云芝液体深层发酵天冬氨酸蛋白酶抑制剂的研究
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摘要
本论文主要对云芝液体深层发酵天冬氨酸蛋白酶抑制剂的发酵条件、分离纯化及基本特性等进行了研究,主要结果如下:
对甜芝、云芝、韩芝、赤芝G-8等4株菌进行平板培养,比较菌丝体生长速度,其中云芝和赤芝G-8生长最快,速度约为13.7mm/d,且云芝菌丝体最密,相比之下生长最慢的韩芝只有5.0mm/d。
对4株菌进行液体深层发酵,生物量以云芝最高,约为15.06g/L,是韩芝的2.83倍;将发酵液进行真空旋转蒸发浓缩干燥成粉及真空冷冻干燥成粉,然后再进行热水抽提、乙醇沉淀,比较所得物质对胃蛋白酶的抑制效果,仍以云芝发酵组最好,冻干粉抽提液30%-80%醇沉区段的沉淀对胃蛋白酶的抑制率可达29.3%,烘干粉同样处理后也能达到20%左右。
比较了云芝发酵液细胞破碎前后对胃蛋白酶的抑制效果,破碎后对胃蛋白酶的抑制率由破碎前的0.53%提高到37.2%,说明云芝胞内确实存对胃蛋白酶起抑制作用的物质。研究了超声波细胞破碎条件对云芝胞内天冬氨酸蛋白酶抑制剂提取的影响,确定最佳超声条件为:超声时间10min、超声功率300W、处理量40mL、超声温度20℃。从细胞破碎液中分离得一种天冬氨酸蛋白酶抑制剂(CVPI),建立了云芝胞内天冬氨酸蛋白酶抑制剂的纯化制备工艺为:摇瓶发酵-细胞破碎-大孔树脂D101脱色-DEAE52离子交换层析-ACA54分子筛层析进行CVPI的纯度鉴定及分子量测定。
凝胶过滤测定CVPI的相对表观分子量约为23442,主要成分为糖和蛋白质,蛋白比糖约为2.5;CVPI热稳定性较好,100℃保温30分钟后抑制活性几乎没有损失;CVPI的pH稳定范围为3.5～7.5;抑制活性受温度影响不大,0～100℃基本稳定;抑制效果在2小时内随反应时间延长上升较快,2小时后基本不再升高;抑制剂底物特异性研究表明,CVPI对天冬氨酸族的蛋白酶抑制活性较高,以对胃蛋白酶的抑制效果最强;CVPI对胃蛋白酶和蛋白酶A作用时的Ki值分别分别为0.82,1.32μmol/L;CVPI对胃蛋白酶和蛋白酶A的半抑制浓度IC50值分别约为15和29μg/mL;动力学方法研究(Lineweaver-Burk作图法)发现,CVPI是胃蛋白酶的非竞争与反竞争混合型抑制剂,是蛋白酶A的竞争与反竞争混合型抑制剂。
利用单因素实验,找到云芝液体深层发酵CVPI的主要影响因子为接种量、起始pH和装液量。
在单因素实验的基础上,采用响应面分析法,对培养条件进行了优化,得最佳培养条件:发酵温度为26℃,发酵时间为120小时,转速170r/min,装液量为71.31mL/250mL,起始pH为5.97,接种量为13.96%。优化后的抑制剂产量为10.13IU/mL,比优化前提高了172.3%。
The research mainly focused on the following aspects about aspartic acid protease inhibitor from Coriolus versicolor by submerged fermentation, including fermentation conditions, extracting, purification and the basic properties.
Compared the mycelium growth rate by plate culture, Coriolus versicolor and Gctnoderma lucidum G-8 grew the most rapidly, about 13.7mm/d, and the mycelium of Coriolus versicolor was the most dense, Gantheram Lucidum grew the most slowly, about 5.0mm/d.
Compared the biomass of different fungi by submerged fermentation, the mycelium dry weight of Coriolus versicolor is the most heavy 15.06g/L, is about 2.83 times of Gantheram Lucidum; Dried the fermentation broth by rotary vacuum drying and vacuum freeze drying into powder, and then by hot water extraction, ethanol precipitation, eventually, compared the inhibition rate of extract effect on pepsin, Coriolus versicolor is still the best one, the inhibition rate of extract from powder by vacuum freeze drying effect on pepsin was about 29.3%, and about 20% by rotary vacuum drying.
Compared the inhibition rate of Coriolus versicolor broth by pre-and post cell disruption effect on pepsin, after cell disruption the inhibition rate improved from 0.53% to 37.2%, it was suggested that the intracellular of Coriolus versicolor contains the pepsin inhibitor. Studied on ultrasonic time, ultrasonic power, ultrasonic treatment capacity and ultrasonic temperature, the best ultrasonic conditions was determined as 10 minutes of ultrasonic time, 300W of ultrasonic power, 40mL of ultrasonic treatment capacity and 20℃of ultrasonic temperature.
One kind of aspartic acid protease inhibitor CVPI was purified, The purification was carried out by shaking flask fermentation-cell disruption-decoloration by macroporous resin D101-DEAE52 ion exchange chromatogram-UltroGel ACA54 gel-filtration chromatogram, respectively.
The molecular mass of CVPI was 23442 as estimated via gel filtration; The inhibitor showed a remarkable heat stability, it retained its initial activity even at up to 100℃for 30 min; The pH stability range of CVPI is about 3.5～7.5; The inhibitor rate improved quickly in 2 hours time of inhibitory reaction, and inproved hardly after 2 hours latter; By investigating the interaction between this inhibitor and variety of proteinases, it was indicated that the inhibitor was more specific against aspartic acid proteinase (such as pepsin and yeast proteinase A) than other proteinases; The dissociation constants (Ki) and concentration required for 50% inhibition (IC50) for pepsin were 0.82μmol/L and 15μg/mL, and the dissociation constants (Ki) and concentration required for 50% inhibition (IC50) for proteinase A were 1.32μmol/L and 29μg/mL; Kinetic studies (Lineweaver-Burk method) showed CVPI is a kind of noncompetitive and anti-compeititve inhibitor to pepsin while competitive and anti-competitive inhibitor to yeast proteinase A.
The main impact factors were determined as inoculum size, initial pH and medium volume using single factor experiment method.
The response surface methodology and single-factor tested for the best culture conditions indicated that fermentation temperature is 26℃, fermentation time is 120 h, rotate speed is 170r/min, medium volume is 71.31 mL/250mL, initial pH is 5.97, inoculum size is 13.96%; The inhibitor units were improved from 3.72IU/mL to 10.15IU/mL.
对甜芝、云芝、韩芝、赤芝G-8等4株菌进行平板培养,比较菌丝体生长速度,其中云芝和赤芝G-8生长最快,速度约为13.7mm/d,且云芝菌丝体最密,相比之下生长最慢的韩芝只有5.0mm/d。
对4株菌进行液体深层发酵,生物量以云芝最高,约为15.06g/L,是韩芝的2.83倍;将发酵液进行真空旋转蒸发浓缩干燥成粉及真空冷冻干燥成粉,然后再进行热水抽提、乙醇沉淀,比较所得物质对胃蛋白酶的抑制效果,仍以云芝发酵组最好,冻干粉抽提液30%-80%醇沉区段的沉淀对胃蛋白酶的抑制率可达29.3%,烘干粉同样处理后也能达到20%左右。
比较了云芝发酵液细胞破碎前后对胃蛋白酶的抑制效果,破碎后对胃蛋白酶的抑制率由破碎前的0.53%提高到37.2%,说明云芝胞内确实存对胃蛋白酶起抑制作用的物质。研究了超声波细胞破碎条件对云芝胞内天冬氨酸蛋白酶抑制剂提取的影响,确定最佳超声条件为:超声时间10min、超声功率300W、处理量40mL、超声温度20℃。从细胞破碎液中分离得一种天冬氨酸蛋白酶抑制剂(CVPI),建立了云芝胞内天冬氨酸蛋白酶抑制剂的纯化制备工艺为:摇瓶发酵-细胞破碎-大孔树脂D101脱色-DEAE52离子交换层析-ACA54分子筛层析进行CVPI的纯度鉴定及分子量测定。
凝胶过滤测定CVPI的相对表观分子量约为23442,主要成分为糖和蛋白质,蛋白比糖约为2.5;CVPI热稳定性较好,100℃保温30分钟后抑制活性几乎没有损失;CVPI的pH稳定范围为3.5～7.5;抑制活性受温度影响不大,0～100℃基本稳定;抑制效果在2小时内随反应时间延长上升较快,2小时后基本不再升高;抑制剂底物特异性研究表明,CVPI对天冬氨酸族的蛋白酶抑制活性较高,以对胃蛋白酶的抑制效果最强;CVPI对胃蛋白酶和蛋白酶A作用时的Ki值分别分别为0.82,1.32μmol/L;CVPI对胃蛋白酶和蛋白酶A的半抑制浓度IC50值分别约为15和29μg/mL;动力学方法研究(Lineweaver-Burk作图法)发现,CVPI是胃蛋白酶的非竞争与反竞争混合型抑制剂,是蛋白酶A的竞争与反竞争混合型抑制剂。
利用单因素实验,找到云芝液体深层发酵CVPI的主要影响因子为接种量、起始pH和装液量。
在单因素实验的基础上,采用响应面分析法,对培养条件进行了优化,得最佳培养条件:发酵温度为26℃,发酵时间为120小时,转速170r/min,装液量为71.31mL/250mL,起始pH为5.97,接种量为13.96%。优化后的抑制剂产量为10.13IU/mL,比优化前提高了172.3%。
The research mainly focused on the following aspects about aspartic acid protease inhibitor from Coriolus versicolor by submerged fermentation, including fermentation conditions, extracting, purification and the basic properties.
Compared the mycelium growth rate by plate culture, Coriolus versicolor and Gctnoderma lucidum G-8 grew the most rapidly, about 13.7mm/d, and the mycelium of Coriolus versicolor was the most dense, Gantheram Lucidum grew the most slowly, about 5.0mm/d.
Compared the biomass of different fungi by submerged fermentation, the mycelium dry weight of Coriolus versicolor is the most heavy 15.06g/L, is about 2.83 times of Gantheram Lucidum; Dried the fermentation broth by rotary vacuum drying and vacuum freeze drying into powder, and then by hot water extraction, ethanol precipitation, eventually, compared the inhibition rate of extract effect on pepsin, Coriolus versicolor is still the best one, the inhibition rate of extract from powder by vacuum freeze drying effect on pepsin was about 29.3%, and about 20% by rotary vacuum drying.
Compared the inhibition rate of Coriolus versicolor broth by pre-and post cell disruption effect on pepsin, after cell disruption the inhibition rate improved from 0.53% to 37.2%, it was suggested that the intracellular of Coriolus versicolor contains the pepsin inhibitor. Studied on ultrasonic time, ultrasonic power, ultrasonic treatment capacity and ultrasonic temperature, the best ultrasonic conditions was determined as 10 minutes of ultrasonic time, 300W of ultrasonic power, 40mL of ultrasonic treatment capacity and 20℃of ultrasonic temperature.
One kind of aspartic acid protease inhibitor CVPI was purified, The purification was carried out by shaking flask fermentation-cell disruption-decoloration by macroporous resin D101-DEAE52 ion exchange chromatogram-UltroGel ACA54 gel-filtration chromatogram, respectively.
The molecular mass of CVPI was 23442 as estimated via gel filtration; The inhibitor showed a remarkable heat stability, it retained its initial activity even at up to 100℃for 30 min; The pH stability range of CVPI is about 3.5～7.5; The inhibitor rate improved quickly in 2 hours time of inhibitory reaction, and inproved hardly after 2 hours latter; By investigating the interaction between this inhibitor and variety of proteinases, it was indicated that the inhibitor was more specific against aspartic acid proteinase (such as pepsin and yeast proteinase A) than other proteinases; The dissociation constants (Ki) and concentration required for 50% inhibition (IC50) for pepsin were 0.82μmol/L and 15μg/mL, and the dissociation constants (Ki) and concentration required for 50% inhibition (IC50) for proteinase A were 1.32μmol/L and 29μg/mL; Kinetic studies (Lineweaver-Burk method) showed CVPI is a kind of noncompetitive and anti-compeititve inhibitor to pepsin while competitive and anti-competitive inhibitor to yeast proteinase A.
The main impact factors were determined as inoculum size, initial pH and medium volume using single factor experiment method.
The response surface methodology and single-factor tested for the best culture conditions indicated that fermentation temperature is 26℃, fermentation time is 120 h, rotate speed is 170r/min, medium volume is 71.31 mL/250mL, initial pH is 5.97, inoculum size is 13.96%; The inhibitor units were improved from 3.72IU/mL to 10.15IU/mL.
引文
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