| N-acetyl transferase(Mpr1) is an intracellular enzyme that catalyzes the transfer of acetyl groups between acetyl Co A and amines. Mpr1 has significant physiological functions in ROS oxidative stress resistance ability. It can protect the activity of the cells in a variety of stress conditions, such as oxidative stress, thermal stress, freezing and thawing. Due to the special physiology in methanol metabolism, Pichia pastoris suffers much more ROS oxidative stress, so it is more meaningful to research MPR1 gene in P. pastoris. A detailed study about the physiological characteristics of MPR1 and its impact on yeast cell growth and production of α-glucosidase were performed. In this research, recombinant strain p QE30-MPR1-E. coli JM109 was constructed, and enzyme properties were studied. And the recombinant P. pastoris KM71/p PIC9K-αglu producing recombinant α-glycosidase enzyme were used as the original strain. Constructing the recombinant P. pastoris KM71/p PIC9K-αglu/p PICZA-MPR1, the impact of Mpr1 on yeast cell growth and production of α-glucosidase was investigated, as well as fermentation optimization in 3.6 L bioreactor. The main results are as follows:(1) The MPR1 gene was amplified by PCR from the genome of P. pastoris GS115, and cloned into the expression vector p QE30, achieved recombinant p QE30-MPR1-E. coli JM109. After induction for 24 h in shake-flask culture, the enzyme activity reached 462.32 m U·m L-1.(2) Shake flask optimization of recombinant strain was performed according to the Response Surface Analysis. Induction temperature was 30°C, IPTG induction concentration was 0.4 mmol·L-1, Initial induction OD600 was 4.5, induction time was 24 h, then enzyme activity reached 610.30±9.50 m U·m L-1. After purification, the research on pure enzyme properties was carry on. The results showed that the optimal p H was 7.5, the optimum temperature of Mpr1 was 30°C, and it had good stability under the condition of 4-20°C. Adding glycerol and sorbitol improved the stability of the enzyme to some extent.(3) MPR1 gene was cloned into the expression vector p PICZA, then the recombinant plasmid was lineared by Sac I and transformed into P. pastoris KM71/p PIC9K-αglu(original strain) to achieve recombinant P. pastoris KM71/p PIC9K-αglu/p PICZA-MPR1(recombinant strain). The influence of Mpr1 coexpression on cell death rate, intracellular ROS levels, α-glycosidase production and stability of α-glycosidase in the fermentation process was performed. After a 96 h induction in flask, the activity of α-glycosidase of original strain was 560 m U·m L-1, and the activity of recombinant strain was 1, 080 m U·m L-1, which was 1.93 times higher than that of the original strain. After 134 h and 158 h induction, cell mortality of recombinant strain reduced respectively by 24% and 61% compared to the original strain. After induction of 160 h in the 3.6 L bioreactor, under conditions of different dissolved oxygen, the recombinant strains cell death rate and intracellular ROS was lower than the original strain. The highest enzyme activities of recombinant strain increase by 40% than the original strain, and specific activity was increased by 34%.(4) The fermentation optimization of recombinant strain P. pastoris KM71/p PIC9K-αglu/p PICZA-MPR1 in 3.6 L bioreactor was performed. The optimum fermentation conditions were: The initial induction OD600 was 100, methanol induction concentration was 0.5%(v/v), p H was 5.0, induction temperature was 30°C, induction time was 160 h, then the enzyme activity was up to 16, 270 m U·m L-1. The enzyme activity was significantly improved... |
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