Spontaneous revertants of the AOX1 defective P.pastoris strain have been isolated,which were identified as phenotypically utilize methanol to grow as the wild type.The promoter region of the AOX2 gene from the reverta...Spontaneous revertants of the AOX1 defective P.pastoris strain have been isolated,which were identified as phenotypically utilize methanol to grow as the wild type.The promoter region of the AOX2 gene from the revertant has been obtained by PCR amplification,and the DNA fragment is 1022 base pair in size.By the analysis of sequencing result and compared with the AOX2 gene sequences recorded in Gene Bank,two point mutations which at positions of 529 and 255(relative to the translation initiation codon)respectively,have been found.Since the positions are located in the AOX2 upstream respression sequences,the mutations may act interfere the repressor to combine with the functional sequences,and increase transcriptional activity.The above result implicated that the P.pastoris’ ability to utilize methanol could be incresed through modification the upper stream transcriptional regulation region in the AOX2 gene.展开更多
The activities of intracellular alcohol oxidase(AOX) in recombinant P.pastoris expressing Pro-UK were determined by a self-designed dissolved oxygen measuring equipment. The enzyme vitality and specific enzyme vitalit...The activities of intracellular alcohol oxidase(AOX) in recombinant P.pastoris expressing Pro-UK were determined by a self-designed dissolved oxygen measuring equipment. The enzyme vitality and specific enzyme vitality were defined nd the condition for detecting the enzyme vitality was also established. The experimental results showed that with a certain quantity of biomass in a phosphate buffer containing methanol, the consuming rate of dissolved-oxygen reflected the enzyme vitality of intracellular AOX. It was also found that the pH of the buffer could be very freely between 4.7 and 7.4 and the suitable optical dersity of cell concentration at 600 nm was between 0.5 and 2.0. Furthermore, the values of q O 2max and \%K\%\-m of AOX versus oxygen consumption, which were 0.409 s -1 and 0.16 respectively, were calculated. It is a simple and sensitive and feasible method for quick measuring of AOX.展开更多
文摘Spontaneous revertants of the AOX1 defective P.pastoris strain have been isolated,which were identified as phenotypically utilize methanol to grow as the wild type.The promoter region of the AOX2 gene from the revertant has been obtained by PCR amplification,and the DNA fragment is 1022 base pair in size.By the analysis of sequencing result and compared with the AOX2 gene sequences recorded in Gene Bank,two point mutations which at positions of 529 and 255(relative to the translation initiation codon)respectively,have been found.Since the positions are located in the AOX2 upstream respression sequences,the mutations may act interfere the repressor to combine with the functional sequences,and increase transcriptional activity.The above result implicated that the P.pastoris’ ability to utilize methanol could be incresed through modification the upper stream transcriptional regulation region in the AOX2 gene.
文摘The activities of intracellular alcohol oxidase(AOX) in recombinant P.pastoris expressing Pro-UK were determined by a self-designed dissolved oxygen measuring equipment. The enzyme vitality and specific enzyme vitality were defined nd the condition for detecting the enzyme vitality was also established. The experimental results showed that with a certain quantity of biomass in a phosphate buffer containing methanol, the consuming rate of dissolved-oxygen reflected the enzyme vitality of intracellular AOX. It was also found that the pH of the buffer could be very freely between 4.7 and 7.4 and the suitable optical dersity of cell concentration at 600 nm was between 0.5 and 2.0. Furthermore, the values of q O 2max and \%K\%\-m of AOX versus oxygen consumption, which were 0.409 s -1 and 0.16 respectively, were calculated. It is a simple and sensitive and feasible method for quick measuring of AOX.
