Secretory expression of a phospholipase A₂ from Lactobacillus casei DSM20011 in聽Kluyveromyces lactis

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• 作者：Hui Wang² ; ³ ; Liang Zhang¹ ; ² ; ^{zhangl@jiangnan.edu.cn" class="auth_mail" title="E-mail the corresponding author} ; Guiyang Shi²
• 关键词：Secretory phospholipase A2 ; Lactobacillus casei ; Heterologous expression ; Real-time quantitative PCR ; Copy number ; Kluyveromyces lactis
• 刊名：Journal of Bioscience and Bioengineering
• 出版年：2015
• 出版时间：December 2015
• 年：2015
• 卷：120
• 期：6
• 页码：601-607
• 全文大小：548 K

文摘

The pla2 gene encoding a phospholipase A₂ (EC 3.1.1.4) of Lactobacillus casei DSM20011 was cloned and expressed in the yeast Kluyveromyces lactis GG799 successfully for the first time. The structural pla2 gene fused in frame with the K. lactis secretion signal 伪-mating factor was integrated into the LAC4 locus and expressed under the control of the LAC4 promoter. sPLA₂ activity was detected in the culture supernatant during shake flask culture of K. lactis/pKLAC1-pla2. In comparison with the control strain K. lactis/pKLAC1, SDS-PAGE analysis revealed a 17-kDa recombinant protein band in K. lactis/pKLAC1-pla2, which was consistent with the predicted molecular weight of the mature protein. Real-time quantitative PCR analysis indicated that the copy number of the integrated pla2 gene ranged from 2 to 6 and positively correlated with sPLA₂ activity. When the inducer galactose was used as the carbon source, the sPLA₂ activity in the culture supernatant of the recombinant that harbored six pla2 gene copies reached 1.96 ± 0.15 U/mL. The influence of the culture composition and conditions on the recombinant sPLA₂ activity in shake flask culture were also studied. When the recombinant was cultured at 30°C in a YPD medium culture volume of 70 mL in a 250-mL shake flask with an initial pH of 7.0, the sPLA₂ activity reached 2.16 ± 0.18 U/mL.