关键词: 酸性果胶裂解酶, 黑曲霉, 高效表达, 酶学性质, 果汁澄清

Abstract: In this study, the overexpression of acidic pectin lyase D (PelD) in Aspergillus niger was achieved by using its own strong promoter PglaA (the glucoamylase gene promoter). The recombinant PelD (rePelD) was purified by Ni-affinity chromatography and the specificity of the purified protein was confirmed by Western blot. The enzymatic properties were also studied. The pectin lyase activity was up to 8 822.6 U/mL in shake flask culture. The crude enzyme was one-step purified to a specific activity of 8 522.7 U/mg with a 79.4% yield. The optimum temperature and pH of rePelD were 50 ℃ and 5.0, respectively. rePelD was stable over a wide pH range, retaining over 50% of its initial activity after treatment at pH 3.0–6.5 and 40 ℃ for 2 h. The purified enzyme was stable in the temperature range from 30 to 50 ℃. At a concentration of 1 mmol/L, Ca2+, Mg2+, Cu2+, Zn2+, Ni2+, Mn2+ and Ba2+ (especially Zn2+, Ni2+ and Ba2+) had an activating effect on rePelD, while sodium dodecyl sulfate (SDS) showed inhibition effect. rePelD showed high specific activity for highly esterified pectin. It showed maximum activity (31 248.0 U/mL) towards citrus pectin (esterification degree ≥ 85%). After treatment with rePelD, the light transmittance of orange, apple and grape juice was increased by 17.9, 11.5 and 5.7 folds, respectively, suggesting that rePelD was very effective in clarifying orange, apple and grape juice.

Key words: acidic pectin lyase, Aspergillus niger, over-expression, characterization, juice clarification