双孢蘑菇多酚氧化酶的研究
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摘要
双孢蘑菇营养丰富,味道鲜美。然而,双孢蘑菇在贮藏和加工过程中极容易发生褐变反应,严重地影响了产品的外观、风味、营养和加工性能,大大降低了商业价值,而多酚氧化酶(Polyphenol oxidase, PPO)是引起酶促褐变的主要酶类。另外,近年来研究发现PPO能催化蛋白发生交联反应,从而可改善蛋白质的功能特性。因此,双孢蘑菇多酚氧化酶值得深入研究。
本论文开展的研究工作主要包括双孢蘑菇PPO的分离纯化及酶学性质的研究、双孢蘑菇PPO对乳蛋白交联的影响、双孢蘑菇PPO新基因的克隆、PP03和PP04成熟酶基因的克隆及其在原核系统中的表达,研究的主要方法、结果及结论如下：
1采用硫酸铵分级沉淀、DEAE-Sepharose Fast Flow离子交换层析和Phenyl sepharose CL-4B疏水层析相结合的方法纯化双孢蘑菇中PPO,经SDS-PAGE检测,该PPO分子量为43 kDa,N端测序得到的氨基酸序列为：Ala-Thr-Asn-Ser-Gly-Thr-Leu-Ile-Ile-Phe.
2 PPO活力的最适温度和pH分别为20℃和6.5-7.0。PPO对热不稳定,当温度达到60℃时,PPO活力几乎完全丧失。Al3+可以使PPO完全失活,Cu2+和Zn2+对PPO活力表现很强的抑制作用。以邻苯二酚为底物时,PPO的Km和Vmax值分别为0.67 mmol/L和3,333 U/mLmin-1.
3采用双孢蘑菇PPO对酪蛋白交联的最佳条件是PPO活力600 U/mL、pH 7、30℃反应6 h,乳清蛋白交联的最佳条件是PPO活力600U/mL、pH3-5、40℃反应4 h。交联后乳蛋白的乳化性及其稳定性和起泡性及其稳定性发生了较大的变化。另外,交联后β-酪蛋白、α-乳白蛋白和β-乳球蛋白的抗原性明显降低。
4通过多序列比对寻找真菌中PPO保守区的氨基酸序列,设计一对简并引物扩增保守区的基因序列,克隆获得了两个新的基因片段,然后通过RACE技术克隆获得了两个编码双孢蘑菇PPO的新基因-PPO3和PPO4. PPO3长度为1,844 bp,开放阅读框为1,731 bp,编码576个氨基酸。PPO4长度为2,042 bp,开放阅读框为1,836 bp,编码611个氨基酸。在核酸水平上,PPO3和PPO4同源性为52%,它们在Genbank数据库中的登录号分别为GQ354801和GQ354802。
5通过生物信息软件和网络服务器对PPO3和PPO4的氨基酸序列、系统进化树、二级结构和三级结构进行分析,结果表明PPO3和PPO4确为编码双孢蘑菇PPO的新基因。
6通过PPO3和PPO4 C-端切割位点的分析可知,PPO3成熟酶基因含有1,173 bp,编码391个氨基酸,分子量为45.3 kDa; PP04成熟酶含有1,134 bp,编码378个氨基酸,分子量为43.2 kDa。采用pGEX-4T-1表达载体和E.coli BL21 (DE3) RIPL表达菌,在原核系统中表达了PPO3和PPO4的成熟酶基因,并对其进行了纯化。得到的两种重组表达蛋白的分子量分别为71 kDa和69 kDa,均能被羊抗双孢蘑菇PPO抗体所识别。
Agaricus bisporus was nutritious and delicious. However, Agaricus bisporus was easy to occur enzymatic browning during processing and storage, seriously affecting the product appearance, flavor, nutrition, processing performance and then resulting in great economic losses. Polyphenol oxidase (PPO) was the main enzyme which was responsible for enzymatic browning. In addition, in recent years it was found that PPO could catalyze enzymatic cross-linking of proteins, which can improve the functional properties of proteins. Therefore, Agaricus bisporus PPO deserves further research.
The research work was composed of four parts, including purification and biochemical characterisation of PPO from Agaricus bisporus, effect of Agaricus bisporus PPO on cross-linking of milk proteins, cloning of new polyphenol oxidase cDNAs from Agaricus bisporus, cloning and expression of mature forms of PPO3 and PPO4 cDNAs in prokaryotic expression system. The main methods, results and conclusions were as follows:
1 Agaricus bisporus PPO was purified by ammonium sulfate fractional precipitation, DEAE-Sepharose Fast Flow ion-exchange chromatography and phenyl Sepharose CL-4B chromatography. The molecular weight of the purified PPO was about 43 kDa by SDS-PAGE, and the N-terminal sequence was identified as Ala-Thr-Asn-Ser-Gly-Thr-Leu-Ile-Ile-Phe by Edman degradation.
2 The optimum temperature and pH for the PPO activity was 20℃and pH 6.5-7.0. PPO was thermal labile. When the temperature was up to 60℃, the PPO activity was completely lost. Moreover, PPO activity was completely inactivated by Al3+, while Cu2+ and Zn2+ showed hearvy inhibition on the PPO activity. The Km and Vmax values of this PPO for catechol were 0.67 mmol/L and 3,333 U/ml min-1, respectively.
3 The optical parameters of cross linking of caseins with Agaricus bisporus PPO were as follows:(1) PPO activity was 600 U/ml, (2) pH value was 7, (3) cross linking performed at 30℃for 6 h. The optical parameters of cross linking of whey proteins were that, (1) pH value was 3-5, (2) PPO activity was 600 U/ml, (3) cross linking performed at 40℃for 4 h. Following the cross linking with PPO, the emulsifying activity index, emulsifying stability index, foam capacity and foam stability of the milk proteins were evaluated to be modified. In addition, the antigenicity ofβ-casein,α-lactalbumin andβ-lactoglobulin was decreased obviously after cross linking by PPO, respectively.
4 Two new conserved regions of PPO cDNAs from Agaricus bisporus were amplified using degenerate primers, which were designed corresponding to well conserved regions of several fungi PPOs. Then two new PPO full length cDNAs (PPO3 and PPO4) were obtained by RACE-PCR. PPO3 was 1,844 bp in length with an open reading frame of 1,731 bp, while that of PPO4 was 2,042 bp with an open reading frame of 1,836 bp. PPO3 and PPO4, with 52% identity at the nucleic acid level, encoded a 596-amino acid protein and 611-amino acid protein respectively. They were deposited at the Genbank database under the accession numbers GQ354801 and GQ354802, respectively.
5 Biotic softwares and network servers were used to analysis the amino acids, phylogenetic tree, secondary and tertiary structures of PPO3 and PPO4. The results indicated that PPO3 and PPO4 were the new encolded genes for Agaricus bisporus PPO.
6 By analysising the cleavage of C-terminal region, we knew that the mature form of PPO3 was 1,173 bp in length encoded a total of 391 amino acids with a molecular weight of 45.3 kDa, and the mature form of PPO4 was 1,134 bp in length encoded a total of 378 amino acids with a molecular weight of 43.2 kDa. Mature forms of PPO3 and PPO4 were both expressed in E. coli BL21 (DE3) RIPL using pGEX-4T-1 vector and the expressed proteins were purified. The molecular weight of the reconbinant proteins were 71 kDa and 69 kDa, respectively. Both of them could be probed by the sheep anti-Agaricus bisporus PPO antibody.
本论文开展的研究工作主要包括双孢蘑菇PPO的分离纯化及酶学性质的研究、双孢蘑菇PPO对乳蛋白交联的影响、双孢蘑菇PPO新基因的克隆、PP03和PP04成熟酶基因的克隆及其在原核系统中的表达,研究的主要方法、结果及结论如下：
1采用硫酸铵分级沉淀、DEAE-Sepharose Fast Flow离子交换层析和Phenyl sepharose CL-4B疏水层析相结合的方法纯化双孢蘑菇中PPO,经SDS-PAGE检测,该PPO分子量为43 kDa,N端测序得到的氨基酸序列为：Ala-Thr-Asn-Ser-Gly-Thr-Leu-Ile-Ile-Phe.
2 PPO活力的最适温度和pH分别为20℃和6.5-7.0。PPO对热不稳定,当温度达到60℃时,PPO活力几乎完全丧失。Al3+可以使PPO完全失活,Cu2+和Zn2+对PPO活力表现很强的抑制作用。以邻苯二酚为底物时,PPO的Km和Vmax值分别为0.67 mmol/L和3,333 U/mLmin-1.
3采用双孢蘑菇PPO对酪蛋白交联的最佳条件是PPO活力600 U/mL、pH 7、30℃反应6 h,乳清蛋白交联的最佳条件是PPO活力600U/mL、pH3-5、40℃反应4 h。交联后乳蛋白的乳化性及其稳定性和起泡性及其稳定性发生了较大的变化。另外,交联后β-酪蛋白、α-乳白蛋白和β-乳球蛋白的抗原性明显降低。
4通过多序列比对寻找真菌中PPO保守区的氨基酸序列,设计一对简并引物扩增保守区的基因序列,克隆获得了两个新的基因片段,然后通过RACE技术克隆获得了两个编码双孢蘑菇PPO的新基因-PPO3和PPO4. PPO3长度为1,844 bp,开放阅读框为1,731 bp,编码576个氨基酸。PPO4长度为2,042 bp,开放阅读框为1,836 bp,编码611个氨基酸。在核酸水平上,PPO3和PPO4同源性为52%,它们在Genbank数据库中的登录号分别为GQ354801和GQ354802。
5通过生物信息软件和网络服务器对PPO3和PPO4的氨基酸序列、系统进化树、二级结构和三级结构进行分析,结果表明PPO3和PPO4确为编码双孢蘑菇PPO的新基因。
6通过PPO3和PPO4 C-端切割位点的分析可知,PPO3成熟酶基因含有1,173 bp,编码391个氨基酸,分子量为45.3 kDa; PP04成熟酶含有1,134 bp,编码378个氨基酸,分子量为43.2 kDa。采用pGEX-4T-1表达载体和E.coli BL21 (DE3) RIPL表达菌,在原核系统中表达了PPO3和PPO4的成熟酶基因,并对其进行了纯化。得到的两种重组表达蛋白的分子量分别为71 kDa和69 kDa,均能被羊抗双孢蘑菇PPO抗体所识别。
Agaricus bisporus was nutritious and delicious. However, Agaricus bisporus was easy to occur enzymatic browning during processing and storage, seriously affecting the product appearance, flavor, nutrition, processing performance and then resulting in great economic losses. Polyphenol oxidase (PPO) was the main enzyme which was responsible for enzymatic browning. In addition, in recent years it was found that PPO could catalyze enzymatic cross-linking of proteins, which can improve the functional properties of proteins. Therefore, Agaricus bisporus PPO deserves further research.
The research work was composed of four parts, including purification and biochemical characterisation of PPO from Agaricus bisporus, effect of Agaricus bisporus PPO on cross-linking of milk proteins, cloning of new polyphenol oxidase cDNAs from Agaricus bisporus, cloning and expression of mature forms of PPO3 and PPO4 cDNAs in prokaryotic expression system. The main methods, results and conclusions were as follows:
1 Agaricus bisporus PPO was purified by ammonium sulfate fractional precipitation, DEAE-Sepharose Fast Flow ion-exchange chromatography and phenyl Sepharose CL-4B chromatography. The molecular weight of the purified PPO was about 43 kDa by SDS-PAGE, and the N-terminal sequence was identified as Ala-Thr-Asn-Ser-Gly-Thr-Leu-Ile-Ile-Phe by Edman degradation.
2 The optimum temperature and pH for the PPO activity was 20℃and pH 6.5-7.0. PPO was thermal labile. When the temperature was up to 60℃, the PPO activity was completely lost. Moreover, PPO activity was completely inactivated by Al3+, while Cu2+ and Zn2+ showed hearvy inhibition on the PPO activity. The Km and Vmax values of this PPO for catechol were 0.67 mmol/L and 3,333 U/ml min-1, respectively.
3 The optical parameters of cross linking of caseins with Agaricus bisporus PPO were as follows:(1) PPO activity was 600 U/ml, (2) pH value was 7, (3) cross linking performed at 30℃for 6 h. The optical parameters of cross linking of whey proteins were that, (1) pH value was 3-5, (2) PPO activity was 600 U/ml, (3) cross linking performed at 40℃for 4 h. Following the cross linking with PPO, the emulsifying activity index, emulsifying stability index, foam capacity and foam stability of the milk proteins were evaluated to be modified. In addition, the antigenicity ofβ-casein,α-lactalbumin andβ-lactoglobulin was decreased obviously after cross linking by PPO, respectively.
4 Two new conserved regions of PPO cDNAs from Agaricus bisporus were amplified using degenerate primers, which were designed corresponding to well conserved regions of several fungi PPOs. Then two new PPO full length cDNAs (PPO3 and PPO4) were obtained by RACE-PCR. PPO3 was 1,844 bp in length with an open reading frame of 1,731 bp, while that of PPO4 was 2,042 bp with an open reading frame of 1,836 bp. PPO3 and PPO4, with 52% identity at the nucleic acid level, encoded a 596-amino acid protein and 611-amino acid protein respectively. They were deposited at the Genbank database under the accession numbers GQ354801 and GQ354802, respectively.
5 Biotic softwares and network servers were used to analysis the amino acids, phylogenetic tree, secondary and tertiary structures of PPO3 and PPO4. The results indicated that PPO3 and PPO4 were the new encolded genes for Agaricus bisporus PPO.
6 By analysising the cleavage of C-terminal region, we knew that the mature form of PPO3 was 1,173 bp in length encoded a total of 391 amino acids with a molecular weight of 45.3 kDa, and the mature form of PPO4 was 1,134 bp in length encoded a total of 378 amino acids with a molecular weight of 43.2 kDa. Mature forms of PPO3 and PPO4 were both expressed in E. coli BL21 (DE3) RIPL using pGEX-4T-1 vector and the expressed proteins were purified. The molecular weight of the reconbinant proteins were 71 kDa and 69 kDa, respectively. Both of them could be probed by the sheep anti-Agaricus bisporus PPO antibody.
引文
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