Affinity Binding of Higher Plant P-Amylases to Starch A Rapid Purification Method K. Subbaramaiah and R. Sharma, Hyderabad (India) zyx A rapid purification method exploiting affinity of higher plant zyxwvuts 0- amylases to its substrate starch is described. Mustard P-amylase on incubation with starch suspension binds rapidly to starch. The enzyme binds tenaciously to starch as various eluants as concentrated salt solutions, a-cyclodextrin and shift in elution pH could not signifi- cantly elute the bound enzyme. Even maltose only partially eluted the bound enzyme at high concentrations zyxwvutsrqp (5% w/v) after a prolonged incubation time zyxwvutsrqpo (24 h). Nevertheless, the enzyme could be rapidly eluted from starch with white dextrin (1% wlv). The eluted enzyme fractions consisted of &amylase purified to homogeneity. The above starch-affinity purification process could also be applied to other higher plant P-amylases such as sweet potato and barley. Affinitatsbindung der 0-Amylasen htiherer Pflanren an Starke - eine schnelle Reinigungsmethode. Es wird eine rasche Methode zur Reinigung von P-Arnylasen aus hoheren Pflanzen durch Ausnut- zung ihrer Affinitat zu ihrem Substrat Stlrke beschrieben. Senf-0- Amylase bindet sich bei der Inkubation mit Starkesuspension rasch an Starke. Das Enzym bindet sich fast an Starke, da verschiedene Elutionsmittel wie konzentrierte Salzlosungen, a-Cyclodextrin sowie die Veranderung des Elutions-pH-Wertes das gebundene Enzym nicht merklich eluieren konnten. Auch Maltose eluierte das gebun- dene Enzym bei hohen Konzentrationen (5% wtv) nur teilweise nach langerer Inkubationszeit (24 h). Demgegenuber konnte das Enzym rnittels Weindextrin (1% w/v) rasch eluiert werden. Die eluierten Enzymfraktionen bestanden aus bis zur Homogenitat gereinigter 0- Amylase. Das zyxwv Starke-Affinations-Reinigungsverfahren lien sich auch auf andere P-Amylasen aus hoheren Pflanzen wie SuRkartoffeln und Gerste anwenden. 1 Introduction The specific binding of an enzyme to its substrate has been exploited in several cases to constitute a useful purification step [l]. The affinity of an enzyme to its insoluble substrate was first employed by zyxwvutsrqpo Sturkenstein (1910) €or purifying a-amylase by binding to its substrate starch [2]. Thereafter, a-amylases from various species have been purified to homogeneity by either binding to starch [2-51 or to glycogen [6] and precipitating the substrate-enzyme complex in ethanolic extracts. In contrast, it has been reported that the other amylolytic enzyme P-amylase does not have affinity for starch [5]. Thuyer (1953) reported that while a-amylase from Pseudomonus sacchurophilu binds to starch, P-amylase has no affinity for starch. In sorghum [4] and malt [3] a-amylase was separated from P-amylase by binding the former to starch. On the contrary, microbial P-amylases have been purified by binding to starch [7-81. Hoshino et al. [7] reported that, while in case of Bacillus sp., a procaryote, more than 60% P-amylase was bound to starch, both barley and soybean P-amylases did not show any binding to starch. In view of lack of affinity of eucaryotic j3-amylases €or starch, P- amylase has been traditionally purified to homogeneity by conventional methods of protein purification. Vretblud [9] reported an affinity purification of sweet potato 0-amylase on a-cyclodextrin-Sepharose column, however, this column does not have affinity for other eucaryotic P-amylases [lo]. Further- more, the above results of Vretblud could not be reproduced in our laboratory [lo]. In this study, we report a simple and efficient method for purification of higher plant P-amylases to homogeneity by binding to starch. 2 P-Amylase Binding to Starch The relative efficiency of various polymeric carbohydrates such as starch, cellulose and glycogen for P-amylase purification was evaluated by directly mixing respective solid substrate with an Table 1. Relative Binding of Mustard a-Amylase to Starch, Cellulose and Glycogen. 0.5 g of respective substrate (solid) with 2 ml of enzyme solution was used for the experiment. The experiment was conducted by mixing as outlined in materials and methods. The control was subjected to identical treatment albeit without any inclusion of above substrates. P-Amylase Activity (nkats) Substrate Initial Super- Control Pellet Loss activity natant zyx ("/I Starch (soluble) 60.00 21.65 59.28 n. d.* 64.10 Cellulose 42.12 37.62 39.39 n.d. 10.60 Glycogen 79.56 76.44 78.15 n.d. 3.90 Native starch 62.40 36.90 60.84 n. d. 40.80 * not determined enriched P-amylase preparation. Table 1 shows that the incuba- tion of enzyme with starch (amylose) or native starch (potato starch grains) caused a significant reduction in a-amylase activity in supernatant. At the same time, the incubation with cellulose and glycogen did not cause a similar reduction in j3- amylase activity. However, no p-amylase activity could be detected in pelleted starch indicating a tenacious binding of enzyme to starch. In contrast to a-amylase [6] where enzymes have been purified by precipitation of enzyme-substrate com- plex with alcohol, in present study, enzyme-substrate complex was collected by centrifugation. Moreover, in presence of alcohol mustard P-amylase was rapidly inactivated, therefore precluding the use of above technique [lo]. The systematic study of P-amylase binding to starch revealed that enzyme binding takes place rapidly within 5 min (Fig. 1) and depends on starch concentration (Fig. 2). The enzyme bound more efficiently on directly mixing with dry powdered 182 starcWst8rke 40 (1988) Nr. zyxwvutsrq 5, S. 182-185 zyxwvutsrq 8 VCH Verlagsgesellschaft rnbH, D-6940 Weinheirn, 1988 0038-9056/88/0505-0182$02.50/0