It has been reported that soybean sprouts accumulated asparagine during sprouting. The accumulation of amide comounds, asparagine and glutamine, in soybean sprouts was discussed as a physiological basis for detoxification of ammonia and reserve substances for the dicarboxylic acid. However, the mechanism of the asparagine synthesis in the soybean sprouts was not elucidated in detail untill the present time.
In this research asparagine biosynthesis in the soybean sprouts was studied. As the results, moisture content and amount of asparagine synthesized in ten day-old soybean sprouts increase to 85-90% and 15.6% on the dry bases, respectively. The effects of nitrogen compunds such as $NH_4Cl$, $(NH_4)_2SO_4$ and urea on asparogine synthesis during soybean sprouting were examined.
Urea was more effective than other two compounds and asparagine content of soybean sprouts, which was cultured with 2% urea solution, increases to 29.4% in ten day-old soybean sprouts, while control which was cultured with distilled water only showed 22.7%. Asparagine synthetase, which catalyses to form L-asparagine from the mixture of aspartic acid, glutamine, ATP and $Mg^{++}$ ion, was purified 8.6 folds from cotyledon extracts of 10 day-old yellow soybean sorouts through the procedures of ammonium sulfate fractionation, Sephadex G-150 gelfilteration. The enzyme preparation, however, revealed seven protein bands by polyacrylamide gel electrophoresis.The glutamine-dependent asparagine synthetase was very labile and required protection by thiol groups and high level of glycerol.
The mixture of ATP and $Mg^{++}$ ion also stabilized the enzyme activity.
The glutamine-depencent enzyme utilized glutamine more effectively than $NH_4^+$ as an amide donor for the formation of asparagine. Using the enzyme preparation of Sephadex G-150, $p^H$ dependence, effects of $NH_4^+$, glutamine, $NH_2OH$, ATP metal ion concentrations and $k_m$ for aspartic acid for asparagine synthesis were examined. The results are as follows;
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$k_m$ for aspartic acid was 3.1mM. The enzyme required $Mg^{++}$ for the activity and $Mn^{++}$, $Fe^{++}$ and $Zn^{++}$ can not replace $Mg^{++}$. This enzyme also catalyzed the formation of β-aspartyl hydroxamate in the presence of aspartic acid, ATP, $Mg^{++}$ and $NH_2OH$ in the reaction mixture.
As the conclusion for the formation of asparagine, asparagine synthetase in the soybean sprouts required aspartic acid, ATP, $Mg^{++}$ and glutamine. The reactions are presented as follows.
Aspartic acid + ATP+$Mg^{++}$+Glutamine
$\longrightarrow$ Asparagine + Glutamic acid + AMP + $PP_i$
The mechanisms of asparagine biosynthesis in the soybean sprouts are very similar to that of lupinus seedlings as reported.
콩을 발아하여 생육시킨 콩나물에 고농도로 존재하는 asparagine의 생합성에 관하여 연구하고자 콩을 물에 침지 발아하여 생육시키면서 시간에 따른 수분함량 및 asparagine 함량의 증가를 조사해본결과 제 10일째에는 수분은 85∼90%에 달했으며 asparagine의 양은 재 결정화한 결과 건물량의 15.6%에 해당하는 1.62 g 이 생성되었다. 또한 콩을 발아하여 생육시키면서 $NH_4C$ℓ, (NH_4)_2SO_4$ 및 urea 를 살포하여 질소화합물에 대한 영향을 고찰하였던바 10일째의 것으로는 증류수를 주어 기른 control 보다 $NH_4C$ℓ 및 $(NH_4)_2SO_4$ 를 주어 기른 실험구는 약간 증가되었으나 $NH_4C$ℓ 보다 $(NH_4)_2SO_4$를 주어 기른 실험구가 높았다. 그러나 urea를 주어 기른 제10일째의 실험구는 control 보다 29.5% 더많은 asparagine이 생성되어 urea가 기타 다른 질소화합물보다 훨씬 효과적으로 aspargine 합성을 촉진시켰음을 알 수 있었다. 제10일째의 콩나물에서 효소를 추출하여 $(NH_4)_2SO_4$ fractionation 및 sephadex G-150 에 의해 gel 여과한 후 이 효소액과 aspartic acid, glutamine (혹은 $NH_4C$ℓ). $Mg^{++}$ 및 ATP와 섞어 반응시키면 2.20 μmoles/mg protein에 해당하는 asparagine이 생성되어 crude extract의 효소보다 8.6배 정제되었으나 gel electrophosis에 분석한 결과 7개의 단백질 band 를 나타내었다. 콩나물에서 추출한 효소는 열에 매우 불안정하나 glycerol, mercaptoefhanol 및 기질인 ATP, $Mg^{++}$ ion에 의해 보호를 받았으며 이 효소에 의해 최고의 asparagine이 생성되는 ATP, $NH_4^+$ 및 glutamine의 농도는 표준 반응용액에서 각각의 구성농도를 변화시키면서 측정했을때 ATP는 10mM, $NH_4^+$는 50mM, glutamine은 10mM이었다. $Mg^{++}$ ion 대신 $Mn^{++},\; Zn^{++}$ 및 $Fe^{++}$ 로는 대치할 수가 없었으며 aspartic acid 에 대한 Km 값은 3.1mM이었고 amide donor로서 $NH_2OH$를 대치한 결과 β-aspartyl hydroxamate가 생성되어, 열 불안정성, amide donor로서 glutamine이용 및 -SH기의 보호에 의한 효소안정성등을 비추어보아 같은 두과작물인 lupinus 및 완두와 비슷하게 β-aspartyl adenylate 의 중간산물을 거치는 반응기작에 의해 asparagine 이 생합성 되리라 추측되어진다.
