Effect Of Signal Peptide,kozak Sequence And N-glycosylation On The Xylanase Expressed In Aspergillus Niger

Xylan is the main component of plant hemicellulose and is widely distributed in nature.Xylanase(D-xylanxylanohydrolase EC3.2.1.8) is the main enzyme in the degradation of xylan,which belongs to the hydrolase. It’s mainly used in wine, feed, paper, food and other industries.Currently, the food-grade xylanase is applied to fermentation production mainly by using Aspergillus niger and other filamentous fungi. However, these methods are generally with low enzyme activity, low yield and so on. Therefore, the effective way to solve this problem is to construct a high level engineering bacteria with high expression of xylanase by gene engineering technology.In the early stage of the laboratory, one strains of Aspergillus niger engineered bacteria were constructed by using CICC2462 as the receptor and the shaking flask fermentation enzyme activity reached to 4495.89U/ml. On the basis of this study, the xylanase gene was modified by changing the signal peptide sequence, adding kozak sequence and N-glycosylation modification method to further improve the expression and stability of xylanase.The main results are as follows:1. The transformation of xylanase gene and construction of expression vectors.Using PCR technology, the Aspergillus niger xylanase gene xyn Bm2 with glucoamylase signal peptide and the Aspergillus niger xylanase gene xyn Bm3 with glucoamylase signal peptide and the kozak sequence were amplified. Aspergillus niger xylanase gene xyn BNG1, xyn BNG2 and xyn BNG3 modified by different N-glycosylation were amplified by overlap extension PCR technique.Then,the Aspergillus niger expression vector(pSZHG-xyn Bm2,pSZHGxyn Bm3,pSZHG-xynBNG1,pSZHG-xynBNG2,pSZHG-xyn BNG3)was constructed by connecting them with pSZHG.2. Transformation of Aspergillus niger and identification of homologous recombination transformant.The expression vector was transformed into Agrobacterium AGLI by freezing thawing method,and the xylanase gene was transferred to Aspergillus niger by Agrobacterium mediated method.Then, we extracted DNA from Aspergillus niger and identified by PCR, finding that pSZHG-xynBm2,p SZHG-xyn Bm3,pSZHG-xynBNG1,pSZHG-xyn BNG2,pSZHG-xyn BNG3 positive transformants are homologous recombinant strain. After subculture, the pSZHG-xyn Bm2 homozygous strains were screened.3. The expression analysis of Xylanase gene in recombinant strains.The fermentation supernatant of recombinant strain was taken to detect the enzyme activity.The results showed that the highest enzyme activity of the recombinant strain pSZHG-xynBm2,pSZHG-xynBm3, pSZHG-xynBNG1, pSZHG-xynBNG2 and pSZHG-xynBNG3 was 2525U/ml,7710U/ml, 5907U/ml, 3074U/ml and 2305U/ml respectively.The optimum temperature of the three genes xyn BNG3, xyn BNG2 and xyn BNG1 modified by glycosylation was 45 ℃, and the optimum pH value was 7. The enzyme activity of xyn BNG1,xyn BNG2, and xynBNG3 decreased significantly when the temperature was higher than 50℃, 55℃and 45℃ respectively. Heat insulation at different temperatures for 1 hours to detect the residual enzyme activity of xylanase. As the heat treatment temperature increased, the enzyme activity began to decrease. After treanment at 40℃ for 1h, the enzyme activity is 60.8%,65.5%,65.1% of the untreated group. Whereas, treatment at 50 ℃ of the same time, the relative enzyme activity was only 60.8%, 65.5%, 65.1%, all have decreased.Quantitative PCR technique was used to compare the gene expression difference of several genes at the transcriptional level. The expression of transformant pSZHG-xyn Bm2 was lower than the control group pSZHG-xynB gene for 45%. Gene integration of the target site of the glycosylated enzyme gene mRNA level reduced to 6.9% of the starting strain. Compared with the control group(pSZHG-xyn B), the expression of pSZHG-xynBm3 was increased two times. Gene integration of the target site of the glycosylated enzyme gene mRNA level reduced to 7.3% of the starting strain.The results showed that the replaced signal peptide of xylanase by signal peptide of glycosylated enzyme reduced the expression of xylanase in Aspergillus niger. Adding kozak sequence can effectively improve the expression of xylanase. The effect of N-glycosylation modification on the thermal stability of xylanase was not obvious, and its effect on improving the expression amount of xylanase needed further evaluated after getting homozygous recombinant strains.