Construction Of Genetic Engineering Strain Penicillium Oxalicum For High β-xylosidase Production And Its Application

Xylan is one of the main components of hemicellulose, and widely presented in the cell walls of hardwood and cereals. Because of its complex structures, xylan from plant was completely hydrolyzed into monosaccharide components by the synergistic action of different enzymes, in which β-xylosidase was a key enzyme in the enzyme system for hydrolysis of xylan backbone. During enzymatic degradation of hemicellulose, the increased β-xylosidase not only reduced the inhibition of product xylobiose to xylosidase, but also formed more xylose by hydrolysis oligosaccharides. Xylosidase has great potential in some industries, for example, biotransformation, pulping and papermaking, and pharmaceutical industy, etc. Therefore, it is very important to increase xylosidase production for enhancing xylosidase applicaiton in the industries.By measuring β-xylosidase activity of crude enzyme from liquor fermentation by wild strain of P. oxalicum 114-2 and its various mutant starins, their ability for β-xylosidase production were evaluated. It was found that the crude enzyme from P. oxalicum AlaeA-gpdA(p):xxlnR had higher β-xylosidase activity (4.5 fold higher) than that from the wild strain P. oxalicum 114-2. The gene transcription levels about the original strain P. oxalicum 114-2 and mutant strains which include ΔlaeA, gpdA(p)::xlnR, ΔlaeA-gpdA(p):xlnR gpdA(p)::clrB, and ΔlaeA-gpdA(p)::clrB were analyzed. The results showed that, xylosidase gene transcript levels of P. oxalicum ΔlaeA-gpdA(p):.xlnR strain have a very significant increase compared to other strains, while the single transcriptional regulatory factor laeA and xlnR had no significant effect on the expression of xylosidase gene. It was supposed that LaeA and XlnR could collectively affect the xylosidase gene expression.Overexpression cassettes of eight xylosidase genes (xyl3A、xyl3C、xyl3D、 xyl43A、xyl43C、xyl43D、abf43D and xyl31A) were constructed and successfully transformed into P. oxalicum ΔlaeA-gpdA(p)::xlnR, respectively. After liquor fermentation by the transformant of xyl3A (named as BZ-1), the xylosidase and xylanase activity of the crude enzyme were 35.7 U/mL and 408 U/mL, approximately increased by 28.4-and 1.9-fold respectively. The production of xylosidase by transformants of abf43D、xyl3D and xyl31A were approximately improved by 1.6-、 1.6-and 2.0-fold, respectively. It was demonstrated that the production of β-xylosidase could be significantly improved by overexpressing β-xylosidase gene in the P. oxalicum.The enzyme properties of purified protein Xyl3A from engineering strain BZ-1 were studied. The optimal substrate for the enzyme Xyl3A was pNPX. The specific activity of the Xyl3A was 25.2 U/mg. The optimal temperature for the enzyme Xyl3A was 50 ℃, and its suitable pH value was pH 3.5 to 5.0. It was stable at pH 4.5 and temperatures of 45℃ and below. Metal ions, for example, Cu2、Co2、Ca2+、Pb2+、 Ba2+、Mg2+、K+、Mn2+ and Zn2+ etc., had no significant influence on the enzyme activity. The addation of Fe3+promoted the P-xylosidase activity, but SDS of 5 mM had a strong inhibit to the enzyme activity which was reduced by about 57%. The Ki value of the Xyl3A enzyme was 5.0 mM to xylose and Km value was 0.36 mM to pNPX.Culture medium components which include carbon sources, nitrogen sources, etc. were optimizated for produciton of xylosidase by the engineering strain BZ-1, and the optimal culture medium were as follows:corncob 2.4%, microcrystalline cellulose 2.4%, wheat bran 2.4%, com steep powder 2.1%, KH2PO40.5%, and MgSO4·7H2O 0.05%. Using the culture medium, the activites of β-xylosidase, xylanase and filter paper by this strain BZ-1 were 67.7 U/mL,557 U/mL and 0.4 U/mL, respectively, in which, the P-xylosidase activity is the highest when compared to that of filamentous fungi reported by literatures until now.The crude enzyme from liquid fermentation by P. oxalicum BZ-1 was used to hydrolyzing NaOH pretreated corn stover for studying its potential application in degradation of hemicellulose and cellulose material. Compared with the crude enzyme from the original strain P. oxalicum AlaeA-gpdA(p)::xlnR and commodity xylanase, the curde enzyme from the P. oxalicum BZ-1 has a higher hydrolysis ability on xyaln in corn stover, and conversion of xylan significantly increased. It was also found that the xylosidase from P. oxalicum BZ-1 exhibited highest hydrolysis efficiency for xylan from corn stover when compared with the xylans from sugarcane bagasse and beechwood.