红曲霉产酯化酶特性及其酶学性质的研究
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摘要
红曲霉(Monascus)其发酵产物红曲古代称丹曲,起源于中国,很早便被用于制酒、酿造食品、医药等。红曲霉能产生多种对人类有益的代谢产物。酯化酶作为其代谢产物之一,具有多向合成功能,既能催化酯的合成,也能催化酯的分解,具有广泛的市场应用前景。本论文首先对四株红曲霉菌株进行产液态酯化酶能力的筛选,筛选出一株最佳菌株。然后对其培养基组成成分、培养条件进行优化,从而获得最大产酶量。最后对分离纯化出酯化酶进行了酶学相关性质的研究。主要试验结果如下：
1.通过对四株红曲霉菌株Monascus sp. ZZ、Monascus ruberZK、Monasucs purpureusGX、Monasucs sp. XM的筛选。得到高产液态酯化酶的菌株Monascus ruberZK,其液体产酶水平为203.4U／mL,总酶活9061.1U。
2.对培养基中碳源、氮源、无机盐进行了单因子以及正交实验。确定红曲霉Monascus ruberZK的最佳培养基组成为可溶性淀粉35g／L,葡萄糖35g／L,蛋白25g/L, NaN032g/L, MgSO41. Og/L, KH2PO41.5g／L；验证实验表明,以最佳组合条件B2A2C2D1发酵时,酯化酶酶活力达293.8 U/mL。
3.对培养条件中的温度、初始pH值、接种量、装液量、摇床转速等影响因素进行了单因子实验。随即选取了温度、初始pH值、接种量、摇床转速四个最佳单因子做四因素三水平正交实验。确定红曲霉Monascus ruberZK的最佳条件为温度32℃,初始pH值5.0、接种量10%、装液量为60mL(250mL三角瓶),摇床转速160r/min。验证实验表明,以最佳组合条件B2A2C2D1发酵时,酯化酶酶活力312.lU/mL.
4.粗酶液经硫酸铵分级盐析,用Sephadex G-25柱层析脱盐,使用Sephadex G-100对酶液进行分离纯化,我们得出酯化酶回收率是25.3%,纯化倍数是7.6.
5.酯化酶最适酶活温度40℃,最适pH为5.3,pH在4.8-5.7范围内,相对酶活力均在90%以上。酸碱稳定性方面,在pH值3.5-6.8之间时,有较好的耐酸性,较强的稳定性。Cacl2、Mgcl2、EDTA对该酯酶都有明显激活作用。Nacl、Kcl对该酯酶有轻度激活作用；而Hgcl2、AgNO3对该酯酶都有明显抑制作用,使其丧失活性。Fecl2、Nicl2、Sncl2等对该酯酶都也有抑制作用。
The fermentation product of Monascus is called Dan Qu in ancient time. originated in China, very early be used in wine, fermentating food, medicine and so on. Monascus can produce many kinds of beneficial products of metabolism for the humanity.The esterification enzyme is one of its metabolites, has the multi-direction synthesis function, can catalyze the synthesis of ester, can also catalyze the decomposition of ester.It Can be widely applied in industrial market. In this paper, first carries four Monascus strains for producing the liquid state esterification enzyme ability screening, screening a best strain. Then we carried on the optimization to its culture medium component and raise condition, in order to obtain the most amount of enzyme production. Finally separation and purification of esterification enzyme-for conducting the zymology related nature research. The main test results are as follows:
1. Through to four Monascus strains Monascus sp. ZZ, Monascus ruberZK, Monasucs purpureusGX, Monasucs sp. XM screening. Obtains high-yield strain of liquid state esterification enzyme.Monascus ruberZK, its production of liquid enzyme level is 203.4U/mL, the total enzyme lives 9061.1U.
2. First, we carried on the single factor to the culture medium in the carbon source, the nitrogen source, the inorganic salt.then,we carried on orthogonal experiment to the single factor results. Determined the best culture medium composition ofMonascus ruberZK are soluble starch 35g/L, glucose 35g/L, protein peptone 25g/L, NaN03 2g/L, MgS04 1.0g/L, KH2P04 1.5g/L; The confirmation experiment indicated that enzyme-activity of the esterification enzyme reached 293.8 U/mL. With the best combination of conditions B2A2C2D1 fermentation,
3. carry on the single factor experiment to temperature, initial pH value, vaccination quantity, attire liquid volume, table rotational speed and other influencing factors of the culture conditions. Then Selected the temperature, the initial pH value, the vaccination quantity, the table rotational speed four best single factors to do four factor three horizontal orthogonal experiments. Determined that Monascus ruberZK the optimum condition for the temperature 32℃, the initial pH value 5.0, vaccination quantities 10%, installs the liquid volume is 60mL (250mL triangle bottle), table rotational speed 160r/min. The confirmation experiment indicated that enzyme-activity of the esterification enzyme reached 312. 1U/mL. With the best combination of conditions B2A2C2D1 fermentation,
4. The rough enzyme fluid are gradually salted out by the ammonium sulfate, enzyme fluid are desalted by Sephadex the G-25column chromatographic analysis. Used Sephadex G-100 to carry on the separation and purification for the enzyme fluid, We obtain esterase receiving rate are 25.3%, the purification multiple is 7.6.
5. The esterification enzyme most suitable enzyme live temperature 40℃, most suitable pH is 5.3, PH in 4.8-5.7 scopes, relative enzyme activity is above 90%. Acid and alkali stable aspect, when the pH value between 3.5-6.8, has the good acid resistance, strong stability. Cacl2, Mgcl2, EDTA have the obvious activation function to this esterase. Nacl, Kcl have the mild activation function to this esterase; But Hgcl2, AgNO3 have the obvious inhibitory action to this esterase, causes it to lose activeness. Fecl2, Nicl2, Sncl2 and so on also have the inhibitory action to this esterase.
1.通过对四株红曲霉菌株Monascus sp. ZZ、Monascus ruberZK、Monasucs purpureusGX、Monasucs sp. XM的筛选。得到高产液态酯化酶的菌株Monascus ruberZK,其液体产酶水平为203.4U／mL,总酶活9061.1U。
2.对培养基中碳源、氮源、无机盐进行了单因子以及正交实验。确定红曲霉Monascus ruberZK的最佳培养基组成为可溶性淀粉35g／L,葡萄糖35g／L,蛋白25g/L, NaN032g/L, MgSO41. Og/L, KH2PO41.5g／L；验证实验表明,以最佳组合条件B2A2C2D1发酵时,酯化酶酶活力达293.8 U/mL。
3.对培养条件中的温度、初始pH值、接种量、装液量、摇床转速等影响因素进行了单因子实验。随即选取了温度、初始pH值、接种量、摇床转速四个最佳单因子做四因素三水平正交实验。确定红曲霉Monascus ruberZK的最佳条件为温度32℃,初始pH值5.0、接种量10%、装液量为60mL(250mL三角瓶),摇床转速160r/min。验证实验表明,以最佳组合条件B2A2C2D1发酵时,酯化酶酶活力312.lU/mL.
4.粗酶液经硫酸铵分级盐析,用Sephadex G-25柱层析脱盐,使用Sephadex G-100对酶液进行分离纯化,我们得出酯化酶回收率是25.3%,纯化倍数是7.6.
5.酯化酶最适酶活温度40℃,最适pH为5.3,pH在4.8-5.7范围内,相对酶活力均在90%以上。酸碱稳定性方面,在pH值3.5-6.8之间时,有较好的耐酸性,较强的稳定性。Cacl2、Mgcl2、EDTA对该酯酶都有明显激活作用。Nacl、Kcl对该酯酶有轻度激活作用；而Hgcl2、AgNO3对该酯酶都有明显抑制作用,使其丧失活性。Fecl2、Nicl2、Sncl2等对该酯酶都也有抑制作用。
The fermentation product of Monascus is called Dan Qu in ancient time. originated in China, very early be used in wine, fermentating food, medicine and so on. Monascus can produce many kinds of beneficial products of metabolism for the humanity.The esterification enzyme is one of its metabolites, has the multi-direction synthesis function, can catalyze the synthesis of ester, can also catalyze the decomposition of ester.It Can be widely applied in industrial market. In this paper, first carries four Monascus strains for producing the liquid state esterification enzyme ability screening, screening a best strain. Then we carried on the optimization to its culture medium component and raise condition, in order to obtain the most amount of enzyme production. Finally separation and purification of esterification enzyme-for conducting the zymology related nature research. The main test results are as follows:
1. Through to four Monascus strains Monascus sp. ZZ, Monascus ruberZK, Monasucs purpureusGX, Monasucs sp. XM screening. Obtains high-yield strain of liquid state esterification enzyme.Monascus ruberZK, its production of liquid enzyme level is 203.4U/mL, the total enzyme lives 9061.1U.
2. First, we carried on the single factor to the culture medium in the carbon source, the nitrogen source, the inorganic salt.then,we carried on orthogonal experiment to the single factor results. Determined the best culture medium composition ofMonascus ruberZK are soluble starch 35g/L, glucose 35g/L, protein peptone 25g/L, NaN03 2g/L, MgS04 1.0g/L, KH2P04 1.5g/L; The confirmation experiment indicated that enzyme-activity of the esterification enzyme reached 293.8 U/mL. With the best combination of conditions B2A2C2D1 fermentation,
3. carry on the single factor experiment to temperature, initial pH value, vaccination quantity, attire liquid volume, table rotational speed and other influencing factors of the culture conditions. Then Selected the temperature, the initial pH value, the vaccination quantity, the table rotational speed four best single factors to do four factor three horizontal orthogonal experiments. Determined that Monascus ruberZK the optimum condition for the temperature 32℃, the initial pH value 5.0, vaccination quantities 10%, installs the liquid volume is 60mL (250mL triangle bottle), table rotational speed 160r/min. The confirmation experiment indicated that enzyme-activity of the esterification enzyme reached 312. 1U/mL. With the best combination of conditions B2A2C2D1 fermentation,
4. The rough enzyme fluid are gradually salted out by the ammonium sulfate, enzyme fluid are desalted by Sephadex the G-25column chromatographic analysis. Used Sephadex G-100 to carry on the separation and purification for the enzyme fluid, We obtain esterase receiving rate are 25.3%, the purification multiple is 7.6.
5. The esterification enzyme most suitable enzyme live temperature 40℃, most suitable pH is 5.3, PH in 4.8-5.7 scopes, relative enzyme activity is above 90%. Acid and alkali stable aspect, when the pH value between 3.5-6.8, has the good acid resistance, strong stability. Cacl2, Mgcl2, EDTA have the obvious activation function to this esterase. Nacl, Kcl have the mild activation function to this esterase; But Hgcl2, AgNO3 have the obvious inhibitory action to this esterase, causes it to lose activeness. Fecl2, Nicl2, Sncl2 and so on also have the inhibitory action to this esterase.
引文
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