| Phospholipase B are a group of hydrolases, which could hydrolyze phospholipids releasing a variety of products, such as lyso-phospholipids, free fatty acids, di-acylglycerols, choline phosphate and phosphatidates. Nowadays, phospholipase B has been used in bread making, egg-yolk industry and refinement of vegetable oils, especially in terms of crude oil degumming. Compared with the traditional physical degumming methods, enzymatic degumming can greatly reduce the consumption of chemicals with the advantage of hardly any waste water, leading to an economical, efficient and stable green oil degumming process with international leading level.In the study, the gene-optimized “lipase”(pho)from Fusarium oxysporum was expressed in Pichia pastoris. We found unexpectedly that this enzyme has the activity of hydrolyzing phospholipids, which was much higher than its lipase activity, thus we defined it as a phospholipase, and the phospholipase B was identified by gas phase experiment. After that, the induction strategy of the recombinant P. pastoris was optimized, and the characterization and the application in oil-degumming of the recombinant phospholipase was investigated. The results of the study are as following:(1) The “lipase” gene from Fusarium oxysporum was optimized according to the codon usage of Pichia pastoris, named pho. Then pho was ligated into p PIC9 K, linearized by Sac I and transformed into P. pastoris, by electroporation, yielding the recombinant strain P. pastoris GS115/p PIC9K-pho and P. pastoris KM71/p PIC9K-pho. After 120 h of methanol induction in the shaking flask, the phospholipase activity of recombinant P. pastoris GS115/p PIC9K-pho and P. pastoris KM71/p PIC9K-pho culture supernatant were 450.5 U·m L-1, 650.4 U·m L-1, the lipase activity were 16.1 U·m L-1, 22.5 U·m L-1. The phospholipase activity was much higher than its lipase activity, thus we defined it as a phospholipase, and the phospholipase B was identified by gas phase experiment.(2) When the inducing temperature was 30℃, the initial cell density was 60 g·L-1, p H was 5.0 and the methanol concentration was 1.0 %(v·v-1), after 120 h of methanol induction the phospholipase B activity of the culture supernatant was 4972.6 U·m L-1 from the recombinant strain P. pastoris KM71/p PIC9K-pho, 2.6-fold of the phospholipase B activity from the recombinant strain P. pastoris GS115/p PIC9K-pho, and the protein concentration in supernatant production of the enzyme were 5.0 mg·m L-1, 1.9 mg·m L-1. To increase the expression level of phospholipase B in P. pastoris KM71/p PIC9K-pho, optimization of fermentation conditions were investigated. When the inducing temperature was 28℃, the initial cell density was 40 g·L-1, p H was 5.0 and the methanol concentration was 0.5 %(v·v-1), after 120 h of methanol induction the phospholipase B activity of the culture supernatant was 6503.8 U·m L-1.(3) Fusarium oxysporum phospholipase B have disulphide bonds. So, to increase the production of recombinant phospholipase B, Mpr1 was selected to coexpress with the phospholipase B. The Mpr1 gene was amplified by PCR and cloned into the expression vector p PICZA, then the recombinant plasmid was lineared and transformed into the strain to achieve recombinant P. pastoris KM71/p PIC9K-pho/p PICZA-Mpr1(recombinant strain). The recombinant strain were investigated in shake flasks and 3.6 L bioreactor. When the inducing temperature was 28℃, the initial cell density was 40 g·L-1, p H was 5.0 and the methanol concentration was 1.0 %(v·v-1), after 120 h of methanol induction the phospholipase B activity of the culture supernatant was 1.3-fold than before.(4) After various purification steps to obtain the pure phospholipase B. Then, the properties of the recombinant enzyme were studied. The recombinant enzyme was purified to the specific activity of 1171 U·mg-1. The optimum temperature of the recombinant enzyme was 55℃. The enzyme retained 52.1 % of its activity after being treated at 55℃ for 4 h and showed good thermostability. The enzyme exhibited the highest activity at p H 5.0, retaining more than 80 % of its initial activity in the range of p H 4.0~5.3. In addition, after 2 h under optimal conditions, which the enzyme dosage 40 U·g-1, 60℃ and p H 5.0, the phosphorus content decreases from 75.9 ppm to 3.3 ppm. The high-level expression and short reaction time described here should reduce production costs, compared with the previous studies. Thus, this study may provide for industrial use of phospholipase B for oil-degumming and other industrial applications. |
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