Enzymatic Degradation of p-Nitrophenyl Esters, Polyethylene Terephthalate, Cutin, and Suberin by Sub1, a Suberinase Encoded by the Plant Pathogen Streptomyces scabies

Abstract

The genome of Streptomyces scabies, the predominant causal agent of potato common scab, encodes a potential cutinase, the protein Sub1, which was previously shown to be specifically induced in the presence of suberin. The sub1 gene was expressed in Escherichia coli and the recombinant protein Sub1 was purified and characterized. The enzyme was shown to be versatile because it hydrolyzes a number of natural and synthetic substrates. Sub1 hydrolyzed p-nitrophenyl esters, with the hydrolysis of those harboring short carbon chains being the most effective. The V_max and K_m values of Sub1 for p-nitrophenyl butyrate were 2.36 mol g^-1 min^-1 and 5.7 10^-4 M, respectively. Sub1 hydrolyzed the recalcitrant polymers cutin and suberin because the release of fatty acids from these substrates was observed following the incubation of the enzyme with these polymers. Furthermore, the hydrolyzing activity of the esterase Sub1 on the synthetic polymer polyethylene terephthalate (PET) was demonstrated by the release of terephthalic acid (TA). Sub1 activity on PET was markedly enhanced by the addition of Triton and was shown to be stable at 37°C for at least 20 d.

Keywords: actinobacteria; common scab; cutinase; esterase; potato.

Fig. 1.

SDS-PAGE gel of the cytoplasmic extract obtained from pET-transformed Escherichia coli strain SHuffle T7, without (E. coli SHuffle T7-pET) or with (E. coli SHuffle T7-pET-sub1) the insert of the sub1 gene, after induction with different concentrations of IPTG.

Fig. 2.

Esterase activity of cytoplasmic extracts from Escherichia coli SHuffle T7 transformed with plasmid pET without (E. coli SHuffle T7-pET) or with (E. coli SHuffle T7-pET-sub1) the sub1 insert and exposed to various concentrations of IPTG. Activity is expressed as the concentration of p-nitrophenol released from p-nitrophenyl butyrate substrate in 5- and 30-min reactions. These results are the means of five replicates±SD. Bar values accompanied by the same lower case letter or upper case letter were not significantly different.

Fig. 3.

SDS–PAGE gel of cytoplasmic soluble proteins obtained from Escherichia coli transformed with SHuffle T7-pET-sub1, after fractionation on the affinity column (IMAC). Lane 1, molecular weight marker; lane 2, cytoplasmic extract; lane 3, flow-through; lane 4, proteins released after washing with buffer A; lanes 5 to 9, proteins released after washing with buffer A supplemented with 4, 5, 10, 50, or 200‍ ‍mM imidazole, respectively.

Fig. 4.

Esterase activity of the purified Sub1 enzyme using p-nitrophenyl substrates of different carbon chain sizes (C4, C8, C10, and C12) in the absence or presence of Triton X-100 (0.5%). Data shown are the mean±SD of three replicates. Bar values accompanied by the same letter are not significantly different.

Fig. 5.

Effects of substrate (p-NPB) concentrations on the initial speed (V₀) of the hydrolysis reaction of the esterase Sub1. (A) Michaelis-Menten kinetic and (B) Lineweaver-Burk plot. Data are the means±SD of three replicates.

Fig. 6.

Degradation of cutin and suberin by enzyme Sub1 at room temperature over a 20-d period, as expressed by the release of fatty acids in the incubation medium. Data are the means±SD of four replicates.

Fig. 7.

Concentrations of terephthalic acid (TA) released following the hydrolysis of ground particles of polyethylene terephthalate by 3 μg of the Sub1 enzyme. (A) Effects of the presence of Triton X-100 (0.5%) on Sub1 performance after 10 and 15‍ ‍d of incubation (at 37°C). (B) Sub1 enzymatic stability (in the presence of 0.5% Triton X-100) during 20‍ ‍d of incubation at 37 and 50°C. TA concentrations were measured every 5 d. Data are the means±SD of four replicates.