ORIGINAL PAPER Cold-adapted yeasts as producers of cold-active polygalacturonases Received: 28 May 2002 / Accepted: 30 November 2002 / Published online: 11 February 2003 Ó Springer-Verlag 2003 Abstract Eight cold-adapted, polygalacturonase-pro- ducing yeasts belonging to four species were isolated from frozen environmental samples in Iceland. They were identified as Cystofilobasidium lari-marini, Cystofi- lobasidium capitatum, Cryptococcus macerans and Cryptococcus aquaticus species by sequence analysis of rDNA regions. Growth behavior of the isolates was investigated. All strains could grow at 2°C. Addition of glucose to pectin-containing culture medium had a repressive effect on enzyme production except for C. aquaticus, which showed increased polygalacturonase activity. Optimal temperature for enzyme production for the Cystofilobasidium strains was 14°C, while that for the Cryptococcus strains was lower. Among the isolates, C. lari-marini S3B produced highest levels of enzyme activity at pH 3.2. Preliminary characterization of the polygalacturonases in the culture supernatant showed the enzyme from Cystofilobasidium strains to be opti- mally active at 40°C and pH 5, and that from the Cryptococcus strains at 50°C and pH 4. The polygalac- turonase from C. macerans started to lose activity after 1 h of incubation at 40°C, while that from the other strains had already lost activity at 30°C. All the strains except C. aquaticus produced isoenzymes of polyglac- turonase. In addition to polygalacturonase, the Cys- tofilobasidium strains produced pectin lyase, C. aquaticus pectin esterase, and C. macerans pectin lyase, pectate lyase and pectin esterase. Keywords Cold adaptation Æ Cryptococcus aquaticus Æ Cryptococcus macerans Æ Cystofilobasidium capitatum Æ Cystofilobasidium lari-marini Æ Polygalacturonase Introduction Pectic substances are complex polysaccharides of plant origin and are grouped into pectic acid, which is a polymer of galacturonic acid subunits linked by a-1,4 glycoside linkages, and pectinic acid with the same basic structure but having some of its carboxyl groups methylated. The latter group includes pectins, which are capable of form- ing gels with sugar and acid under suitable conditions (Kulp 1975; Fogarty and Kelly 1983). Enzymes that de- grade pectic substances, are classified into pectin esterases which de-esterify pectinic acid, and depolymerases which cleave the main chain. Depolymerases for both pectinic acid and pectic acid are further divided into polygalac- turonases, which hydrolyze the glycosidic bonds by endo- (at random within the chain) and exo- (at terminal end of the chain) action, respectively, and lyases, which break the glycosidic bonds by b-elimination (Kulp 1975). The main application of pectinases is in the fruit and vegetable processing industry. Today, the main source of pectinases used in these industries is from fungi, mainly Aspergillus niger, since it produces high amounts of these enzymes and is a GRAS (generally recognized as safe) microorganism (Alkorta et al. 1998; Kashyap et al. 2001). Scientists have recently been exploring the possi- bility of using pectinases from yeasts for these applica- tions (Blanco et al. 1999). Yeasts that could be declared GRAS microorganisms and which produce high levels of the enzymes might be an interesting alternative as a pectinase source for these industries. Furthermore, de- sirable enzymes could be cloned into a GRAS microor- ganism and overexpressed (Blanco et al. 1999). Extremophiles (2003) 7:185–193 DOI 10.1007/s00792-002-0310-7 Ha´kon Birgisson Æ Osvaldo Delgado Leticia Garcı´a Arroyo Æ Rajni Hatti-Kaul Bo Mattiasson Communicated by K. Horikoshi H. Birgisson Æ O. Delgado Æ L. Garcı´a Arroyo R. Hatti-Kaul (&) Æ B. Mattiasson Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Box 124,22100 Lund, Sweden E-mail: Rajni.Hatti-Kaul@biotek.lu.se Tel.: +46-46-2224840 Fax: +46-46-2224713 Present addresses: H. Birgisson Prokaria, Gylfaflot 5, 112 Reykjavik, Iceland O. Delgado PROIMI-CONICET, Av. Belgrano y Pasaje, Caseros, 4000 Tucuma´n, Argentina