Title: Effect of Senna Leaves on Urine Volume in Mice

Research status:

First, pullulanase

Amylase (EC 3.2. 1.4 1) is an enzyme that can specifically cleave the branching point of amylopectin. -1.6 glycosidic bond, thus cutting off the whole side branch and forming amylose debranching enzyme. Pullulanase can also decompose pullulan. Pullulanase comes from microorganisms, while R- enzyme comes from plants. Pullulanase was first obtained by Bender and Wallenfels in 196 1 by aerogenes (Klebsiella pneumoniae is a typical bacterium), and they reported the good enzymatic properties of this enzyme. After that, researchers from all over the world obtained pullulanase from different regions and microorganisms through extensive and in-depth research, which set off the climax of developing pullulanase.

Amylase is the most commonly used in starch processing industry. Its function is to hydrolyze starch. -1, 4 glycosidic bond, when used alone, the product contains a large number of branched dextrins, including a large number of? -1, 6 glycosidic bond. If you don't put starch? If the glycosidic bond of-1 6 is completely decomposed, it will inevitably cause great waste. In nature, what else can decompose starch? -1, 6 glucosidase, commonly known as debranching enzyme. Widowed? -1 6 glucosidase (e.c3.2. 1.68, oligo-l, 6- glucosidase), pullulanase (e.c3.2.1.4/pullulanase), isoamylase. Among them, pullulanase has the smallest requirement on the molecular structure of substrate, so many countries are scrambling to develop it, but so far, only NOVO company in Denmark has the production capacity of pullulanase. China only imports, but its high price limits the application of pullulanase in China. In fact, a pullulanase producing strain was developed in China as early as 1970s, but its enzymatic properties are not suitable for production. So far, there is no report on the localization of pullulanase in China.

In the starch processing industry, the enzymatic properties of pullulanase are required to be acid-resistant and heat-resistant, because the external enzymatic method is usually used. Due to the limitation of the enzyme used, pullulanase can be added in either step of the two-step reaction, but the above reaction conditions must be met. Therefore, the enzymatic properties of pullulanase must meet the existing conditions of enzymatic hydrolysis to make sugar, namely acid resistance and heat resistance.

Second, the research status of pullulanase

1. Microorganism producing pullulanase

Pullulanase was first produced by Bender and Wallenfels in 196 1 year by Aerobacter.

Gas production) is obtained by fermentation. After they reported the good performance of the enzyme, researchers from all over the world obtained the enzyme from microorganisms in different regions after extensive and in-depth research, which set off the climax of developing pullulanase. But so far, although it has been found that many microorganisms can produce pullulanase, due to the limitations of current industrial production conditions (acidity and temperature), pullulanase produced by most microorganisms has no commercial value. Here is a brief introduction of pullulanase producing bacteria.

1. 1 Bacillus cereus fungus variety

It was discovered by ToshiyukiTakasaki of Japan in 1975. The strain produces two amylases at the same time:? -Amylase and pullulanase. The optimum reaction conditions are pH 6 ~ 6.5, temperature 50℃, and the maximum conversion rate (maltose produced by starch hydrolysis) is about 95%. Enzymatic studies showed that the enzyme still maintained most of its activity at pH5 and 60℃. The vegetative cells of the strain were rod-shaped, aggregated and grew into short and unequal disordered chains, which were motionless and Gram-positive, and the cells did not expand obviously when budding. The optimum growth temperature of this strain is 30℃ ~ 37℃, and the highest growth temperature is 4 1℃ ~ 45℃. Glucose, mannose, maltose, trehalose, starch and glycogen can be used.

1.2 acidophilic pullulan decomposes Bacillus pullulan.

In the early 1980s, the strain was obtained by Novo Company in Denmark, and the pullulanase produced by the strain was thermostable.

(60℃), acid resistance (pH4.5). The company has invested heavily in research and development, 1983 1 1 month. The company sells commercially in Japan and Europe under the trade name Prornozyme. Today, it is the most widely used pullulanase with the largest yield. Bacillus. Lactobacillus acidolyticus is rod-shaped. After several hours of submerged fermentation, we can observe the expanded cells of protoplast-like cells, which are relatively stable and full of cylinders or ellipsoids. Gram reaction was positive, and it grew well at 37℃, not long above 45℃, and pI- 1 was above 6.5, and it grew well on the medium (pH4.8 ~5.2) with pullulan as carbon source.

1.3 Bacillus subtilis

In 1986, Japan's Yushiyuki Takasaki reported a strain which can produce heat-resistant and acid-resistant pullulanase and named it Bacillus subtilis TU. The enzyme produced by this strain is a mixture of pullulanase and amylase, the hydrolyzable starch is maltotriose and maltotriose, and the hydrolyzed pullulanase is maltotriose. The optimum pH of pullulanase is 7.0 ~ 7.5, but it also has about 50% enzyme activity at pH5.0, and the optimum temperature of this pullulanase is 60℃.

1.4 heat-resistant chlorsulfuron

1987. E.madi of Germany reported a strain that can produce α-amylase, pullulanase and glucoamylase at the same time: Clostridium thermotolerant and sulfur-producing bacteria. Pullulanase produced by this strain has a wide temperature adaptation range (40℃ ~ 85℃) and high activity at pH 4.5 ~ 6.0. It will undoubtedly expand the application field of this pullulanase.

1.5 naganoensis, deramificans, Bacillus. Acid amylopectin

In the 1990s, Deweer discovered a kind of Bacillus producing pullulanase. Fucun screened out mycobacterium tuberculosis. The enzymatic properties of pullulanase produced by these two strains are similar to those of Bacillus. These two strains are moderately acidophilic and will not grow above pH6.5 or above 45℃. The discovery of these two pullulanase producing strains further broadened the application scope of pullulanase.

1.6 Thermophilic bacteria producing pullulanase

Since 1980s, people have gradually realized that it is difficult to screen pullulanase-producing bacteria with extreme heat tolerance under normal natural conditions, so scientists all over the world have turned their attention to the screening of thermophilic bacteria in hot springs, and now more achievements have been made. The optimum temperature for producing pullulanase by Bacillus such as vorcaldarius is 75 ~ 85℃ and the optimum pH is 6.3. The optimum temperature and pH of marine thermophilic thermophilic thermophilic bacteria are 90℃ and 6.0, respectively. The optimum temperature and pH of thermophilic thermophilic thermophilic thermophilic bacteria are 75℃ and 5.5, respectively. The optimum temperature and pH of killing algae by Bonavos are 80 ~ 85℃ and 6.0, respectively.

2. Molecular structure of pullulanase

Up to now, many genes of pullulanase have been cloned, but there is no report on the structure of pullulanase. However, according to the classification of sugar bond hydrolases based on sequence similarity, pullulanase belongs to 13 family. Amylase family contains more than 30 kinds of enzymes, which can be divided into hydrolase and transferase. Isomerases are divided into three categories. These enzymes can be hydrolyzed and synthesized? ~ 1.4,? ~ 1.6,? ~ 1.2,? ~ 1.3,? ~ 1.5,? ~ 1. 1 glycoside bond. The structures of many of these enzymes have been reported, and they all adopt (? /? ) 8. Through bioinformatics research, all protein in this family have the same structure, and the active center of the enzyme is (? /? The structure of) 8 folded tube is named domain A.. 13. Most enzymes in the family also have domain B, which is located in (? /? ) 8 in the folding tube, the third one? Layered and third? The sequence between helices has the characteristics of great difference in structure and length, and it is speculated that its function is related to the combination of substrates. Followed by (? /? After 8 folds, it is still C domain, followed by C domain, and some family members also have D domain.

3. The application of pullulanase

Pullulanase is an enzyme preparation and processing aid widely used in food industry. It can specifically decompose amylopectin and glycogen molecules in starch and its derived oligosaccharide branches. ~ l, 6 glycosidic bonds break the branching structure and form amylose with different lengths. Therefore, when this enzyme is combined with other amylases, starch can be completely saccharified. In recent years, pullulanase, as a new type of amylase, has been applied to food and other industrial sectors with starch as raw material, and has the following functions in the food industry:

3. 1 Use pullulanase alone to convert amylopectin into amylose.

Amylose has the characteristics of coagulation and easy formation of gel with stable structure, which can be used as flexible food packaging film. This film is suitable as a protective layer for food, because it has good isolation from oxygen and grease and good coating performance. It is also suitable for the manufacture of starch soft candy, and can also be used as jam thickener for filling high-oil food, preventing oil leakage and processing meat food. In recent years, biodegradable films have been advocated in food industry, and amylose has great development prospects in these aspects. The amylose content of beans is high, so the toughness of vermicelli made of mung bean starch is better than other starches. If cereal starch is treated with pullulanase and then made into amylose, high-quality vermicelli can be made. In general cereal starch, amylose content is only 20%, and amylopectin content is about 80%. For every 1 ton of amylose produced in industry, there are 4 tons of by-product amylopectin. Although a new maize variety containing 60% amylose has been obtained by genetic breeding in the United States, it is not suitable for mass production. Pullulanase has been used to change starch structure abroad, which can transform amylopectin into amylose. It is reported that the yield can reach 100% by this method. The preparation method of amylose is as follows: firstly, the liquefied branches are decomposed by pullulanase to convert them into amylose, and then the starch is precipitated by butanol or slow cooling. Then the crystalline precipitate containing a small amount of water is recovered, and finally the powdery amylose is prepared by low-temperature spray drying.

3.2 pullulanase and? ~ (13) amylase to produce malt pulp

Maltose is a traditional starch sugar product in China, which contains a certain amount of maltose and is widely used in candy, pastry and other food industries. What is the production method now? ~ amylase liquefaction and reuse? ~ (13) amylase hydrolyzes amylopectin, only side chain. Approaching the cross position? When the glycosidic bond is ~ 1.6, the hydrolysis reaction stops. However, if pullulanase is used for simultaneous hydrolysis, the branched chain can be broken, the hydrolysis degree of amylase can be improved and the hydrolysis degree of amylase can be reduced. The content of limit dextrin greatly improves the yield of maltose, which is beneficial to the production of maltose syrup. At present, pullulanase saccharification has been carried out on a large scale.

The test conditions are as follows. The feeding amount of each batch is about 900kg of broken rice, and the slurry concentration is 15 ~ 16be? The skin counting dose is 1.5% (for broken rice). The activity of ~ (13) amylase is more than 2000 units/g, and the activity of pullulanase is 45000 ~ 55000 units/g, which is produced by Aeromonas, and the dosage of each batch is 1 kg. The results showed that compared with the control sugar, the reducing sugar increased by 65438 04.8, the maltose content increased by 45.6, and the dextrin content decreased by 26.7. Compared with high-concentration grape pulp, high-concentration malt syrup is not easy to crystallize and has low hygroscopicity, so it is widely used in food industry. The combination cost of pullulanase and β -amylase is low, and the maltose yield reaches about 70%, or even higher.

3.3 Used for enzymatic beer saccharification.

Malt in beer production is not only the main raw material for brewing beer, but also provides a rich source of enzymes for the brewing process. What are the main enzymes that decompose starch in malt during saccharification of beer brewing? ~ amylase,? ~ amylase and decomposed starch? ~ 1.6 R- enzyme (plant pullulanase or plant pullulanase). ? ~ (2+) amylase decomposes starch into maltose (including a small amount of maltotriose and a very small amount of glucose) and low molecular dextrin through the synergistic effect with the other two amylases. So that wort has ideal sugar content. In order to save the amount of malt in industrial production, the so-called extra enzymatic saccharification is adopted, that is, on the premise of reducing the amount of malt, the proportion of starch auxiliary raw materials is increased, and appropriate kinds of enzyme preparations are added for enamel. In order to saccharify barley and other auxiliary materials completely, α -amylase needs to be added for decomposition? ~ 1.6 glycosidic bond pullulan enzyme preparation, etc. When α -amylase is used alone, it is incomplete to produce maltose and malt three layers. What should I do if the starch is decomposed? If the enzyme activity of ~ 1.6 glycosidic bond is insufficient and the starch decomposition is not complete, the fermentable sugar content is low and the fermentation degree of beer can not meet the requirements. If it can be broken down? ~ 1.4 and? The reaction product of saccharified amylase with glycosidic bond of ~ 1.6 is glucose, which easily weakens the taste of wine. Use pullulanase and? ~ (13) amylase has a good effect, and its decomposition products are mainly maltose and a small amount of maltopolysaccharide. When saccharifying with external enzyme method, the dosage of enzyme preparation is: amylase 6-7 units/g barley and rice: protease 60-80 units/g, and bromelain 10ppm and pullulanase 50 units/g barley are added. The above three enzyme preparations are added at the beginning of saccharification or alcoholization.

In a word, pullulanase, as an enzyme preparation and processing aid, has broad development prospects in the food industry.

Research purpose and significance:

Enzyme preparation industry is an important industry formed since 1970s. At present, the total output value of enzyme preparations in the world is US$ 654.38+000 billion, and the output value in China is about RMB 654.38+000 billion. With the continuous expansion of its application field and the development of new enzyme species, this market has developed rapidly. However, the global enzyme preparation industry is almost monopolized by several foreign companies, among which Novartis in Denmark accounts for almost half of the total global sales. This study is of great significance to the development of pullulanase and enzyme preparation industry.

Secondly, China began to research and develop pullulanase in 1970s, but the developed pullulanase is neither heat-resistant nor acid-resistant, which limits its industrial application. In order to change China's dependence on imported products, fill the gap in this field and find a localized road, the purpose of this study is to improve the utilization rate of starch raw materials in China by using natural microbial resources-pullulanase, thus improving the productivity of the whole starch processing industry, which is of self-evident significance to China's starch processing industry.

Research content (content, structural framework, key points and difficulties):

1. Screening of Pullulanase-producing Bacteria

(1) sample collection;

(2) Preliminary screening of strains;

(3) strain re-screening;

(4) the preservation method of strains;

(5) Establishment of enzyme activity determination method.

2. Study on the production conditions of pullulanase.

Effects of (1) carbon and nitrogen sources on enzyme production by fermentation;

(2) Effect of initial PH on enzyme production;

(3) Effect of inoculation amount on enzyme production;

(4) Effect of fermentation temperature on enzyme production;

(5) Effect of metal ions on enzyme production.

Key points or key technologies:

Isolation of (1) pure strain;

(2) Selection of strain identification methods.

Research methods and means:

1. Screening of Pullulanase-producing Bacteria

(1) Sample collection: Select the appropriate place (flour mill, vegetable field, orchard, etc.). ) collect soil samples.

(2) Primary screening of strains: the collected soil samples were diluted with sterile water, coated on a flat plate in a starch-containing culture medium, cultured at 37℃ for 48 hours, and reacted with iodine solution for color development, and colonies containing amylase were inoculated on an inclined plane for preservation.

(3) Re-screening of strains: The amylase-producing strains isolated in the early stage were coated on a Pullulan plate, cultured at 37℃ for 48 hours, and then transparent circle experiments were conducted with 95% ethanol. The production of transparent circle indicates that the strain produces pullulanase, and the colony producing transparent circle is picked on the inclined culture medium for culture.

(4) strain preservation method: low temperature preservation at 4℃.

(5) Establishment of enzyme activity determination method: centrifuge the fermentation broth, determine the absorbance at 520nm by DNS color development method, and determine the standard curve of glucose, and calculate the activity of pullulanase from the standard curve.

2. Study on the production conditions of pullulanase.

(1) Effects of carbon source and nitrogen source on enzyme production by fermentation: Different carbon sources and nitrogen sources were cultured for a period of time to determine enzyme activity. Other conditions are the same: inoculation amount, bottling amount, initial PH value, rotation speed and culture time. )

(2) Effect of initial PH on enzyme production: Using the same fermentation medium, inoculating the same amount of seed liquid at different initial pH ... and culturing under the same conditions to determine the enzyme activity of the fermentation liquid. Other conditions are the same: inoculation amount, bottling amount, rotating speed, optimal culture temperature and optimal culture time. )

(3) Effect of inoculation amount on enzyme production: Inoculate into fermentation medium with inoculation amount of 2%, 4%, 6% and 8% respectively,

The seed culture media of 10%, 14% and 18% were cultured in the medium with the best carbon source, nitrogen source and the best initial PH under the same conditions, and the enzyme activities were detected respectively. (Using the best carbon source, nitrogen source and the best initial PH determined above. )

(4) Effect of fermentation temperature on enzyme production: Using the same culture medium, culture at different temperatures (25℃, 30℃, 35℃, 40℃, 45℃) for a certain period of time, and measure the enzyme activity.

(5) Effect of metal ions on enzyme production: A small amount of different metal ions were added to the basic medium, and the enzyme activity was determined after fermentation. (Metal ions include: manganese ions, calcium ions, zinc ions, magnesium ions, iron ions and copper ions. )

research progress

30% of the overall progress of the project, collection and preliminary preparation of sample soil samples, and preliminary screening of strains, including (coating culture and shaking table culture of sample soil stock solution, selection of strains producing pullulanase and slant culture).

50% of the overall progress of the project, strain re-screening, including (screening of pullulanase-producing strains and slant culture), determination of glucose standard curve, establishment of enzyme activity determination method, and strain re-screening according to enzyme activity.

Complete 80% of the overall progress of the project and study the conditions for enzyme production. Including carbon source, nitrogen source, initial PH value, inoculation amount, fermentation temperature and metal ions. The optimum carbon source, nitrogen source, initial PH value, inoculation amount, fermentation temperature and metal ions of fermentation medium were determined by single factor experiment.

2009、4? May 2009: overall progress of the project 100%, project summary and paper writing.

Literature review (including: domestic and foreign research theories, research methods, progress, existing problems, reference basis, etc.). )

Pullulanase was first discovered in 196 1 year when Bender H. et al. studied an aerobacterium (Klebsiella pneumoniae is a typical bacterium). After a lot of research on the microorganisms producing this enzyme, it was found that many microorganisms can produce this enzyme, and some excellent strains suitable for industrial production were screened out. With the application and development of this enzyme, the research of heat-resistant pullulanase is gradually increasing, and the gene of this enzyme has been successfully cloned and expressed. A pullulanase strain of aerogenes 100 16 was studied in China from 65438 to 0976. The production conditions, separation and extraction of the enzyme and its enzymatic properties were reported, and the food-grade extraction technology of the enzyme was studied. In addition, Chen et al. screened a thermophilic strain producing pullulanase from hot spring water samples in Yunnan. Through induction and other experiments, the enzyme activity increased from 0.069u/mL to 1.70 U/mL, and the enzyme yield increased by about 2500 times. The optimum temperature and pH of the enzyme are 75℃ and 4.5, and it has certain heat and acid resistance.

Chen Jinquan and others screened a wild strain producing heat-resistant and acid-resistant pullulanase from hot spring water samples. According to morphological characteristics, physiological and biochemical characteristics, cytochemical composition analysis, 16SrDNA sequence comparison, genomic DNA G+C mole percentage, homology comparison and other experiments, it was identified as a new species of Bacillus alicyclic acid, and the optimum temperature for enzyme production was 60℃. Yang Yunjuan and others successfully constructed a genetically engineered strain with high expression of pullulanase by Pichia pastoris. Under the optimum fermentation conditions, the activity of pullulanase in shake flask fermentation can reach 350.8U/mL, and the yield can reach 504.5-5 10. 1U/mL. The optimum temperature and pH of the enzyme are 60℃ and 4.5, respectively, and it has good heat resistance and acid resistance. At present, there is no manufacturer that can independently produce pullulanase in China, and there is still a long way to go to achieve low-cost and localized production.

The application of technology in the research of heat-resistant debranching enzyme has made great progress in the research of heat-resistant isoamylase. Coleman et al. cloned the pullulanase gene of thermophilic anaerobic bacterium T. brockii into Bacillus subtilis, and the amount of pullulanase secreted by the clone was higher than that of the original strain. Okada et al. cloned the gene encoding thermostable isoamylase of Bacillus stearothermophilus into Bacillus subtilis, and the isoamylase of the transformed strain was stable at 60℃ 15 minutes. Burchadf will. 38% of the thermophilic isoamylase gene was cloned and expressed in Escherichia coli. The optimum pH and temperature of the obtained enzyme were the same as those of the original strain, and it could still maintain its activity at high temperature. Antranikiam et al. cloned the isoamylase gene from Pyrococcus riousous into E.coli and isolated the enzyme protein. Nevertheless, there is no report on the application of transgenic thermostable isoamylase engineering bacteria in industrial production. As we all know, treating microbial cells with physical and chemical mutagens alone or in combination is an effective classical method for breeding high-yield mutant strains. It has played an extremely important role in cultivating high-yield mutant strains of various antibiotics, amino acids and nucleotide kinases (especially proteases and amylases), and it is still one of the convenient and effective methods.

Main references:

[1][ America] edited by Whistler, translated by Wang Xiaowen et al. Starch Chemistry and Technology [M]. Beijing: China Food Publishing House, 1988.

[2] Zhang Shuzheng. Enzyme preparation industry [M]. Beijing: Science Press, 1998

[3] Wu. Industrial production technology of enzyme [M]. Jilin: Jilin Science and Technology Press, 1988

[4] Taniguchi H, Sakano Y, Ohnishi M, Okada G( 1985) pullulanase [J].TanpakushitsuKakusan Koso. ju 1； 30(8):989-992. Japanese

[5] B.F. Zhan Sen and B.E. Norman. 1984. pullulanase from Bacillus acidolyticus [J]. It is used in the application and supervision of food industry. Dealing with biochemistry. 19:35 1-369

[6]Tomimura E，Zeman NW，Frankiewicz JR，Teague WM .[J]。 Description of Bacillus

Nagano algae Nov.Int Journal of Bacterial Systems. I 990 Apr40(2): 123- 125

Wu Yanping, et al. Study on microbial production of pullulanase [J]. Biotechnology, 2003,8 (6):14-17.

Jin Qirong, et al. Preliminary study on pullulanase [J]. Bulletin of Microbiology, 200 1, 28 (1): 39-43

Chengchi Pullulanase Promozyme 200L And its production strain [J]. Food and Fermentation Industry, 1992, (6)

[10] Tang Baoying et al. Screening of acid-tolerant and heat-tolerant pullulanase strains and study on fermentation conditions [J]. Bulletin of Microbiology, 200 128 (10): 39-43.

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