A Preliminary Study on the Extracellular Amylase of a Bucillus sp . that Increases the Permeability of Water - Stored

It has been observed that storage of softwoods in water often serves to increase their permeability to subsequent treatment with preservatives. Vasil’ev (1965), Ellwood & Ecklund (1959), Lutz e t a / . (1966) and Dunleavy & Fogarty (1971a, 1972) all associated microbial activity with this process. Dunleavy & McQuire (1970) have established that during water storage of Sitka spruce (Picea sitchensis) destruction of the tori and bordered pit membranes occurs, together with partial degradation of the cross-field pits. They concluded that this was responsible for the observed permeability changes. There is considerable evidence to suggest that permeability increases are the result of degradation of some wood substances by bacterial enzymes. Ellwood & Ecklund (1959) and also Knuth & McCoy (1962) traced the better permeability of water-stored sapwood to degradation of pectin, starch and perhaps hemicellulose and cellulose. Seifert (1967) similarly found a 5 % decrease in cellulose and a 13 % decrease in hemicellulose. Liese & Karnop (1968) isolated organisms from water-stored wood and noted their ability to elaborate extracellular hydrolases. Bacterial isolates (W. M. Fogarty, unpublished work) from the sap of wood stored in a freshwater lake were shown to elaborate amylase, xylanase and pectinase activities (Fogarty & Ward, 1972~) . Two of the bacterial species elaborating these enzymes were also found to increase the permeability of Sitka-spruce sapwood blocks (Dunleavy & Fogarty, 19716). Reports that pectinolytic enzymes play an important role in the above processes have been given by Souhlati & WallBn (1958), Nicholas & Thomas (1968) and Fogarty & Ward (1972b). Studies in our laboratory have also confirmed the association of amylase activity with the increased permeability of Sitka spruce (Ward & Fogarty, 19726). When the pattern of amylase activity was followed in the expressed sap of ponded Sitka spruce, a peak for enzyme activity was found to occur between the tenth and thirteenth weeks, at a time when bacterial numbers were also high. Two of the bacterial isolates, shown to be responsible for the alterations in permeability, elaborate amylase activity in sapwood blocks in laboratory experiments (Fogarty & Ward, 1972d). We have investigated the production, purification and properties of the amylase of one of these bacteria, a Bacillus sp. In nutritional experiments 1 ml volumes of an active cell suspension were inoculated into 250ml Erlenmeyer flasks, each containing 50ml of medium. These were then shaken at 150 oscillations/min in a New Brunswick orbital incubator (model G25) at 27°C. The effect of nitrogen source on enzyme elaboration was determined by incorporating various nitrogen sources into a basal salts medium, containing 0.5% (w/v) of sodium polypectate (Sigma Chemical Co., St. Louis, Mo., U.S.A.) as carbon source. The complex nitrogen sources peptone, casein hydrolysate, casein and yeast extract gave the best amylase induction. In contrast, induction of amylase by inorganic nitrogen sources was very low. The effect of carbon source on enzyme production was evaluated by addition of various carbon sources (0.5 %, w/v) to a basal salts medium containing peptone as nitrogen source. Sodium polypectate and raffinose induced the highest amylase activity in these experiments. The effect of pH on enzyme production was also investigated. Amylase activity was elaborated by the organism over the pH range 5.8-7.8. Although the rate of enzyme production was higher at pH5.8, highest amylase activity was obtained at pH7.8. The addition of complex nitrogen sources to the growth medium also resulted in the induction of high extracellular protease activity (Ward & Fogarty, 1971~) . By omitting CaCI, from the growth medium it was possible to eliminate most of the protease activity. The