Studies On Diversities Of Extracellular β-glucosidases From Penicilhum Oxalicum

With the economic development as well as continuous consumption of non-renewable resources, the increasing attention has been paid to the exploitation of renewable resources to develop second-generation biofuels. Lignocellulosic biomass is the largest reserves of renewable resource on the earth lignocellulose degradation can be achieved by acidich or enzymatic hydrolysis. Comparing with acid hydrolysis, enzymatic hydrolysis has the advantages of mild reaction conditions, without generating toxic products, high production yield of sugar and low investment in equipments. Enzymatic hydrolysis of cellulose requires the synergistic action of cellulases, such as endoglucanase(EC3.2.1.4), cellobiohydrolase (EC3.2.1.91), and β-glucosidase (EC3.2.1.21). Filamentous fungi are a major cellulase-producer, playing an important role in the lignocellulose degradation. Consequencely the researches on the composition of their lignocellulolytic enzyme system and the regulation of enzyme expression are very important for improvement of cellulase production and enzyme system composition.Trichoderma reesei is considered an indisputable champion in cellulase production, but the proportion of the secreted β-glucosidase is very low, resulting in a decreased ability of degrading cellulose. Our laboratory screened a filamentous fungus Penicillium oxalicum114-2can produce a balanced extracellular lignocellulolytic enzyme system with more β-glucosidase, which is very efficient for lignocellulose degradation. β-glucosidase is essential in cellulases by catalyzing hydrolysis of cellobiose or cello-oligomers to glucose, reducing the inhibition effect of cellobiose on cellulose. BGL is considered as the rate-limiting enzyme for the reason of it could be inhibited by its substrate (cellobiose) as well as its product (glucose), once their concentrations reach to a critical point. It shows that, as an important component of lignocellulolytic enzyme system, β-glucosidase can also participate in the regulation of cellulase gene expression. The genome of Penicillium oxalicum has been recently sequenced which suggested that there were at least five extracellular and six intracellular β-glucosidase genes. Multiple P-glucosidase genes present in filamentous fungi is a common phenomenon. For instance both Trichoderma reesei and Aspergillus niger have several extracellular β-glucosidase genes according to genome annotation, whereas only one β-glucosidase protein spot on the DIGE gels was successfully identified. Penicillium oxalicum has the same phenomenon. Basing on this information, we compared the diversities of five BGLs’s biological functions, attempting to discover new genes to improve the degradation efficiency of cellulosic biomass. Reasonable manipulate on strains to improve enzyme production and further build a foundation platform for high-yielding of cellulase.The main results of the thesis are as follows:1. Cloning and recombinant expression of extracellular β-glucosidases from Penicillium oxalicum in Pichia pastoris GS115These β-glucosidase genes were amplified by PCR with corresponding primers, forward and reverse primers introduce the restriction sites, Penicillium oxalicum cDNA as template, pPIC9K as a shuttle vector for the expression of target genes. The amplified DNA fragments and pPIC9K plasmid were double-digested with the corresponding restriction endonuclease to construct pPIC9k-bgls. Transformation of P. pastoris GS115was performed by electroporation and expression was induced by adding1%methanol every24h. The result of SDS-PAGE suggested that all of the recombinant proteins were expressed, although the realistic molecular weight is greater than the theoretical molecular weight. When taken salicin as substrate, the activity of rBGL4is the highest, although the protein concentration of rBGL3is the highest we can not detect the activity. We predicted that bgl3may be not a β-glucosidase encoding gene. 2. Purification and characterization of the five recombinant β-glucosidasesPurification of recombinant rBGLs using the1mL HisTrapTMFF. Detection by SDS-PAGE demonstrated that five rBGLs were successfully purified. When taken salicin as substrate, the specific activity of rBGL1=168.9U/mg, rBGL3=0, rBGL4=331.4U/mg, rBGL5=130.0U/mg, rBGL6=96.9U/mg. Surprisingly, the specific activity of rBGL4was significantly superior to rBGL1. We selected aryl glycosides and saccharides to examine the substrate specificity of rBGLs. Among the aryl glycosides, esculin was the most preferable substrate, among the saccharides, gentiobiose was the most preferred substrate for rBGL1and rBGL4, cellobiose was the most preferred substrate for rBGL5, sophorose was the most preferred substrate for rBGL5. The specific activity of rBGL4is the best towards the same substrate. The hydrolysis properties of rBGLs towards cello-oligosaccharides were monitored by HPLC. rBGL1and rBGL4exhibit high affinity toward cello-oligosaccharide and could convert it to glucose and cellobiose. For rBGL5and rBGL6, their efficiency for the hydrolysis of cello-oligosaccharides were far less than rBGL4, they were capable of converting cello-oligosaccharide to a mixture of shorter oligosaccharides and glucose. The optimum temperature of rBGL4is80℃higher than rBGL1. Thermostability of rBGL4is the best, more than94%of its maximum activity was retained at80℃after2h of incubation. For rBGL1, more than75%of its maximum activity was retained at60℃after2h of incubation, it was inactivated rapidly above70℃. After endoglycosidase H digestion, rBGLs’s molecular weights were in accordance with theoretical value. After deglycosylation experiment, the activity of rBGL4and rBGL6were reduced, rBGL5was increased while rBGL1almost no change; for thermostability, rBGL5was decreased while the thermostability of rBGL4, rBGL1and rBGL6was good.3. Efficiency of rBGLs in boosting enzymatic degradation of corncob residueJU-A10-T was cultured for5days then collected crude enzyme and measured the filter paper activity (FPA) and β-glucosidase activity (BGL). Different amounts of purified rBGLs were added into the crude enzyme of JU-A10-T with the ratio of the FPA to (3-glucosidase activity as1:1or1:2. All the enzymes could improve the saccharifying ability of JU-A10-T cellulase toward different corncob residues, and the higher the proportion of p-glucosidase, the more ability of improving the saccharification. When the ratio of the FPA and β-glucosidase activity was1:2by adding rBGLl or rBGL4, the saccharifying abilities toward delignified corncob residue were improved46%and44%, respectively.