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Enzymatic phospholipid removal (degumming) is a fast-growing and environmentally friendly process for vegetable oil refining. Type C phospholipases (PLC) are the preferred enzymes since they provide an extra yield in the oil recovery. Bacillus cereus PLC can hydrolyze phosphatidylcholine (PC) but has a limited efficiency at removing phosphatidylethanolamine (PE), which together represent ∼70% of the phospholipids present in crude soybean oil. In the present work, we show that the B. cereus PLC mutant F66Y can remove up to 90% of PE while retaining its efficiency at hydrolyzing PC. Oil treatment with the engineered enzyme provides an extra yield of 1.84% making the B. cereus PLC F66Y mutant an attractive candidate for its industrial use. [ABSTRACT FROM AUTHOR]

Crude vegetable oils are refined to remove impurities that adversely impact in their stability, color and flavor. In recent years, enzymatic degumming methods using phospholipase C (PLC) enzymes provide an environmentally friendly process for phospholipids removal with improved oil recovery yields. In this study, Pichia pastoris was used as the expression system for the production of PLC-Y, a modified PLC enzyme derived from Bacillus cereus . Production of secreted PLC-Y driven by the methanol inducible AOX1 promoter was optimized by genetic strain engineering which included gene codon optimization, generation of multi-copy chromosomal integrations and the co-expression of helper factors supporting protein folding, processing and secretion processes. In addition, tunable promoters directing helper factor expression were tested. In batch cultures, a strain harboring seven integrated copies of the PLC-Y expression cassette and co-expressing the HAC1 transcription factor under an attenuated AOX1 promoter showed a 6.2 fold increase in the production titers compared to the strain harboring a single gene copy. A fed-batch fermentation process developed using this engineered strain produced 4.5 g/l of this enzyme. The results presented in this work show the viability of using PLC-Y for oil degumming and provide a manufacturing process for its cost effective production. [ABSTRACT FROM AUTHOR]