Constitutive and extracellular expression of pectin methylesterase from Pectobacterium chrysanthemi in Pichia pastoris

Abstract

Pectin methylesterase (PME) which is widely used in the cosmetic, food and pharmaceutical industries catalyses the hydrolysis of the methyl ester of pectin to yield methanol and free carboxyl groups. This study was performed to produce active pectin methylesterase (PME) extracellularly from Pectobacterium chrysanthemi in Pichia pastoris. Firstly, pGKBα was constructed for the secretion of heterologous protein. After it was cloned in Escherichia coli cells and the sequence was affirmed, PME gene was inserted into pGKBα. So, pGKBα-PME carried the PME gene in correct position was cloned in E. coli cells. Then, P. pastoris X-33 cells were transformed with linearized pGKBα-PME and six transformants were cultivated for recombinant PME production. It was observed that one of them had a high-capacity secretion of active PME. The molecular mass of extracellular PME enzyme was found to be about 59 kDa. The PME enzyme from P. chrysanthemi was produced by P. pastoris for the first time in this study. This recombinant enzyme might be produced in a large scale and also purified from the culture medium. Then, the purified enzyme might be used for clarification and increasing yield of juice in food industrial applications.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-022-03291-3.

Keywords: Expression; Pectin methylesterase; Pectobacterium chrysanthemi; Pichia pastoris; pGKBα.

Fig. 1

Obtained recombinant vectors (a) pGKBα, and (b) pGKBα-PME

Fig. 2

Agarose gel electrophoresis results of PCR (a) with gene-specific primers, and (b) geneticin-resistance gene primers (M: Marker, 1–6: gDNAs of 1st, 2nd, 3rd, 6th, 7th, and 10th colonies)

Fig. 3

a Agarose plate activity staining analysis at pH 5 (1, 2, 3, 6, 7, and 10 (number of colonies): concentrated culture supernatant from cells harboring pGKBα-PME, C (control): concentrated culture supernatant from cells harboring pGKBα). b, c Agarose plate activity staining analysis at pH 5 and pH 7, respectively, of recombinant P. pastoris cells forming 1st colony (1: concentrated culture supernatant from cells harboring pGKBα, 2: soluble fraction from lysis of cells harboring pGKBα, 3: concentrated culture supernatant from cells harboring pGKBα-PME, 4: soluble fraction from lysis of cells harboring pGKBα-PME), (d) Growth of recombinant P. pastoris cells forming 1st colony in YPD plate containing pectin and plate activity staining analysis (1: yeast cells forming the first colony harboring pGKBα-PME, 2: yeast cells harboring pGKBα)

Fig. 4

SDS-PAGE analysis of recombinant PME expressed in P. pastoris (lane M: protein molecular weight marker, lane 1: culture supernatant from cells harboring pGKBα, lane 2, 3, and 4: fivefold, tenfold, and 20-fold concentrated culture supernatant from cells harboring pGKBα-PME, respectively)