High-level Expression Pectate Lyase In Pichia Pastoris And Enzymatic Determination

Pectin was a type of polysaccharide that was widely present in plant cell walls and has important influence on the structural stability of cells.Pectin backbone was polygalacturonic acid with different degrees of methyl esterification,acetylation and other modifications.Pectin had significant effects on the processing of beverages such as fruit juices,fruit wines,feeds,and degumming of papermaking and bast fiber.Pectinase can degrade pectin effectively and has broad prospects in food,beverage,feed and papermaking.In this paper,three pectin lyase genes pelA,pelB and pelC of Bacillus subtilis were cloned into Pichia pastoris,respectively,and has obtained the expressed protein.The enzymatic properties of the expressed products were further analyzed.In order to improve the expression level,the codon of pelA gene was optimized,and multi clone expression cassettes of pelAO and pelC were constructed.The main contents of this study:(1)Pectin lyase pelA gene was cloned in this study.The results showed that the enzyme activity was 285 U/mL induction by low temperature of 16°C,in Escherichia coli BL2;however,expression level of pelA in P.pastoris was very low,and the enzyme activity was only 250 U/mL after about 50-fold concentration of the fermentation broth.The pelA sequence was optimized with reference to the P.pastoris codon and synthesized the optimized gene pelAO.The expression level of pelAO was significantly higher than pelA,and specific enzyme activity of fermentation supernatant reached 330 U/mg in shake flask.Some multi-copy vectors of pelAO were constructed.Analysis of obtained recombinants expression level showed that the enzyme activities of two copies,three copies,and seven copies were 300%,320%,and 350% of the single copy,respectively.The specific activity of multi-copy strain reached 3620 U/mg in 14 L bioreactor.Analysis of the enzymatic properties indicated that PelA belongs to the endo-alkaline pectate lyase.(2)In this study,pectinase gene pelB and pelC were cloned,they were expressed in P.pastoris,respectively,and their enzymatic properties were studied.The expressed proteins of pelB and pelC were 38 kDa and 45 kDa,respectively.The optimum temperatures were 50?and 60?,and the optimum pH were 9.0 and 9.6.The enzyme activities of PelB and PelC were 3.6 U/mg and 1.7 U/mg,respectively,which were significantly lower than those of PelA.Three-dimensional structural simulations indicate that the low activity of PelB may be due to the fact that the active center was too shallow;the low activity of PelC may be due to the high glycosylation.In this study,the pectinase genes pelA,pelB and pelC of B.subtilis were cloned and high expression level in P.pastoris was successfully achieved.Although the specific activity of PelB and PelC was very low,the high specific activity of PelA laid the foundation for the large-scale application of this enzyme.