Aspergillus Niger ATCC1015 Extracellular Glycoside Hydrolase Enzymes And Dynamic Analysis Of A Specific Enzyme Function

Liquid biofuels produced from lignocellulosic biomass are anenvironmentally clean and renewable source of energy. The main components of the lignocellulose including cellulose, hemicellulose, pectin and lignin. After years of research, the chemical efficiency and the enzymatic hydrolysis efficiency of biomass still very low, and the cost is expensive. The reason is that the structure of natural biomass is not well known. The cell wall of plant is complicated in structure and constitution, which is known as "biomass recalctrance".Themajority of cellulose is degraded by microorganism. Filamentous fungi have the capacity to secrete large amounts of lignocellulosic enzymes that release fermentable sugars from plant cell walls. Our study demonstratethat oligosaccharides released from polymers and their derivatives function as the actual molecules that trigger enzyme induction, and it still is the key point to study. However, it is not clear about that how microorganismregulate the expression of glucoside hydrolases. In order to understand those problems, we use dynamic zymography assay and LC-MS to study the expression pattern of glucoside hydrolases of A.niger ATCC1015and the function of some important lignocellulosic enzymes. Based on the above background knowledge, the following work was performed:1. A.niger ATCC1015regulated the expression of lignocellulosic enzymes by means of sensing the reducing sugar in cultural medium.We found that A.niger ATCC1015presented different expressional patterns with different kinds of carbons and different concentrations of carbon resource. A.niger ATCC1015secreted glucoside hydrolases in turn. Xylanases was detected at48h firstly, then endoglucanase at72h, and β-glucosidase was at about96h. Glycerol, glucose and cellobiose can’t induce the expression of glucoside hydrolases. The Xyn2and Egl5can be induced by xylose, xylan, CMC and Avicel PH105. The mixer of xylose and cellobiose can increase the expression of Xynl, XynB, Egl1, Egl3and EglA. The activity of Xyn1and Xyn2increased with the increase of the concentration of inducing carbons, the same as Egl5. However with the accumulation of reducing sugar, the expression of glucoside hydrolases was inhibited and the expression time was delayed. Multiple oligosaccharides released from lignocellulosic feedstock during the degration process can induce the expression of xylanase and β-1,4-endoglucanase.2. The identification of lignocellulosic enzymes secreted by A.niger ATCC1015by LC-MSBy LC-MS, we can analyze the secreted glycoside hydrolases more quickly. We found that the kinds and the quantity of glycoside hydrolases under CSWB cultured condition was much more abundant than other conditions. Among them, pectinhydrolase was the most obvious. The hemicellulases were abundant under all conditions, and hemicellulases were the main glucoside glycosidases when cultured with xylose, xylan, xylose+cellobiose, it demonstated that A.niger was hemicellulases high-producing strain. Comparing with dynamic zymography assay, we detected EglA, Bgl, XynA, XynB and XynD by LC-MS. The expression of xylanase is consistent with dynamic zymography assay,we detceted three kinds of xylanase. XynB was efficiently expressed continuely. By LC-MS, we can also detected XynD which was less obvious indynamic zymography assay. The results also demonstated the expression of glucoside hydrolases can be highly reduced by complicatedly mixed oligosaccharide. In addition, we detected high content of amylase protein, it illustrated that A.niger was amylase high-producing fungi.3. The heterogeneous expression of key glycoside hydrolases and the functional analysis By dynamic zymography assay and LC-MS, we found that A.niger can secret several kinds of isoenzyme, among them, XynB were detected firstly and was always on high-level on most kinds of carbons. EglA was expressed preferentially. We study why fungi secreted so many isoenzyme. The XynB (EC3.2.1.8) of GH11and EglA (EC3.2.1.4) of GH12were obtained. By mass spectrometry, Xynl is the XynA of GH10, Xyn2is the XynB. Xylan can be hydrolyzed by XynB to produce a series of xylo-oligosaccharides. In addition, XynB can also hydrolyze RAC to produce monosacchride, disaccharide and trisacchride, providing oligosaccharides inducer for other glycoside hydrolases. RAC can be hydrolyzed by EglA to produce disaccharide, trisacchride and tetrose, CMC was hydrolyzed to produce a series of oligosaccharide. We speculated the reason why XynB and EglA was preferentially expressed was that both XynB and EglA was small molecular, it was energy saving for fungi to compound. Both of them have low substrate specificity, they can produce a series of oligosaccharides and these oligosaccharides can play an important role in inducing other glucoside hydrolases.