A Preliminary Study on the Production, Purification and Properties of a Xylan-Degrading Enzyme from a Bacillus sp. Isolated from Water-Stored Sitka Spruce (Picea sitchensis)

Abstract

The natural resistance of Sitka spruce (Picea sitchensis) to penetration by preservatives such as creosote can be altered by storage of the wood in water (Dunleavy & Fogarty, 1971~). During this process bacterial degradation of the tori and bordered pit membranes occurs, and this results in increased permeability of the wood. Since these pit structures contain mainly pectic and hemicellulose materials, bacterial enzymes that degrade these materials are thought to be primarily responsible for aspiration of the pit membranes. This suggestion is substantiated by the fact that both xylanase and polygalacturonate lyase activity have been detected in the sapwood of Sitka spruce during water storage (Ward & Fogarty, 1972b). Further, two bacterial isolates from water-stored sapwood that have the ability to increase the permeability of the sapwood (Dunleavy & Fogarty, 1971b, 1972) also produce both of these enzymes when grown on synthetic media (Ward & Fogarty, 1971~). In a previous communication (Ward & Fogarty, 1972a) we have given information on the polygalacturonate lyase of one of the isolates, a Bacillus sp. The following is a preliminary survey on the production, purification and properties of the xylanase produced by this organism. For enzyme-production studies Iml volumes of an active cell suspension were inoculated into 250ml Erlenmeyer flasks each containing 50ml of medium. The flasks were then shaken at 150 oscillations/min in a New Brunswick Orbital Incubator Shaker (model G25) at 27°C. After various incubation times samples were removed from the flasks and the cell-free supernatants were assayed for enzyme activity. Xylanase activity was determined by measuring the production of reducing groups from 1 % xylan (Nutritional Biochemicals Corp., Cleveland, Ohio, U.S.A.) in 5Om~-sodium phosphate buffer, pH7.0. Activity is expressed in pmol of glucose reducing equivalents/ min at 30°C. The effect of carbon source on xylanase production was determined by incorporating various carbon sources (0.5 %) into a basal salts medium containing 0.2% (NM,),SO, as sole nitrogen source. Under these conditions the highest activity was induced by either xylan or cellobiose. Because of the insoluble nature of xylan, cellobiose was found to be more suitable as a carbon source and was consequently used in all subsequent experiments. In evaluating the effect of nitrogen source on enzyme elaboration, various nitrogen-containing substances were added to a basal salts medium containing cellobiose. The highest enzyme production was obtained from casein, skim milk, yeast extract and (NH4)2S04, in that order. In these nutritional experiments the initial pH of the growth medium was 7.0. To determine the effect of pH on xylanase production the initial pH (pHi) of the medium was varied over the range 5.5-7.4. Although the growth rate of the organism was optimal at pHi 5.8, higher and more stable xylanase activity was produced at pH,7.3. Fig. 1 demonstrates the relationship between pHi and enzyme production. The ability of this Bacillus sp. to elaborate xylanase activity in Sitka-spruce sapwood was also investigated. The organism was incubated with small sapwood blocks floated in sterile lake water. Sample blocks were removed from the flasks at daily intervals, and the xylanase activity in the sap expressed from the wood was measured. Xylanase activity was detected in the sapwood after 24h and this rose to a maximum on the fifth day of the experiment. For enzyme-purification studies (NH4)2S04 was used as nitrogen source. Although this substance induced slightly less xylanase activity than did the complex nitrogen compounds, the activities of other enzymes, particularly amylase and protease, were relatively much lower in the cell-free supernatant when inorganic nitrogen was used