecyl sulfate and corn starch, the additive can fix the pullulanase, improve the flowability of the pullulanase, the pullulanase can promote the decomposition of the branched chain, improve the flowability of the substrate, further promote the mixing of the saccharifying enzyme and the substrate, and promote saccharification.

[0054] (2) The present application can improve the saccharification efficiency and the purity of the saccharification liquid, the precipitant is prepared by using tannic acid, corn starch and sodium dodecyl sulfate as raw materials, the cost is low, when using high-concentration starch slurry and ultra-high-concentration starch slurry as the substrate, it can still achieve high saccharification efficiency and obtain high-purity saccharification liquid. After removing the transglycosidase from the saccharifying enzyme by the method of the present application, the saccharifying enzyme activity change rate is -0.07% to 0%, and the transglycosidase removal rate is 93.2-94.6%; according to the method of the present application, when treating corn starch liquid with a mass concentration of 40-45%, the DX value of the obtained saccharification liquid is 97.0-97.3%, the DE value is 99.1-99.6%, and the trisaccharide content in the saccharification liquid is 0.19-0.21%. DETAILED DESCRIPTION

[0055] In order to have a clearer understanding of the technical features, objectives and effects of the present application, the specific embodiments of the present application will be described.

[0056] When testing the enzyme activity of saccharifying enzyme and transglycosylase in saccharifying enzyme, the method disclosed in the research on removal of transglycosylase in saccharifying enzyme by precipitation method by Lin Weixuan, Yun Xia, Liu Yingxin and Lin Chunlai, Journal of Dalian Institute of Light Industry, March 1996 is referred to.

[0057] When testing the DX value and DE value in the saccharifying liquid, the method disclosed in the research on significant improvement of efficiency of preparation of glucose by amylase method by Cong Huihui, Zhang Yanan, Niu Dandan, Zhao Jihua and Wang Zhengxiang, Food and Fermentation Industries, June 2022 and the research on influencing factors of double-enzyme method corn starch saccharification reaction by Ke Xinqing, Cereals and Oils, February 2019 is referred to.

[0058] Example 1

[0059] The present embodiment provides a compound saccharifying enzyme, and the preparation method thereof specifically comprises the following steps:

[0060] 1. Removing transglycosylase: mixing saccharifying enzyme and purified water according to a mass ratio of 1:15, stirring at a stirring speed of 30 rpm for 10 min at room temperature to obtain diluted saccharifying enzyme, adding hydrochloric acid with a molar concentration of 1 mol / L to adjust the pH value to 3, adding a precipitant, controlling the mass ratio of the precipitant and purified water to be 1.5:100, continuing to stir for 30 min, filtering, taking the filtrate, and obtaining saccharifying enzyme after removing transglycosylase;

[0061] The saccharifying enzyme is in a liquid dosage form, and the enzyme activity is 100,000 U / mL;

[0062] The preparation method of the precipitant is: mixing corn starch and the first buffer according to a mass ratio of 2:98, stirring at 90 DEG C at a stirring speed of 30 rpm for 60 min, cooling to room temperature, adding tannic acid, controlling the mass ratio of corn starch to tannic acid to be 2:1.2, stirring at room temperature at a stirring speed of 30 rpm for 60 min, standing at room temperature for 25 h, centrifuging at a centrifugal speed of 8000 rpm for 15 min, removing supernatant, taking precipitate, washing the precipitate with anhydrous ethanol twice, freeze-drying at-40 DEG C for 34 h, and obtaining a complex; mixing all the obtained complexes with the second buffer according to a mass ratio of 2:20, stirring at room temperature at a stirring speed of 30 rpm for 10 min, adding sodium dodecyl sulfate, controlling the mass ratio of the complex to sodium dodecyl sulfate to be 2:0.8, stirring for 60 min, adding 10% sodium bicarbonate aqueous solution to adjust the pH value to 6, centrifuging at a centrifugal speed of 8000 rpm for 15 min, removing supernatant, taking precipitate, and freeze-drying at-40 DEG C for 34 h to obtain a precipitate;

[0063] The first buffer and the second buffer are both acetic acid-sodium acetate buffers, the pH value of each is 4, and the molar concentration of each is 0.2 mol / L;

[0064] 2. Mixing: mixing pullulanase and adjuvant according to a mass ratio of 100:1, stirring at 25 DEG C at a stirring speed of 30 rpm for 2 h, adding saccharifying enzyme after removing transglycosidase, controlling the mass ratio of pullulanase to saccharifying enzyme after removing transglycosidase to be 100:2500, and continuing to stir for 60 min to obtain compounded saccharifying enzyme;

[0065] The pullulanase is a liquid preparation, and the enzyme activity is 5000 U / mL;

[0066] The preparation method of the aid is as follows: corn starch and hydrochloric acid with a molar concentration of 1 mol / L are mixed at a mass ratio of 10:28, then stirred at 60 DEG C at a stirring speed of 30 rpm for 120 min, sodium hydroxide aqueous solution with a molar concentration of 10 mol / L is added to adjust the pH value to 7, then stirred at 85 DEG C at a stirring speed of 30 rpm for 50 min, the temperature is lowered to 60 DEG C, the stirring speed is unchanged, sodium dodecyl sulfate is added, the mass ratio of corn starch to sodium dodecyl sulfate is controlled to be 10:0.9, hydrochloric acid with a molar concentration of 1 mol / L is added to adjust the pH value to 4, then stirred for 30 min, sodium bicarbonate aqueous solution with a mass concentration of 10% is added to adjust the pH value to 6, calcium chloride aqueous solution with a mass concentration of 10% is added, the mass ratio of corn starch to calcium chloride aqueous solution is controlled to be 10:0.9, then stirred for 60 min to obtain a feed liquid, 4 times the volume of ethanol with a volume concentration of 95% is added to the feed liquid, then stirred for 30 min, placed for 25 h, centrifuged at a centrifugal speed of 8000 rpm for 15 min, the supernatant is removed, the precipitate is taken out, frozen and dried at-40 DEG C for 34 h to obtain the aid.

[0067] The embodiment also provides a saccharification method of the complex saccharifying enzyme, in particular:

[0068] 1. liquefaction: hydrochloric acid with a molar concentration of 1 mol / L is added to corn starch feed liquid with a mass concentration of 40% to adjust the pH value to 5.6, then medium-temperature alpha-amylase and anhydrous calcium chloride are added, the amount of the medium-temperature alpha-amylase is controlled to be 14 U / g corn starch, the mass ratio of corn starch to anhydrous calcium chloride is controlled to be 40:0.2, then stirred at 70 DEG C at a stirring speed of 50 rpm for 25 min, the temperature is raised to 92 DEG C, the stirring speed is unchanged, high-temperature alpha-amylase is added, the amount of the high-temperature alpha-amylase is controlled to be 12 U / g corn starch, then stirred for 18 min to obtain a liquefied liquid;

[0069] The medium-temperature alpha-amylase is in a powder form, and the enzyme activity is 2000 U / g;

[0070] The high-temperature alpha-amylase is in a liquid form, and the enzyme activity is 20,000 U / mL;

[0071] 2. saccharification: the liquefied liquid is stirred at 62 DEG C at a stirring speed of 50 rpm, meanwhile, hydrochloric acid with a molar concentration of 1 mol / L is added to the liquefied liquid to adjust the pH value to 4.3, then the complex saccharifying enzyme is added, the amount of saccharifying enzyme in the complex saccharifying enzyme is controlled to be 80 U / g corn starch, then stirred for 40 h, filtered, the filtrate is taken out, and a saccharified liquid is obtained.

[0072] Embodiment 2

[0073] The embodiment provides a complex saccharifying enzyme, and a preparation method of the complex saccharifying enzyme specifically includes the following steps:

[0074] 1. removing transglycosylase: mixing saccharifying enzyme and purified water according to a mass ratio of 1:16, stirring at a stirring speed of 60 rpm for 30 min at room temperature to obtain diluted saccharifying enzyme, adding hydrochloric acid with a molar concentration of 1 mol / L to the diluted saccharifying enzyme to adjust the pH value to 3.2, adding a precipitant, controlling the mass ratio of the precipitant to purified water to be 1.6:100, continuing to stir for 40 min, filtering, taking the filtrate, and obtaining saccharifying enzyme after removing transglycosylase;

[0075] The saccharifying enzyme is in a liquid dosage form, and the enzyme activity is 100,000 U / mL;

[0076] The preparation method of the precipitant is as follows: mixing corn starch and a first buffer according to a mass ratio of 2:100, stirring at a stirring speed of 60 rpm for 70 min at 92℃, cooling to room temperature, adding tannic acid, controlling the mass ratio of corn starch to tannic acid to be 2:1.4, stirring at a stirring speed of 60 rpm for 90 min at room temperature, standing for 30 h at room temperature, centrifuging at a centrifugal speed of 9000 rpm for 20 min, removing the supernatant, taking the precipitate, washing the precipitate with anhydrous ethanol for 3 times, freeze-drying at-35℃ for 36 h, and obtaining a complex; mixing all the obtained complexes with a second buffer according to a mass ratio of 2:25, stirring at a stirring speed of 60 rpm for 30 min at room temperature, adding sodium dodecyl sulfate, controlling the mass ratio of the complex to sodium dodecyl sulfate to be 2:0.9, stirring for 80 min, adding sodium bicarbonate aqueous solution with a mass concentration of 10% to adjust the pH value to 6.2, centrifuging at a centrifugal speed of 900 rpm for 20 min, removing the supernatant, taking the precipitate, and freeze-drying at-35℃ for 36 h to obtain a precipitate;

[0077] The first buffer and the second buffer are both acetic acid-sodium acetate buffers, the pH values are both 4, and the molar concentrations are both 0.2 mol / L;

[0078] 2. mixing: mixing pullulanase and an auxiliary agent according to a mass ratio of 100:1.1, stirring at a stirring speed of 60 rpm for 2.5 h at 30℃, adding saccharifying enzyme after removing transglycosylase, controlling the mass ratio of pullulanase to saccharifying enzyme after removing transglycosylase to be 100:2600, and continuing to stir for 80 min to obtain compounded saccharifying enzyme;

[0079] The pullulanase is in a liquid preparation, and the enzyme activity is 5000 U / mL;

[0080] The preparation method of the aid is as follows: corn starch and hydrochloric acid with a molar concentration of 1 mol / L are mixed at a mass ratio of 10:30, then stirred at 65 DEG C at a stirring speed of 60 rpm for 150 min, sodium hydroxide aqueous solution with a molar concentration of 10 mol / L is added to adjust the pH value to 7, then stirred at 90 DEG C at a stirring speed of 60 rpm for 60 min, the temperature is lowered to 65 DEG C, the stirring speed is unchanged, sodium dodecyl sulfate is added, the mass ratio of corn starch to sodium dodecyl sulfate is controlled to be 10:1, hydrochloric acid with a molar concentration of 1 mol / L is added to adjust the pH value to 4.5, and stirring is continued for 40 min, sodium bicarbonate aqueous solution with a mass concentration of 10% is added to adjust the pH value to 6.2, calcium chloride aqueous solution with a mass concentration of 10% is added, the mass ratio of corn starch to calcium chloride aqueous solution is controlled to be 10:1, and stirring is continued for 70 min to obtain a feed liquid, 5 times the volume of ethanol with a volume concentration of 95% is added to the feed liquid, stirring is continued for 40 min, and then standing for 30 h, centrifugation is carried out at a centrifugal speed of 9000 rpm for 20 min, the supernatant is removed, the precipitate is taken out, and freeze-drying is carried out at-35 DEG C for 36 h to obtain the aid.

[0081] The embodiment also provides a saccharification method of the compound glucoamylase.

[0082] 1. liquefaction: hydrochloric acid with a molar concentration of 1 mol / L is added to corn starch feed liquid with a mass concentration of 45% to adjust the pH value to 5.8, then medium-temperature alpha-amylase and anhydrous calcium chloride are added, the amount of the medium-temperature alpha-amylase is controlled to be 15 U / g corn starch, and the mass ratio of corn starch to anhydrous calcium chloride is controlled to be 45:0.21, then stirring is carried out at 75 DEG C at a stirring speed of 100 rpm for 30 min, the temperature is raised to 94 DEG C, the stirring speed is unchanged, high-temperature alpha-amylase is added, the amount of the high-temperature alpha-amylase is controlled to be 13 U / g corn starch, and stirring is continued for 19 min to obtain liquefied liquid;

[0083] The medium-temperature alpha-amylase is in a powder form, and the enzyme activity is 2000 U / g;

[0084] The high-temperature alpha-amylase is in a liquid form, and the enzyme activity is 20,000 U / mL;

[0085] 2. saccharification: the liquefied liquid is stirred at 65 DEG C at a stirring speed of 100 rpm, hydrochloric acid with a molar concentration of 1 mol / L is added to the liquefied liquid to adjust the pH value to 4.4, then compound glucoamylase is added, the amount of the glucoamylase in the compound glucoamylase is controlled to be 82 U / g corn starch, and stirring is carried out for 42 h, then filtration is carried out, the filtrate is taken out, and saccharified liquid is obtained.

[0086] Comparative example 1

[0087] The comparative example provides a compound saccharifying enzyme, and a preparation method thereof is as follows: on the basis of the preparation method of the compound saccharifying enzyme in example 1, the first step is changed to:

[0088] The saccharifying enzyme is mixed with purified water at a mass ratio of 1:15, stirred at room temperature at a stirring speed of 30 rpm for 10 min to obtain diluted saccharifying enzyme, 1 mol / L hydrochloric acid is added to the diluted saccharifying enzyme to adjust the pH value to 3, corn starch, tannic acid and sodium dodecyl sulfate are added, the mass ratio of corn starch, tannic acid, sodium dodecyl sulfate and purified water is controlled to be 0.8:0.5:0.2:100, and stirring is continued for 30 min, filtration is performed, the filtrate is taken, and the saccharifying enzyme after removing the transglycosidase is obtained.

[0089] The saccharifying enzyme is in a liquid dosage form, and the enzyme activity is 100,000 U / mL.

[0090] The remaining technical solutions are consistent with those in example 1.

[0091] The comparative example also provides a saccharification method of the compound saccharifying enzyme, and the same saccharification method as in example 1 is adopted.

[0092] Comparative example 2

[0093] The comparative example provides a compound saccharifying enzyme, and a preparation method thereof is as follows: on the basis of the preparation method of the compound saccharifying enzyme in example 1, the second step is changed to:

[0094] The pullulanase is mixed with the saccharifying enzyme after removing the transglycosidase at a mass ratio of 100:2200, stirred at 25℃ at a stirring speed of 30 rpm for 60 min to obtain the compound saccharifying enzyme.

[0095] The pullulanase is in a liquid preparation, and the enzyme activity is 5,000 U / mL.

[0096] The remaining technical solutions are consistent with those in example 1.

[0097] The comparative example also provides a saccharification method of the compound saccharifying enzyme, and the same saccharification method as in example 1 is adopted.

[0098] Test example 1

[0099] With the same production batch of saccharifying enzyme as raw material, the complex saccharifying enzyme was prepared according to the method of Example 1-2 and Comparative Example 1 respectively, and the amount of saccharifying enzyme in the first step of removing transglycosidase was controlled to 100 mL. At the same time, the enzyme activity of saccharifying enzyme and transglycosidase in the diluted saccharifying enzyme obtained in the first step of removing transglycosidase was tested, and then the enzyme activity of saccharifying enzyme and transglycosidase in the saccharifying enzyme after removing transglycosidase was tested. The change rate of saccharifying enzyme activity and the removal rate of transglycosidase were calculated, and the calculation formula and results were as follows:

[0100] The change rate of saccharifying enzyme activity = (the enzyme activity of saccharifying enzyme in the saccharifying enzyme after removing transglycosidase - the enzyme activity of saccharifying enzyme in the diluted saccharifying enzyme) / the enzyme activity of saccharifying enzyme in the diluted saccharifying enzyme * 100%;

[0101] The removal rate of transglycosidase = (the enzyme activity of transglycosidase in the diluted saccharifying enzyme - the enzyme activity of transglycosidase in the saccharifying enzyme after removing transglycosidase) / the enzyme activity of transglycosidase in the diluted saccharifying enzyme * 100%;

[0102]

[0103] Test Example 2

[0104] The complex saccharifying enzyme was prepared according to the method of Example 1-2 and Comparative Example 1-2, and saccharification was carried out using the prepared complex saccharifying enzyme. The corn starch, enzyme and other raw materials used were of the same production batch. Then the DX value and DE value of the obtained saccharification liquid were tested, and the trisaccharide content in the saccharification liquid was also tested. The test results were as follows:

[0105]

[0106] From the results of Test Example 1 and Test Example 2, it can be seen that compared with Example 1, the method of removing transglycosidase in Comparative Example 1 has the problems of large influence on saccharifying enzyme activity and low removal rate of transglycosidase. Moreover, Comparative Example 1 and Comparative Example 2 also have the problems of low DX value and DE value of the saccharification liquid obtained after saccharification, and high trisaccharide content.

Claims

1. A complex saccharifying enzyme characterized in that, It is prepared by the following steps: removing transglycosidase, mixing; The process for removing transglycosidase involves mixing glucoamylase with purified water, stirring at room temperature to obtain diluted glucoamylase, adjusting the pH of the diluted glucoamylase to 3-3.2, adding a precipitant, stirring, filtering, and collecting the filtrate to obtain glucoamylase after removing transglycosidase. The precipitant is prepared by mixing corn starch with a first buffer solution, stirring at 90-92°C, cooling to room temperature, adding tannic acid, stirring at room temperature, allowing to stand at room temperature, centrifuging, removing the supernatant, taking the precipitate, washing with anhydrous ethanol, and freeze-drying for the first time to obtain the complex; mixing the complex with a second buffer solution, stirring at room temperature, adding sodium dodecyl sulfate, stirring, adjusting the pH to 6-6.2, centrifuging, removing the supernatant, taking the precipitate, and freeze-drying for the second time to obtain the precipitate; In the preparation of the precipitant, the first and second buffer solutions are both acetate-sodium acetate buffer solutions, both with a pH of 4 and a molar concentration of 0.2 mol / L. The mixing process involves mixing pullulanase with the adjuvant, stirring at 25-30°C, adding glucoamylase after removing transglycosides, and stirring to obtain a compound glucoamylase. The preparation method of the additive is as follows: corn starch is mixed with hydrochloric acid, stirred at 60-65℃, the pH value is adjusted to 7, stirred at 85-90℃, cooled to 60-65℃, sodium dodecyl sulfate is added, the pH value is adjusted to 4-4.5, stirred, the pH value is adjusted to 6-6.2, calcium chloride aqueous solution is added, stirred, ethanol is added to precipitate, centrifuged, the supernatant is removed, the precipitate is taken, and freeze-dried to obtain the additive.

2. The complex saccharifying enzyme according to claim 1, characterized by, In the process of removing transglycosidase, the mass ratio of glucoamylase to purified water is 1:15-16; The mass ratio of precipitant to purified water is 1.5-1.6:100; The saccharifying enzyme is in liquid form with an enzyme activity of 100,000 U / mL.

3. The complex saccharifying enzyme according to claim 1, characterized by, In the preparation of the precipitant, the mass ratio of corn starch to the first buffer solution is 2:98-100; The mass ratio of corn starch to tannic acid is 2:1.2-1.4; The mass ratio of the complex to the second buffer solution is 2:20-25; The mass ratio of the complex to sodium dodecyl sulfate is 2:0.8-0.

9.

4. The complex saccharifying enzyme according to claim 1, characterized by, In the preparation of the precipitant, the temperature of the first freeze-drying and the second freeze-drying are -40℃ to -35℃, and the time is 34-36h.

5. The complex saccharifying enzyme according to claim 1, characterized by, In the mixture, the mass ratio of pullulanase to the adjuvant is 100:1-1.1; The mass ratio of pullulanase to glucoamylase after the removal of transglycosidase is 100:2500-2600; The pullulanase is a liquid preparation with an enzyme activity of 5000 U / mL.

6. The complex saccharifying enzyme according to claim 1, characterized by, In the preparation of the auxiliary agent, the mass ratio of corn starch to hydrochloric acid is 10:28-30; The mass ratio of corn starch to sodium dodecyl sulfate is 10:0.9-1; The mass ratio of corn starch to calcium chloride aqueous solution is 10:0.9-1.

7. The complex saccharifying enzyme according to claim 1, characterized by, In the preparation of the auxiliary agent, the molar concentration of the hydrochloric acid is 1 mol / L; The mass concentration of the calcium chloride aqueous solution is 10%. The volume concentration of the ethanol is 95%; The freeze-drying temperature is -40℃ to -35℃, and the time is 34-36h.

8. A saccharification method using the complex saccharifying enzyme according to any one of claims 1 to 7, characterized by, The method comprises the following steps: liquefaction and saccharification; In the liquefaction, the mass concentration of the high-concentration corn starch material liquid is 40-45%; The mesophilic alpha-amylase is in a powder form, and the enzyme activity is 2000U / g; 9. The saccharification method of claim 8, wherein, The high-temperature alpha-amylase is in a liquid form, and the enzyme activity is 20,000U / mL; The mesophilic alpha-amylase is used in an amount of 14-15U / g of corn starch; The mass ratio of corn starch to anhydrous calcium chloride is 40-45:0.2-0.21; The high-temperature alpha-amylase is used in an amount of 12-13U / g of corn starch. In the saccharification, the molar concentration of the hydrochloric acid is 1mol / L; The saccharifying enzyme in the compound saccharifying enzyme is used in an amount of 80-82U / g of corn starch.

10. The saccharification method of claim 8, wherein, ​ ​

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