Recombination And Expression Of Glucose Oxidase Gene From A.Niger In Trichoderma Reesei

Glucose Oxidase has the capability of catalyzing the oxidation of β-D-glucose to gluconic acid and hydrogen peroxide. Due to its efficiency in removal of glucose and oxygen, bacteriostasis and gluconic acid production etc., glucose oxidase has gained considerable commercial importance in the food, beverage, textile, pharmaceutical and other industries. So far, glucose oxidase is mainly produced by fermentation of Aspergillus niger and molds. However, GOD produced by these strains is mostly distributed in intracellular locations or cell walls, which makes it difficult to recover the enzyme directly from the liquid stream, causing a relatively low enzyme yield. To fix this problem, construction of a genetically modified microorganism with high GOD activity is needed. Trichoderma reesei, characterized for producing high level of extracellular proteins, is an ideal host for heterogeneous expression of GOD.In this work, the glucose oxidase gene from Aspergillus niger was first optimized according to codon bias of T.reesei. The new sequence was then synthesized and inserted between the strong promoter Pcbhl and the terminator Tcbhl to construct the expression cassette of Pcbhl-ss-GOD-Tcbhl. Using pCAMBIA1300 as vector backbone, the recombinant expression vector pCB-GOD containing both the GOD gene expression cassette and hygromycin B resistance marker was constructed. The plasmid pCB-GOD was introduced into T.reesei by Agrobactrium tumefaciens-mediated transformation (AMT), producing 45 transformants in total. Double-layer screening plates were applied to narrow down potential recombinants with high GOD activity. Four transformants, as indicated by their larger blue circle areas, were selected for further analysis. After 72h shaking-flask fermentation, the GOD activity of recombinant T. reesei ZU-G3 reached 100.20U/mL, which makes it most qualified for further investigation. Stable integration of glucose oxidase gene into chromosomal DNA of host strain T. reesei was confirmed by PCR analysis. An obvious protein band (approximately 80kDa) of the target enzyme was detected by SDS-PAGE from fermentation broth, suggesting that the GOD gene in recombinant T. reesei was successfully expressed and extracellularly secreted.Fermentation conditions for glucose oxidase production by recombinant T. reesei ZU-G3 was studied in shaking flask cultivation. The optimized culture medium contained(1L):lactose 20g, microcrystalline cellulose(MCC) 20g, tryptone 9g, (NH4)2SO4 2.8g, KH2PO4 4g, MgSO4 · 7H2O 0.6g, CaCl2 0.6g, trace element 2mL. The optimized fermentation condition was:Temperature 28℃, initial pH 5.5. Under optimal conditions, the maximum glucose oxidase activity was achieved at 72h, reaching 154.87U/mL,1.5 times higher than that of the basal medium.The properties of glucose oxidase produced by recombinant T. reesei ZU-G3 was investigated. The optimum temperature and pH value for enzymatic reaction were 40℃ and pH 5.5, respectively. When incubated at pH 5.0～6.5 for 24h, glucose oxidase remained over 80% of initial enzyme activity. And after lh-incubation at 20～ 45℃, approximately 85% of initial enzyme activity was retained, indicating that GOD was intrinsically thermostable. Metal ions like Ca2+、Cu2+、Zn2+、Mn2+ were able to activate GOD activity while Pb2+、Ag+、Fe2+ profoundly inhibit the enzyme, and K+、 Na+、Mg2+ showed no significant influence on its activity.Recombination and expression of glucose oxidase gene from A.niger in Trichoderma reesei was achieved in this study, which is meaningful for the construction of filamentous fungi expression system, scale-up production of GOD and further application of GOD in industries.