Xylan-digesting Mechanism Of Anoplophora Chinensis Larvae,and Purification Of β-1,4-xylanase And Enzymatic Properties

Most of the larvae of Cerambycidae, a large group insect in Coleoptera, bore in wood materials and use the polysaccharides as their energy source. Some of them are economically important pest. The distribution, origin and properties of the enzyme of xylan hydrolysis namely 13-i, 4-xylanase in the gut of wood-boring larvae of Anoplophora chinensis (Coleoptera: Cerambycidae) were studied. A xylanase was purified from the gut extract. 1. Distribution of xylanase activity in the gut of A. chinensis larvae The distribution of xylanase in the gut of A. chinensis larvae was measured from the freshly collected and the laboratory-maintained on the diet free of active microorganisms by the serially frozen section and the exam of the enzyme activity. It showed that the majority (99%) of the total xylanase 2 activity contained in the midgut. About 60% of the total xylanase activity was localized in the anterior section of the midgut. A little of the enzyme activity (0.3%) lay in the foregut and was hardly detected in the hindgut. This indicated that xylan digestion was mainly occurred in the midgut of larvae, especially in the anterior-midgut. 2. Isolation and identification of xylan-degrading bacteria A strain of xylan-degrading bacteria (XYI) was isolated from the borers? guts by xylan medium. According to the morphological, physiological and biochemical characteristics, XY1 was grouped to the genus Cellulomonas. Isolation rate (number of the larvae in which bacteria were isolated/number of the larvae detected) of XY1 was 56% and its concentration was very low (under 1 x 1 0~ cells per larva) in the gut. XY 1 could be isolated from the midgut and hindgut but foregut, and the isolation rate increased steadily from anterior to hind. The xylan-degrading bacteria could also be isolated from the boring tunnels of larvae. Combining with the localization of the xylanase activity in the guts of borers, it seemed that the xylan-degrading bacteria in the gut were probably acquired from the wood, on which larvae fed. 3.The effect of different carbon sources on the enzyme production of XV1 The xylanase activity induced by different carbon sources in mediums, which are xylan, straw powder, CMC-Na, and CM, was measured. The highest xylanase activity occurred in the xylan liquid fermentation. XY 1 could produce endo-f3-1, 4-glucanase (Cx-cellulase) degrading CMC-Na and the highest Cx-cellulase activity was also induced by xylan. In other three carbon sources fermentations, both xylanase and Cx-cellulase activities were obviously low. 4.Purification of xylanase from the gut of A. chinensis larvae A xylanase component was purified from the gut extract of A. chinensis larvae by 80% acetone precipitation,Q-Sepharose ion-exchange 3 chromatography and preparative PAGE. A recovery of 5.80% was achieved. The molecular weight of purified xylanase was 25kD, and the p1 was 4.0. Its specific activity was 4.45U/mg. The optimum temperature and pH were SOC and 5.4, respectively. The enzyme was stable in the pH range 3.0-7.8. About 60% of the activity remained after the enzyme had been incubated at 50C fo...