Cloning and heterologous expression of a thermostable pectate lyase from Penicillium occitanis in Escherichia coli.

The entire pectate lyase cDNA (Pel1) of Penicillium occitanis was cloned from a cDNA bank and sequenced. The ORF exhibited a great homology to Penicillium marneffei and conservation of all features of fungal pectate lyases such as the barrel structure with "eight right-handed parallel β-helix" architecture. The structure modeling also showed the interesting resemblance with thermostable pectate lyases since several specific residues were also shared by Pel1 and these thermostable enzymes.

The constitutive production of pectinase by the CT1 mutant of Penicillium occitainis is modulated by pH.

The aim of the present study was to investigate pectinases production by CT1 mutant of Penicillium occitanis on glucose based media. Two main groups of pectinases were followed: lyases (pectin and pectate lyases) and hydrolases (polygalacturonases and polymethylgalacturonases). When cultivated in different liquid media, where either the starting glucose concentration or the nature of nitrogen sources used was varied, the CT1 mutant secreted either lyases or hydrolases.

Studies on the zymogram method for the detection of pectinolytic activities using CTAB.

Zymogram analysis is a useful tool for the identification of several enzymes. The present study was undertaken to investigate the efficiency gains from the characterization of pectic enzymes on zymograms by staining of pectin-agarose overlays using cetyl trimethyl ammonium bromide also known as cetrimide or CTAB. The method is based on the fact that the enzymatic hydrolysis of the pectic substrates included in the agarose matrix gel inhibited their precipitation by CTAB, leading to the appearance of cleared zones in front of the pectin hydrolases and lyases.

Constitutive over-expression of pectinases in Penicillium occitanis CT1 mutant is transcriptionally regulated.

The CT1 mutant of Penicillium occitanis hyperproduces extracellular pectinases constitutively since it secretes pectinases even on glucose-containing medium. We show here that all other hydrolytic enzymes remain at low activities in CT1, confirming the specificity of the regulatory mutation towards pectinases. We isolated, by RT-PCR and through the construction of a cDNA library, three fragments coding for: a pectin lyase (pnl1), a polygalacturonase (pga1) and a pectate lyase (pal1).

Genomic organization of a polygalacturonase gene from a hyperpectinolytic mutant strain of Penicillium occitanis.

The pga1 gene encoding an endopolygalacturonase was isolated from a hyperpectinolytic mutant strain of Penicillium occitanis. It consists of an ORF of 1.155 kbp encoding a putative protein of 346 amino acids with a predicted molecular mass of 39 kDa, belonging to the family 28 of glycosyl hydrolases.