FOOD SCIENCE ›› 2020, Vol. 41 ›› Issue (8): 100-107.doi: 10.7506/spkx1002-6630-20190903-029

• Bioengineering • Previous Articles     Next Articles

Recombinant Expression of Phospholipase D from Streptomyces septatus in Pichia pastoris and Analysis of Its Enzymatic Properties

HUANG Lin, MA Jieying, WANG Shuang, WANG Nan, MORADANA GAMAGE Rasadi Sajeewa Rajakaruna, LU Fuping, LIU Yihan   

  1. (Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China)
  • Online:2020-04-25 Published:2020-04-20

Abstract: The phospholipase D (PLD) encoding sequence (pld) was amplified from the genomic DNA of Streptomyces septatus TCCC 21057. The optimized pldm sequence was synthesized according to the codon usage bias in Pichia pastoris and then integrated into the expression vector pPIC9K. The resulting recombinant plasmid was then transformed into P. pastoris GS115 competent cells to obtain the recombinant strain GS115/pPIC9K-pldm. The transphosphatidylation activity of recombinant PLD (rPLDM) could reach 2.37 U/mL after shaking flask fermentation. The recombinant enzyme was purified and characterized. Its optimal activity was observed at 60 ℃ and pH 6.5. The yield of phosphatidylserine (PS) could reach 33% after 6 h reaction in a purely aqueous system under the following conditions: molar ratio between the substrates soybean phosphatidylcholine (PC) and L-serine substrate 1:10, rPLDM amount 4.0 U/mL, temperature 40 ℃, pH 6.5, and Ca2+ concentration 10 mmol/L. This study lays a foundation for enzymatic synthesis of PS as a novel food ingredient.

Key words: Streptomyces, phospholipase D, Pichia pastoris, expression, enzymatic properties, phosphatidylserine

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