两种天冬氨酸蛋白酶抑制剂的制备及特性表征
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摘要
本论文主要研究云芝深层发酵液和马铃薯中两种天冬氨酸蛋白酶抑制剂(CVPI、PPI)的分离纯化过程和两种抑制剂的基本特性表征及作用机理,对云芝深层发酵CVPI的条件进行了优化,并对两种抑制剂的应用潜力进行了初步考察。
考察云芝发酵产天冬氨酸蛋白酶抑制剂(CVPI)发酵培养基中碳氮源的组成和利用正交试验法确定碳氮源的最佳发酵浓度,确定最佳碳氮源为葡萄糖、马铃薯汁、豆渣粉和蛋白胨,含量分别为1%、1.5%、1%和0.4%,在此培养基基础上进行单因素实验得优化后的培养条件:pH 6.0、装液量70mL/250mL、转速160r/min、接种量10%、温度28℃,优化后CVPI对胃蛋白酶的抑制单位提高到13.1 IU/mL,是优化前的2.01倍。CVPI由云芝深层发酵液经脱色、超滤、DEAE-52、MonoQ离子交换层析等方法得到了纯化,纯化倍数达到21.1倍,比抑制活力达到295.6 IU/mg,经SDS-PAGE鉴定为一种分子量约为22.3 kDa的单亚基蛋白。马铃薯中天冬氨酸蛋白酶抑制剂(PPI)由马铃薯块茎经匀浆、超滤、40-60%乙醇沉淀、DEAE-52、MonoQ离子交换层析得到纯化,纯化倍数为36.6,比抑制活力为149.6 IU/mg,结合SDS-PAGE和UltroGelACA-54分析确定该抑制剂是单亚基,分子量约为16.1 kDa。
对PPI基本性质的研究发现,PPI氨基酸组成中酸性氨基酸的含量较高,占32.6%;对胃蛋白酶的最佳抑制反应时间为1h;具有较高的温度稳定性,在20-80%范围内基本保持稳定;在pH稳定范围均为3.0～7.0;属性分析表明PPI本身不是蛋白酶也不能被胃蛋白酶水解;抑制特异性分析表明PPI只对天冬氨酸族的胃蛋白酶具有较高的抑制活性,而对其他家族的蛋白酶基本无抑制活性;动力学分析确定PPI的半抑制浓度IC50为26.67μg/mL,为非竞争性与竞争性混合型抑制剂。
将MALDI-TOF MS应用于CVPI及PPI的蛋白质肽谱分析,由数据库搜索结果知,两种抑制剂在氨基酸水平上与其它已知的天冬氨酸蛋白酶抑制剂没有一定的相似性,可能是目前数据库中未报道的抑制剂类型。
CD光谱分析推测CVPI与胃蛋白酶属于相伴型的结合类型,即抑制剂分子与酶分子并列相伴,不占据靶酶结合位点,通过形成氢键的方式封锁酶与底物的结合部位使其失活。而PPI可能是以覆盖型的方式与胃蛋白酶结合,即为阻止底物与酶的活性中心接触以线性分子的形式覆盖到酶活性中心附近的区域上。
荧光探针法研究CVPI及PPI与DNA的相互作用,在此基础上依据结果初步判断两者可能具有一定的抗癌效果。抗氧化作用实验表明PPI具有很强的清除羟自由基的作用,具有作为减少自由基对人体伤害的抗氧化剂的潜力。
In this thesis, two aspartic protease inhibitors (CVPI, PPI) were purified from versicolor fermentation broth and potatoes, and characterization of two inhibitors and the role of the basic mechanism were been studied, the submerged fermentation CVPI of versicolor conditions were optimized, and the application potential of the two inhibitors were preliminarily investigated. Study carbon and nitrogen sources in versicolor fermentation medium composition and though the orthogonal design to optimize the concentration of carbon and nitrogen source medium of coriolus versicolor submerged fermentation, The best concentration of carbon and nitrogen source as follow: 1% glucose, 1.5% potatoes juice, 1% soybean residue powder and 0.4% peptone respectively. On the basis of single factor experiments in the best culture conditions: initial pH6.0, liquid volume 70mL/250mL, rotation speed 160r/min, 10% inoculum, the fermentation temperature 28℃, optimized inhibitors yield 13.1IU/mL, compared with 2.01 times before optimization.
CVPI was pretreated by decolorization, ultrofilition, and then isolated by ion-exchange chromatography DEAE-52 and MonoQ, the purification fold was 21.1 and the inhibition of activity was 295.6 IU /mg, identified by SDS-PAGE as a single subunit with molecular weight of about 22.3 kDa. The PPI was purified by cut potato homogenate, ultrafiltration, ethanol precipitation, DEAE-52 and Mono Q , the purification fold was 36.6. Identified by SDS-PAGE was a single band with molecular weight of 16.1 kDa.
PPI analysis showed that acidic amino acids of PPI were higher in the amino acids, accounting for 32.6%; Inhibition of the best response time is 1 h; The high thermostability of PPI has also been observed at 20℃~ 80℃; pH stability range was 3.0 to 7.0; attribute analysis showed that PPI itself is not a protease and can not be hydrolyzed by pepsin; the PPI is more specific against pepsin than other proteinases; Concentration required for 50% pepsin inhibition (IC50) were 26.26μg/mL; it were non-competitive and competitive mixed type inhibitor.
Used MALDI-TOF MS peptide mapping of CVPI and PPI analysis, database search, we found that two inhibitors of the amino acid level with other known aspartic protease inhibitors without a certain similarity, is an inhibitor type of the database not reported.
CD spectra analysis pepsin combined with CVPI is accompanied by type, that is the inhibitor molecules do not occupy the binding sites of target enzyme, but tied with the enzyme molecule, and enzyme activity in the group with the formation of hydrogen bonds, while blockade of enzyme and substrate binding site, making it inactive. Type of PPI with the combination of pepsin may belong to "coverage", it may be similar to the linear molecule in the form of coverage to the target area around the active site, thereby preventing the enzyme's active site and substrate contacts.
Fluorescent probe CVPI and PPI interaction with DNA, based on the results of this preliminary judgments on the basis of two may have some anti-cancer effect. Anti-oxidation experiments show that PPI has a strong scavenging effect on hydroxyl radical, can be used as antioxidants to slow the potential of hydroxyl radical damage to the human body.
考察云芝发酵产天冬氨酸蛋白酶抑制剂(CVPI)发酵培养基中碳氮源的组成和利用正交试验法确定碳氮源的最佳发酵浓度,确定最佳碳氮源为葡萄糖、马铃薯汁、豆渣粉和蛋白胨,含量分别为1%、1.5%、1%和0.4%,在此培养基基础上进行单因素实验得优化后的培养条件:pH 6.0、装液量70mL/250mL、转速160r/min、接种量10%、温度28℃,优化后CVPI对胃蛋白酶的抑制单位提高到13.1 IU/mL,是优化前的2.01倍。CVPI由云芝深层发酵液经脱色、超滤、DEAE-52、MonoQ离子交换层析等方法得到了纯化,纯化倍数达到21.1倍,比抑制活力达到295.6 IU/mg,经SDS-PAGE鉴定为一种分子量约为22.3 kDa的单亚基蛋白。马铃薯中天冬氨酸蛋白酶抑制剂(PPI)由马铃薯块茎经匀浆、超滤、40-60%乙醇沉淀、DEAE-52、MonoQ离子交换层析得到纯化,纯化倍数为36.6,比抑制活力为149.6 IU/mg,结合SDS-PAGE和UltroGelACA-54分析确定该抑制剂是单亚基,分子量约为16.1 kDa。
对PPI基本性质的研究发现,PPI氨基酸组成中酸性氨基酸的含量较高,占32.6%;对胃蛋白酶的最佳抑制反应时间为1h;具有较高的温度稳定性,在20-80%范围内基本保持稳定;在pH稳定范围均为3.0～7.0;属性分析表明PPI本身不是蛋白酶也不能被胃蛋白酶水解;抑制特异性分析表明PPI只对天冬氨酸族的胃蛋白酶具有较高的抑制活性,而对其他家族的蛋白酶基本无抑制活性;动力学分析确定PPI的半抑制浓度IC50为26.67μg/mL,为非竞争性与竞争性混合型抑制剂。
将MALDI-TOF MS应用于CVPI及PPI的蛋白质肽谱分析,由数据库搜索结果知,两种抑制剂在氨基酸水平上与其它已知的天冬氨酸蛋白酶抑制剂没有一定的相似性,可能是目前数据库中未报道的抑制剂类型。
CD光谱分析推测CVPI与胃蛋白酶属于相伴型的结合类型,即抑制剂分子与酶分子并列相伴,不占据靶酶结合位点,通过形成氢键的方式封锁酶与底物的结合部位使其失活。而PPI可能是以覆盖型的方式与胃蛋白酶结合,即为阻止底物与酶的活性中心接触以线性分子的形式覆盖到酶活性中心附近的区域上。
荧光探针法研究CVPI及PPI与DNA的相互作用,在此基础上依据结果初步判断两者可能具有一定的抗癌效果。抗氧化作用实验表明PPI具有很强的清除羟自由基的作用,具有作为减少自由基对人体伤害的抗氧化剂的潜力。
In this thesis, two aspartic protease inhibitors (CVPI, PPI) were purified from versicolor fermentation broth and potatoes, and characterization of two inhibitors and the role of the basic mechanism were been studied, the submerged fermentation CVPI of versicolor conditions were optimized, and the application potential of the two inhibitors were preliminarily investigated. Study carbon and nitrogen sources in versicolor fermentation medium composition and though the orthogonal design to optimize the concentration of carbon and nitrogen source medium of coriolus versicolor submerged fermentation, The best concentration of carbon and nitrogen source as follow: 1% glucose, 1.5% potatoes juice, 1% soybean residue powder and 0.4% peptone respectively. On the basis of single factor experiments in the best culture conditions: initial pH6.0, liquid volume 70mL/250mL, rotation speed 160r/min, 10% inoculum, the fermentation temperature 28℃, optimized inhibitors yield 13.1IU/mL, compared with 2.01 times before optimization.
CVPI was pretreated by decolorization, ultrofilition, and then isolated by ion-exchange chromatography DEAE-52 and MonoQ, the purification fold was 21.1 and the inhibition of activity was 295.6 IU /mg, identified by SDS-PAGE as a single subunit with molecular weight of about 22.3 kDa. The PPI was purified by cut potato homogenate, ultrafiltration, ethanol precipitation, DEAE-52 and Mono Q , the purification fold was 36.6. Identified by SDS-PAGE was a single band with molecular weight of 16.1 kDa.
PPI analysis showed that acidic amino acids of PPI were higher in the amino acids, accounting for 32.6%; Inhibition of the best response time is 1 h; The high thermostability of PPI has also been observed at 20℃~ 80℃; pH stability range was 3.0 to 7.0; attribute analysis showed that PPI itself is not a protease and can not be hydrolyzed by pepsin; the PPI is more specific against pepsin than other proteinases; Concentration required for 50% pepsin inhibition (IC50) were 26.26μg/mL; it were non-competitive and competitive mixed type inhibitor.
Used MALDI-TOF MS peptide mapping of CVPI and PPI analysis, database search, we found that two inhibitors of the amino acid level with other known aspartic protease inhibitors without a certain similarity, is an inhibitor type of the database not reported.
CD spectra analysis pepsin combined with CVPI is accompanied by type, that is the inhibitor molecules do not occupy the binding sites of target enzyme, but tied with the enzyme molecule, and enzyme activity in the group with the formation of hydrogen bonds, while blockade of enzyme and substrate binding site, making it inactive. Type of PPI with the combination of pepsin may belong to "coverage", it may be similar to the linear molecule in the form of coverage to the target area around the active site, thereby preventing the enzyme's active site and substrate contacts.
Fluorescent probe CVPI and PPI interaction with DNA, based on the results of this preliminary judgments on the basis of two may have some anti-cancer effect. Anti-oxidation experiments show that PPI has a strong scavenging effect on hydroxyl radical, can be used as antioxidants to slow the potential of hydroxyl radical damage to the human body.
引文
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