Effect of Carbon Sources on Formation of Α-Amylase And

J. Gen. App!. Microbiol,, 21, 51-59 (1975)

EFFECT OF CARBON SOURCES ON FORMATION OF a-AMYLASE AND GLUCOAMYLASE BY CLOSTRIDIUM ACETOBUTYLICUM

BURT ENSLEY, JOHN J. McHUGH, AND LARRY L. BARTON

Department of Biology, University of New Mexico, Albuquerque, New Mexico 87131, U. S. A.

(Received November 2, 1974)

As determined by enzyme levels and by differential rate of enzyme formation, the synthesis of a-amylase and glucoamylase by Clostridium acetobutylicum is under separate regulatory systems. Induction of a- amylase occurs when starch is the carbon source while induction of glucoamylase accompanies growth with glucose. Minimal production of both enzymes is associated with growth on fructose, and intermediate levels result from growth with maltose. While both a-amylase and glucoamylase are elaborated into the culture fluid during the logarithmic growth phase, 41-44% of total amylase and 22-33 % of total glucoamylase remain associated with the bacterial cells. Studies involving cell fra- ctionation, cell washing, and pH-dependent adsorptions indicate that cell- associated a-amylase and glucoamylase are localized primarily with the surface of the cells and only to a small extent with the intracellular region.

Many bacteria produce starch-hydrolyzing enzymes (1). Saccharification is usually attributed to a-amylase; however, a few bacteria are reported to have other types of starch-degrading enzymes. Pseudomonas stutzeri does not form an a- amylase but produces an enzyme which converts starch to maltotetrose (2) while Streptococcus equinus produces both a-amylase and a starch-hydrolyzing transgly- cosylase (3). Clostridium acetobutylicum hydrolyzes starch by two types of a- mylase (4). One is a-amylase [a-D-(l, 4)-glucan glucanohydrolase, EC 3.2.1.1] which converts starch to maltose and low molecular weight dextrins. The other is glucoamylase [a-D-(l, 4)-glucan glucohydrolase, EC 3.2.1.3], originally referred to as a maltase by HoCKENHULLand HERBERT(5), which splits starch to glucose by hydrolyzing both a-D-(1--4) and a-D-(l->6) glucosidic linkages.

Index Words : a-amylase, formation by C. acetobutylicum; glucoamylase, formation by C. acetobutylicum; Clostridium acetobutylicum, amylases of.

51 52 ENSLEY, MCHUGH, and BARTON VOL. 21

The partially purified a-amylase from C. acetobutylicum has been reported to have a broad pH optimum curve with a midpoint at pH 4.8 and to hydrolyze starch, dextrin, and glycogen (5). The glucoamylase from C. acetobutylicum has not been purified but has been reported to release glucose from the non-reducing end of a starch chain. The rate of hydrolytic attack by glucoamylase is similar for maltose, amyloheptose, starch, and limit dextrin (6). Additionally, this glucoamylase is stable for several weeks at 37°, catalyzes quantitative hydrolysis of maltose to glucose, and has a pH optimum of 4.63 (5). Although the metabolism and physiology of C. acetobutylicum has been ex- tensively studied (7), the formation of starch-hydrolyzing enzymes has not been examined. This paper describes the formation of a-amylase and glucoamylase by C. acetobutylicum as influenced by the carbon source.

MATERIALSAND METHODS Organism and media. C. acetobutylicum NRRL B 592, obtained from C. E. Georgi at the University of Nebraska, was maintained by periodic transfers in fluid thioglycolate medium (BBL). Media used to evaluate amylase production contained 0.8 % yeast extract (Difco), 0.05 % sodium thioglycolate (Difco), and 0.5 % glucose, maltose, fructose, or starch. When starch was the carbon source, 0.01 % glucose was added along with 0.49 % starch to decrease the length of the lag phase. Growth of organism for enzyme production. Media, 100 ml, were sterilized in 300 ml side-arm culture flasks fitted with serum stoppers. The flasks were thoroughly flushed with sterile nitrogen gas before being inoculated with 2 ml of a 6-hr culture grown in fluid thioglycolate. Incubation of the stationary culture was at 37°. At specific intervals, 4-ml samples were withdrawn from the culture and centrifuged at 8,000><9 for 20 min. The resulting centrifugate was used for enzyme measurements. Growth was determined by measuring the absorbance of the culture at 540 nm using a Bausch and Lomb Spectronic 20. The application of a slight negative pressure to the spectrophotometer tube enabled recording of cell densities without interference from gas bubbles. Increase in optical density of the culture was directly proportional to an increase in cellular dry weight. Glucoamylase measurements. The assay for glucoamylase was based on the quantity of glucose liberated from starch by enzymic hydrolysis. The culture fluid, 0.25 ml, was added to an equal volume of 2 % starch (Difco) in 0.1 M sodium acetate buffer, pH 4.5. Following aerobic incubation at 37° for 2 hr, the glucoamylase activity was destroyed by placing the reaction tubes in boiling water for 2 min. Glucose liberated was quantitated by the colorometric glucose oxidase- peroxidase system previously described (8). A unit of glucoamylase was defined as the quantity of enzyme which liberated 1pmol of glucose per minute from 1975 Formation of Amylases by C. acetobutylicum 53 starch under the described conditions. a-Amylase measurements. Assays for a-amylase was based on the quantitation of reducing sugars resulting from enzymic hydrolysis of starch. The culture fluid, 1 ml, was mixed with an equal volume of 1 % starch (Difco) in 0.1 M sodium acetate, pH 4.6, which also contains 0.02 M CaC12. Incubation was at 37° for 20 min. Reducing sugars were measured using the 3, 5-dinitrosalicyclic acid reagent as described by WELKERand CAMPBELL(9) and calculated, in umol, from a standard curve previously calibrated against maltose. One unit of a-amylase activity was defined as the amount of enzyme releasing 1 pmol of reducing sugar per minute under the reaction conditions defined. Differential rate of enzyme formation. The growth rate is expressed as k, where k=A/t and A is the absorbance of the culture and t is the time of incubation in minutes. The values for determining k were taken from the growth curve corresponding to the logarithmic phase. The expression of the rate of a-amylase and glucoamylase synthesis with respect to rate of growth is referred to as the differential rate of enzyme formation. The method for calculation of differential rate of enzyme synthesis follows previously established methods (9). The differential rate of a-amylase synthesis is referred to as Ka, where Ka= units of a- amylase/A. Similarly the differential rate of glucoamylase formation is expressed as Kg, where Kg= units of glucoamylase/A. Cellfree extracts. Following incubation periods of 4 and 10 hr, C. acetobutylicum was removed from 200 ml of the glucose medium by centrifugation and washed twice with 0.01 N acetate buffer, pH 4.6. The cells were suspended in 20 ml of 0.01 N acetate buffer, pH 4.6, and disrupted by a single passage through a French pressure cell at 10,000 lb/in2 moval of cell debris was accomplished by centrifugation at 8,000 x g for 20 min. Enzyme adsorption study. From a 4-hr culture growing in the glucose medium, 50 ml was withdrawn and divided into 10 ml fractions and adjusted to the desired pH with 0.02 N NaOH. Following incubation at room temperature for 15 min, the cells were removed by centrifugation and the centrifugate was dialyzed for 24 hr against 0.001 M sodium acetate, pH 4.6. The dialysate was assayed for both glucoamylase and a-amylase.

RESULTS a-Amylase and glucoamylase formation accompanying growth Both a-amylase and glucoamylase are produced by C. acetobutylicum during the logarithmic growth phase (Fig. 1). With glucoamylase, production occurs in the early portion of the logarithmic phase with maximum amount formed by the time the culture reaches the middle of this exponential growth period. On the other hand, elaboration of a-am lyase into the extracellular medium appears constant throughout the logarithmic growth phase. Metabolic activity of the culture is 54 ENSLEY, MCHUGH, and BARTON VOL. 21

Fig. 1. Characteristics of growth of C. acetobutylicum with glucose as a carbon source, including formation of glucoamylase and a-amylase. • Concentration of glucose in the medium, 0 absorbance of culture, extracellular glucoamylase, o extracellular a-amylase, and x pH of medium. very high during the time of formation of the amylases as evidenced by the rapid disappearance of glucose and corresponding production of organic acids. Spore formation, as determined by microscopic examination, did not occur until after 12 hr of incubation; therefore, neither formation nor liberation of the starch- hydrolyzing enzymes is attributed to sporulation. The type of carbohydrate used to support the growth of C. acetobutylicum greatly affects the quantity and time of synthesis of a-amylase and glycoamylase (Fig. 2). With glucoamylase, two patterns of enzyme formation are apparent. Early synthesis of glucoamylase leading to high levels of enzyme occurs with growth on glucose and maltose, while latter formation resulting in markedly lower levels of enzyme results with fructose and starch. With a-amylase, greatest levels of enzyme occurred with growth on starch, while an intermediate level resulted with glucose, and lowest levels resulted when growth was on maltose and fructose. None of the four carbohydrates tested produced maximum levels of both enzymes; however, both enzymes are produced in appreciable levels. Lowest levels of both amylases are found with growth on fructose. The amount of growth resulting from the utilization of the carbohydrates does not effect formation of the amylases since glucose, maltose, and fructose have comparable levels of growth but different rates and amounts of enzyme formed. Growth is slowest with starch but resulted in the production of greatest levels of a-amylase. Additionally, considerable elaboration of a-amylase was observed between 3 and 8 hr of incubation even though little increase in growth was observed. The different amounts of a-amylase and glucoamylase formed, as influenced 1975 Formation of Amylases by C. acetobutylicum 55

Fig. 2. Formation of glucoamylase and a-amylase accompanying growth of C. acetobutylicum on different carbon sources. A and B are extracellular glucoamylase and extracellular a-amylase levels, respectively, while C is absorbance of the cultures. Carbon sources included: 0 glucose, o maltose, x fructose, and starch.

by the type of carbon source, is presented in Table 1. Under conditions where neither a-amylase nor glucoamylase synthesis is induced such as with fructose, a-amylase production exceeds that of glucoamylase by 100-fold. Even though the levels of glucoamylase and a-amylase were greater with growth on glucose and maltose than with fructose, ratios of glucoamylase : a-amylase near 1:100 resulted. The 1: 570 glucoamylase : a-amylase ratio obtained when growth occurs with starch resulted from stimulation of only a-amylase. 56 ENSLEY, MCHUGH, and BARTON VOL. 21

Table 1. Effect of carbon source on the quantity of a-amylase and glucoamylase produced by C. acetobutylicum.

Table 2. Effect of different carbohydrates on growth rate (k) and differential rate of glucoamylase (Kg) and a-amylase (Ka) synthesis by C. acetobutylicum.

Differential rate of enzymeformation The differential rate of a-amylase and glucoamylase formation during logarithmic phase of growth on various carbohydrates is recorded in Table 2. There appears to be no relationship between rate of growth and rate of enzyme formation. Induction of glucoamylase occurs when growth is on glucose. Slight stimulation of glucoamylase synthesis following growth on starch could reflect partial hydrolysis of starch since glucose and reducing sugars were detected between 5 and 10 hr of incubation. Of the four carbohydrates examined, induction of a-amylase results only when growth is on starch.

Adsorption of amylases onto cells As shown in Table 3, the amount of a-amylase and glucoamylase absorbed onto the cells remains constant over the physiological range of pH 6.5-4.5. In alkaline environment, the enzyme-cell association is slightly diminished as evidenced by the increased amount of a-amylase and glucoamylase present in the culture fluid at pH 7.5 and 8.5, respectively. 1975 Formation of Amylases by C, acetobutylicum 57

Table 3. Effect of pH on adsorption of a-amylase and glucoamylase onto cells of C. acetobutylicum.

Table 4. Localization of a-amylase and glucoamylase in C. acetobutylicum.

Localization of glucoamylase and a-amylase in C. acetobutylicum Each of the amylases is associated with cells of C. acetobutylicum but to a different degree (Table 4). With glucoamylase, 67-78 % of the enzyme is secreted into the culture fluid while 56-59% of a-amylase is found in the extracellular media. About 40% of the a-amylase and 20% of the glucoamylase is loosely associated with the cells and can be removed by washing the cells twice in buffer. The extracel- lularity of the amylases is indicated by the minimal levels of enzyme activity observed in the centrifugate of the cell-free extract. In fact, enzyme activity associated with both fractions of the cell-free extract may reflect the presence of cell wall-associated enzymes not fully removed by the previous two washings of the cells. 58 ENSLEY, MCHUGH, and BARTON VOL. 21

DISCUSSION Composition of the growth medium has been shown to greatly effect the formation of glucoamylase by fungi and the synthesis of a-amylase by bacteria. Of noted importance is the carbon source. Induction of glucoamylase in Aspergillus niger is attributed to maltose and isomaltose (8) while induction of a-amylase in Bacillus subtilis (10), B, stearothermophilus (11), Pseudomonas saccharophila (12), and A. niger (8) is due to maltodextrins or starch. Using the criteria of levels of enzymes produced and differential rates of enzyme synthesis, it is apparent that synthesis of a-amylase and glycoamylase by C. acetobutylicum is influenced by the type of carbohydrate added to the growth medium. The induction of glucoamylase occurred when glucose was present in the culture medium while a-amylase formation was stimulated when starch was employed as the carbon source. The stimulation of a-amylase formation, by cultivation of C. acetobutylicum on starch, is consistent with previous reports (10-12) ; however, the inability for maltose to stimulate glucoamylase formation in C. acetobutylicum differs from the A. niger system (8). One may assume that hydrolytic products of starch are responsible for the induction of a-amylase, since starch cannot pene- trate the bacterial cell, but it is not known if glucose or a product of glucose metabolism is responsible for increased synthesis of glucoamylase. The formation of a-amylase and glucoamylase by C. acetobutylicum is apparently under separate biosynthetic control. The association of low levels of a-amylase and glucoamylase with clostridial cells is consistent with published observations. Cell-bound a-amylase has been reported in bacterial (13,14) and fungal (15) cultures. Adsorption of a-amylase and glucoamylase onto starch was described by FRENCHand KNAPP(6), and interactions of yeast mannans and glucoamylase have been observed (Barton, unpublished results). It is speculated that glucoamylase and a-amylase are associated with carbohydrate moieties at the surface of the C, acetobutylicum cell (Table 4) and this interaction can be reduced by washing the cells in buffer. Since a-amylase and glucoamylase are produced in C. acetobutylicum under different nutritional conditions, both enymes should not be considered as starch- hydrolyzing enzymes (amylases) but enzymes having distinct physiological roles. Perhaps a-amylase should be considered a "true" amylase and glucoamylase should be considered a general carbohydrase capable of hydrolyzing compounds containing a-D(1-->4) and a-D(1---6) glucosidic linkages. The precise role of glucoamylase and the regulatory system associated with synthesis of this enzyme in C. acetobutylicum is yet to be established.

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