豆浆脂肪氧合酶测定方法的优化及酶动力学的研究
|
摘要
本文以前人所确定的大豆中LOX酶活力测定方法为基础,对生豆浆中LOX活力测定方法进行稳定性方面的优化,研究热处理过程、不同浓度豆浆对LOX活力的影响,并确定LOX的最大反应速率、米氏速率常数和不同金属离子抑制剂及激活剂等酶反应动力学参数,研究结果表明:待测样品稀释300倍时,LOX活力值的变化趋势较为明显,且结果稳定;对10个不同地域大豆进行制浆,在300倍稀释倍数下测定不同加热温度的豆浆发现,LOX活力在40℃时最高,当加热至80℃时完全失活,故选择对LOX测定方法进行样品稀释300倍,加热至40℃的优化处理。优化后的方法较其它已报到方法相比,能够快速、准确地测出不同豆种所制豆浆中LOX活力值,且试验结果重复性好,稳定性高。通过对LOX酶促反应动力学的研究,确定了LOX酶促反应最大反应速率V_(max)=1. 99ΔA·min~(-1),R~2=0. 942 8,米氏速率常数K_m=452. 49μmol·L~(-1);金属离子中,钙离子、镁离子对LOX起抑制作用,当锌离子在浓度为0. 002 mol·L~(-1)时,对LOX起抑制作用,而当锌离子浓度大于0. 004 mol·L~(-1)时,则对LOX起激活作用。
Based on the method of determining the activity of LOX enzyme in soybean,we optimized the stability of the method of determining the activity of LOX in raw soybean milk,and studied the effect of heat treatment and different concentration of soybean milk on the activity of LOX in this paper. The maximum reaction rate of LOX,the Michlet rate constant and the kinetic parameters of enzyme reaction such as different metal ion inhibitors and activators were determined. The results showed that the change trend of LOX activity was obvious when the sample was diluted 300 times. The results were stable,and the results of soybean pulping in 10 different regions were determined at 300 times dilution times by different heating temperatures. The activity of LOX in soybean milk was highest at 40℃ and completely inactivated when heated to 80℃. Compared with other reported methods,the optimized method can measure the activity of LOX in soybean milk of different soybean varieties quickly and accurately,and the experimental results are reproducible and stable. By studying the kinetics of enzymatic reaction of LOX,the maximum reaction rate of LOX was determined: V_(max)= 1. 99ΔA·min~(-1),R~2= 0. 942 8,K_m= 452. 49 μmol·L~(-1). In metal ions,Ca~(2+) and Mg~(2+) had an inhibitory effect on LOX. At the concentration of 0. 002 mol·L~(-1),the LOX was inhibited,and when the concentration of zinc ion was greater than0. 004 mol·L~(-1),the LOX was activated.
Based on the method of determining the activity of LOX enzyme in soybean,we optimized the stability of the method of determining the activity of LOX in raw soybean milk,and studied the effect of heat treatment and different concentration of soybean milk on the activity of LOX in this paper. The maximum reaction rate of LOX,the Michlet rate constant and the kinetic parameters of enzyme reaction such as different metal ion inhibitors and activators were determined. The results showed that the change trend of LOX activity was obvious when the sample was diluted 300 times. The results were stable,and the results of soybean pulping in 10 different regions were determined at 300 times dilution times by different heating temperatures. The activity of LOX in soybean milk was highest at 40℃ and completely inactivated when heated to 80℃. Compared with other reported methods,the optimized method can measure the activity of LOX in soybean milk of different soybean varieties quickly and accurately,and the experimental results are reproducible and stable. By studying the kinetics of enzymatic reaction of LOX,the maximum reaction rate of LOX was determined: V_(max)= 1. 99ΔA·min~(-1),R~2= 0. 942 8,K_m= 452. 49 μmol·L~(-1). In metal ions,Ca~(2+) and Mg~(2+) had an inhibitory effect on LOX. At the concentration of 0. 002 mol·L~(-1),the LOX was inhibited,and when the concentration of zinc ion was greater than0. 004 mol·L~(-1),the LOX was activated.
引文
[1] Gardner H W. Recent investigations into the lipoxygenase pathway of plants[J]. Biochemica et Biophysica Acta,1991,1084(3):221-239.
[2] Feussner I,Wasternack C. The lipoxygenase pathway[J]. Annud Review of Plant Biology,2002,53:275-297.
[3]何婷,赵谋明,崔春.脂肪氧合酶的酶学特性及其活性抑制机理的研究进展[J].食品工业科技,2008(2):291-293,298.(He T,Zhao M M,Cui C. Advances in studies on enzymatic properties and inhibition mechanism of lipoxygenase[J]. Food Industry Science&Technology,2008(2):291-293,298.)
[4]杨淑媛.新编大豆食品[M].北京:中国商业出版社,1989:45-60.(Yang S Y. New soybean food[M]. Beijing:China Commercial Press,1989:45-60.)
[5]李笑梅,马永强,段善海.几种大豆脂肪氧化酶抑制物的作用研究[J].中国粮油学报,2001(5):47-49.(Li X M,Ma Y Q,Duan S H. Studies on the effects of several soybean fatty oxidase inhibitors[J]. Chinese Journal of Cereals and Oils,2001(5):47-49.)
[6]石胜尧,张延坤,郭大发,等.大豆脂肪氧化酶活性的测定[J].营养学报,1996,18(3):354-357.(Shi S Y,Zhang Y K,Guo D F,et al. Determination of the activity of soybean fat oxidase[J]. Nutrition Journal,1996,18(3):354-357.)
[7]郑兵福,廖卢艳,蒋立文.不同处理对四棱豆脂肪氧化酶活性的抑制作用[J].食品科学,2011,32(1):82-85.(Zheng B F,Liao L Y,Jiang L W. Inhibitory effects of different treatments on the activity of lipoxygenase in tetragonia bean[J]. Food Science,2011,32(1):82-85.)
[8]岗田真由美,陈葆新.加热处理对豆浆风味的影响[J].中国调味品,1980(6):41-43.(Okada Y,Chen B X. Effects of heating treatment on the flavor of soybean milk[J]. Chinese Condiments,1980(6):41-43.)
[9] And S Y,Chang K C. Selected odor compounds in soymilk as affected by chemical composition and lipoxygenases in five soybean materials[J]. Journal of Agricultural&Food Chemistry,2007,55(2):426.
[10] Jin G,Zhang J,Yu X. Crude lipoxygenase from pig muscle:Partial characterization and interactions of temperature,Na Cl and pH on its activity[J]. Meat Science,2011,87(3):257-263.
[2] Feussner I,Wasternack C. The lipoxygenase pathway[J]. Annud Review of Plant Biology,2002,53:275-297.
[3]何婷,赵谋明,崔春.脂肪氧合酶的酶学特性及其活性抑制机理的研究进展[J].食品工业科技,2008(2):291-293,298.(He T,Zhao M M,Cui C. Advances in studies on enzymatic properties and inhibition mechanism of lipoxygenase[J]. Food Industry Science&Technology,2008(2):291-293,298.)
[4]杨淑媛.新编大豆食品[M].北京:中国商业出版社,1989:45-60.(Yang S Y. New soybean food[M]. Beijing:China Commercial Press,1989:45-60.)
[5]李笑梅,马永强,段善海.几种大豆脂肪氧化酶抑制物的作用研究[J].中国粮油学报,2001(5):47-49.(Li X M,Ma Y Q,Duan S H. Studies on the effects of several soybean fatty oxidase inhibitors[J]. Chinese Journal of Cereals and Oils,2001(5):47-49.)
[6]石胜尧,张延坤,郭大发,等.大豆脂肪氧化酶活性的测定[J].营养学报,1996,18(3):354-357.(Shi S Y,Zhang Y K,Guo D F,et al. Determination of the activity of soybean fat oxidase[J]. Nutrition Journal,1996,18(3):354-357.)
[7]郑兵福,廖卢艳,蒋立文.不同处理对四棱豆脂肪氧化酶活性的抑制作用[J].食品科学,2011,32(1):82-85.(Zheng B F,Liao L Y,Jiang L W. Inhibitory effects of different treatments on the activity of lipoxygenase in tetragonia bean[J]. Food Science,2011,32(1):82-85.)
[8]岗田真由美,陈葆新.加热处理对豆浆风味的影响[J].中国调味品,1980(6):41-43.(Okada Y,Chen B X. Effects of heating treatment on the flavor of soybean milk[J]. Chinese Condiments,1980(6):41-43.)
[9] And S Y,Chang K C. Selected odor compounds in soymilk as affected by chemical composition and lipoxygenases in five soybean materials[J]. Journal of Agricultural&Food Chemistry,2007,55(2):426.
[10] Jin G,Zhang J,Yu X. Crude lipoxygenase from pig muscle:Partial characterization and interactions of temperature,Na Cl and pH on its activity[J]. Meat Science,2011,87(3):257-263.