马铃薯22-KD球蛋白的分离纯化及其结构测定的研究
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摘要
以荷兰马铃薯为原料,采用Osborne法提取马铃薯球蛋白(tuberin),通过单因素试验,对其提取工艺进行优化。试验结果表明：料液比1：12,温度35℃,浸提时间1h, NaCl浓度为1mol/L,提取次数为2次的条件下,马铃薯球蛋白提取率较高,达85%。
采用半微量凯氏定氮法与等电聚焦技术分别测定粗球蛋白中的蛋白含量及其等电点,试验结果表明：粗品中球蛋白含量为96.7%,该蛋白存在3个等电点,分别为5.83、6.0、6.7。
马铃薯球蛋白(tuberin)是马铃薯中三大主要贮藏蛋白之一,主要分布在马铃薯块茎中,占整个马铃薯蛋白的25%左右。它是一种易溶于盐的蛋白质,其含有较高的亮氨酸、赖氨酸、缬氨酸等,其必需氨基酸明显高于FAO/WHO的推荐值,因此,对食品加工来说它是一种很好的蛋白质原料来源,具有很好的应用前景。
将提取得到的马铃薯粗球蛋白,采用Sephadex G-50和DEAE-Cellulose 52阴离子交换剂纯化技术分离22-KD马铃薯球蛋白,纯化后在280nm下测得一个吸收峰,经SDS-PAGE测定其相对分子量为22-KD,此时洗脱液中盐离子浓度为0.67 mol/L。
将纯化后的22-KD球蛋白进行氨基酸成分分析、X-粉末晶体衍射、圆二色谱分析、红外光谱分析等结构分析,结果表明：必需氨基酸中缬氨酸、亮氨酸、赖氨酸含量较高,分别为5.69g/100g、5.54g/100g、5.51 g/100g,甲硫氨酸、胱氨酸的含量很低,分别为0.92g/100g,0.61g/100g,表明含硫氨基酸是22-KD马铃薯球蛋白的限制性氨基酸；22-KD马铃薯球蛋白二级结构主要是无规则卷曲,占85%以上,β折叠和α螺旋含量接近,β转角含量最低。
The crude tuberin was extracted from the Netherlands potato by Osborne method, optimization its extraction craft by single factor experiment. The results showed that when the dosage liquor ratio is 1:12, temperature is 35℃, diffusion time is 1h, the concentration of NaCl is lmol/L, extracted twice, the extraction ratio was 85%.
Take semimicro Kjeldah method and isoelectric focusing technology determined the protein level and isoelectric points of crude tuberin, the results showed that the protein level is 96.7% and it exists 3 isoelectric points, dicern as 5.83、6.0.6.7.
Tuberin is one of third main storage protein in potato, most distribution in its tubers, holds 25% of all potato protein. It is a kind of salting protein, contains higher essential amino acid of Leucine, Lysine and Valine, and the essential amino acid higher obviously than the recommendation value of FAO/WHO. Therefore, it is a good kind of protein source to food processing.
The crude tuberin was purified by Sephadex G-50 and DEAE-Cellulose 52 anion. The results showed that the max absorption peak of 280nm, and the molecular weight was 22-KD, when the concentration of salt is 0.67mol/L. These results will provide help for the relationship between the structure and function of tuberin.
The purified 22-KD tuberin for structural analysis of amino acid analysis, X-powder diffraction pattern, circular dichroism analysis and IR analysis, the results showed that the essential amino acids of valine, leucine, lysine Higher levels, respectively is 5.69 g/100g,5.54 g/100g,5.51 g/100g, but methionine, cystine content is very low, namely 0.92 g/100g,0.61 g/100g, this indicated that the sulfur-containing amino acids is the limiting amino acid in 22-KD tuberin; 22-KD tuberin secondary structure is mainly unordered, accounting for more than 85%,βsheet and a helix content close,βturns is the lowest.
采用半微量凯氏定氮法与等电聚焦技术分别测定粗球蛋白中的蛋白含量及其等电点,试验结果表明：粗品中球蛋白含量为96.7%,该蛋白存在3个等电点,分别为5.83、6.0、6.7。
马铃薯球蛋白(tuberin)是马铃薯中三大主要贮藏蛋白之一,主要分布在马铃薯块茎中,占整个马铃薯蛋白的25%左右。它是一种易溶于盐的蛋白质,其含有较高的亮氨酸、赖氨酸、缬氨酸等,其必需氨基酸明显高于FAO/WHO的推荐值,因此,对食品加工来说它是一种很好的蛋白质原料来源,具有很好的应用前景。
将提取得到的马铃薯粗球蛋白,采用Sephadex G-50和DEAE-Cellulose 52阴离子交换剂纯化技术分离22-KD马铃薯球蛋白,纯化后在280nm下测得一个吸收峰,经SDS-PAGE测定其相对分子量为22-KD,此时洗脱液中盐离子浓度为0.67 mol/L。
将纯化后的22-KD球蛋白进行氨基酸成分分析、X-粉末晶体衍射、圆二色谱分析、红外光谱分析等结构分析,结果表明：必需氨基酸中缬氨酸、亮氨酸、赖氨酸含量较高,分别为5.69g/100g、5.54g/100g、5.51 g/100g,甲硫氨酸、胱氨酸的含量很低,分别为0.92g/100g,0.61g/100g,表明含硫氨基酸是22-KD马铃薯球蛋白的限制性氨基酸；22-KD马铃薯球蛋白二级结构主要是无规则卷曲,占85%以上,β折叠和α螺旋含量接近,β转角含量最低。
The crude tuberin was extracted from the Netherlands potato by Osborne method, optimization its extraction craft by single factor experiment. The results showed that when the dosage liquor ratio is 1:12, temperature is 35℃, diffusion time is 1h, the concentration of NaCl is lmol/L, extracted twice, the extraction ratio was 85%.
Take semimicro Kjeldah method and isoelectric focusing technology determined the protein level and isoelectric points of crude tuberin, the results showed that the protein level is 96.7% and it exists 3 isoelectric points, dicern as 5.83、6.0.6.7.
Tuberin is one of third main storage protein in potato, most distribution in its tubers, holds 25% of all potato protein. It is a kind of salting protein, contains higher essential amino acid of Leucine, Lysine and Valine, and the essential amino acid higher obviously than the recommendation value of FAO/WHO. Therefore, it is a good kind of protein source to food processing.
The crude tuberin was purified by Sephadex G-50 and DEAE-Cellulose 52 anion. The results showed that the max absorption peak of 280nm, and the molecular weight was 22-KD, when the concentration of salt is 0.67mol/L. These results will provide help for the relationship between the structure and function of tuberin.
The purified 22-KD tuberin for structural analysis of amino acid analysis, X-powder diffraction pattern, circular dichroism analysis and IR analysis, the results showed that the essential amino acids of valine, leucine, lysine Higher levels, respectively is 5.69 g/100g,5.54 g/100g,5.51 g/100g, but methionine, cystine content is very low, namely 0.92 g/100g,0.61 g/100g, this indicated that the sulfur-containing amino acids is the limiting amino acid in 22-KD tuberin; 22-KD tuberin secondary structure is mainly unordered, accounting for more than 85%,βsheet and a helix content close,βturns is the lowest.
引文
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[2]张泽生,郭宝芹,刘素稳,不同酶水解马铃薯蛋白的分析、评价[J].食品研究与开发,2009,30(1)：112-114
[3]巩慧玲,赵萍,杨俊峰.马铃薯块茎贮藏期间蛋白质和维生素C含量的变化[J].西北农业学报,2004,13(1)：45-51.
[4]谢庆华,吴毅歆.马铃薯品种营养成分分析测定[J].云南师范大学学报,2002,22(2)：50-52.
[5]REXEN B. Studies of protein of potatoes[J]. Potato Res,1976,19:189-202.
[6]KALDY M S. Protein yield in various crops as related to protein value[J]. Economical Botany, 1972,26:142-144.
[7]KALDY M S,MARKAKIS P. Amino acid composition of selected potato varieties[J]. Food Sci,1972,37(3):375-377.
[8]VAN GELDER W M J, VONK C R. Amino acid composition of coagulable protein from tubers of 34 potato varieties and its relationship with protein content[J]. Potato Research,1980,23: 427-434.
[9]马莺.马铃薯加工业的现状及发展前景[J].中国马铃薯,2001,15(2)：123-125.
[10]张泽俊,苏春元,刘期成.马铃薯淀粉厂工艺废水的综合处理及利用研究[J].食品科学,2004,(增1)：134-137.
[11]ALUKO R E,YADA R Y. Structure-Funtion Relationship of Cowpea(Vigna ungui-culata) Globulin Isolates:Influence of pH and NaCl on Physicochemical and Functional Properties[J]. Food Chemistry,1995,53:259-265.
[12]Fennema O R.'食品化学[M].3版.王璋,许时婴,江波,等译.北京：中国轻工业出版社,2003：302-313.
[13]PHILLIPS L G,YANG S T,KINSELLA J E. Neutral Salt Effects on Stability of Whey Protein Isolate Foams[J]. Journal of Food Science,1991,56:588-589.
[14]MWASARU M A,MUHAMMAD K,BAKAR J. Influence of Altered Solvent Enviroment on the Functionality of Pigeon pea(Cajanus cajan) and Cowpea (Vigna unguiculata) Protein Isolates[J]. Food Chemistry,2000,71:157-165.
[14]李玉珍,肖怀秋,兰立新.大豆分离蛋白功能特性及其在食品工业中的应用[J].中国食品添加剂,2008,1：121-122.
[15]朴金苗,都凤华,齐斌.马铃薯分离蛋白的溶解性和乳化性研究[J].食品科学,2009,17(30)：91-94
[16]张泽生,刘素稳,郭宝芹,王浩,马铃薯蛋白质的营养评价[J].食品科技,2007,11：71-74.
[17]常志敏.马铃薯的生长特性、营养价值及加工利用[J].安徽农学通报,2007,
[18]杜启艳,路淑霞,常重杰等马铃薯蛋白酶抑制因子的特性研究[J].河南师范大学学报(白然科学版),1998,26(1)：107-110
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