J Ind Microbiol Biotechnol (2008) 35:159–166 DOI 10.1007/s10295-007-0277-6 123 ORIGINAL PAPER Overexpression of the plg1 gene encoding pectin lyase in Penicillium griseoroseum Patrícia Gomes Cardoso · João Batista Ribeiro · Janaina Aparecida Teixeira · Marisa Vieira de Queiroz · Elza Fernandes de Araújo Received: 1 July 2007 / Accepted: 27 October 2007 / Published online: 21 November 2007  Society for Industrial Microbiology 2007 Abstract The pectin lyase (PL) is an industrially important enzyme since it is used for maceration and clariWcation in the process of fruit juice production in food industries. In order to increase the yields of pectin lyase we cloned the plg1 (pec- tin lyase 1) from Penicillium griseoroseum gene under the control of the strong constitutive promoter of the glyceralde- hyde-3-phosphate dehydrogenase gene (gpdA) and the termi- nator region of the tryptophan synthetase (trpC) gene from Aspergillus nidulans (plasmid pAN52-Plg1) and transformed this construct into the P. griseoroseum strain PG63. One of the pAN52-Plg1 multi-copy transformants (strain 105) grown in culture medium containing glucose or sugar cane juice showed PL activities of 4,804 or 5,202 U ml ¡1 respec- tively, which represented 57- and 132-fold increases. In addi- tion, the apparent speciWc activity of PL produced by this strain was much higher than the one observed for a commer- cial pectinase preparation. Evaluation of the extracellular proteins in the culture supernatant of strain 105 by SDS- PAGE showed the presence of a clear and strong band of approximately 40 kDa that probably corresponds to PL. The enzyme yields reported here demonstrate that the system we developed is able to express pectin lyase at levels comparable to, or exceeding, previously reported data. Keywords Penicillium griseoroseum · Pectin lyase genes · Transformation · Overexpression · Gpd promoter Introduction Pectinases are a group of enzymes that hydrolyze pectin by diVerent mechanisms and are divided into two broad clas- ses: pectinesterases and depolymerases. The pectinesterases remove methoxy groups from methylated galacturonides. The depolymerases catalyze the cleavage of glycosidic bonds via hydrolysis (hydrolases) or via -elimination (lyases) [42]. These enzymes are widely applied in fruit juice industries, in the processing of ramie, hemp, Xax and jute Wbers, tea, coVee, oil extraction, treatment of industrial wastewater, and in the making of paper [18, 19]. Pectin lyase is the most important enzyme involved in pectin depolymerization, since it is the only enzyme capable of cleaving the internal glycosidic bonds of highly methylated pectin, such as fruit pectin, without the prior action of other enzymes [1]. Thus, the development of PL overproduction systems is of great importance for the fruit juice industry. Fungi of the genus Penicillium are of signiWcant indus- trial importance and species of this genus are among the microorganisms able to produce the enzymatic complex involved in pectin degradation [35]. Penicillium griseoro- seum CCT6421 has proven to be a good pectinase producer [4]. Multiple forms of pectin lyases are produced by Wla- mentous fungi and several pectin lyase genes have been isolated and characterized, which constitute gene families in many fungal species, such as, Aspergillus niger [14, 15, 23, 24], Aspergillus oryzae [20, 21], and P. griseoroseum [6]. Two pectin lyase-encoding genes (plg1 and plg2) from P. griseoroseum CCT6421 were isolated, characterized, P. G. Cardoso Departamento de Biologia/Setor de Microbiologia, Universidade Federal de Lavras, Lavras-MG, Brazil J. B. Ribeiro EMBRAPA Suínos e Aves, Concórdia-SC, Brazil J. A. Teixeira · M. V. de Queiroz · E. F. de Araújo (&) Departamento de Microbiologia/BIOAGRO, Universidade Federal de Viçosa, Viçosa-MG CEP 36570-000, Brazil e-mail: ezfa@ufv.br Downloaded from https://academic.oup.com/jimb/article/35/3/159/5993569 by guest on 11 June 2023