Keywords

1 Introduction

The unique properties of the enzymatic apparatus of psychrophilic and psychrotrophic microorganisms, resulting from the special environmental challenges they face, make them an interesting subject of scientific research and lead to their increasingly widespread use in industry (Cavicchioli et al. 2011). The advantages of cold-active enzymes are unquestionable. Being thermolabile, they can be selectively inactivated. Thanks to low-temperature processing, they allow for the reduction of the energy costs of technological processes and prevent infection with mesophilic bacteria thus ensuring a better quality of the final product (Margesin and Schinner 1994).

Interest in psychrophilic microorganisms, which are a source of unique cold-active biocatalysts, has grown considerably over the past twenty years. However, it should be noted that the majority of works on the structure and properties of these proteins, the general mechanism of low-temperature catalysis, and its biological potential, concern enzymes derived from prokaryotic organisms, mostly psychrophilic and psychrotrophic bacteria (Feller and Gerday 1997; Russel 2000; Zecchinon et al. 2001; D’Amico et al. 2002; Feller and Gerday 2003; Feller 2003; Hoyoux et al. 2004; Georlette et al. 2004; Siddiqui and Cavicchioli 2006; Struvay and Feller 2012), while there are few papers dealing with the enzymatic properties of psychrophilic eukaryotes (Shivaji and Prasad 2009; Cavicchioli et al. 2011; Buzzini et al. 2012). Undoubtedly, the least information is available on cold-adapted yeasts, even though they are a versatile group of eukaryotic microorganisms characterized by varied feeding preferences and a surprising ability to survive in extreme environments with diverse physical and geochemical parameters (Buzzini et al. 2012). It has even been suggested that in psychrophilic habitats yeasts are better adapted to low temperatures than bacteria (Margesin et al. 2003). To date, research into these yeasts has mainly focused on their growth conditions, interactions with the abiotic environment, and taxonomical classification. Over the last two decades, there has been increasing interest in psychrophilic yeasts, as it is believed that due to their exceptional ability to grow and maintain metabolism at low temperatures, they produce unique biocatalysts that may be valuable for industrial applications. Examples include lipases A and B synthesized by Candida antarctica (now Pseudozyma antarctica), which are used in many biotransformations in the food, pharmaceutical, and cosmetic industries (Shivaji and Prasad 2009).

2 Biotechnological Potential of Cold-Active Yeast Enzymes

The potential commercial users of cold-active enzymes are primarily the food and household chemical industries. Some of these biocatalysts may also prove a valuable tool for low-temperature biotransformations and in molecular biology (Cavicchioli et al. 2011). Cold-active enzymes may also find applications in the chemical industry for manufacture of extremely volatile organic compounds that can only be modified at low temperatures. Low temperature may facilitate product separation and reduce the process costs (Brenchley 1996).

Among the psychrophilic yeast enzymes explored to date, most attention has been given to hydrolases. However, these enzymes (perhaps except for lipases, discussed at length in Chap. 16) have been studied to varying extent: some authors report characteristics of purified homogeneous protein preparations, while others determine crude enzymatic preparations. Some other works are limited to enzymatic activity screening without in-depth analysis (Brizzio et al. 2007; Khan Pathan et al. 2010; Carrasco et al. 2012; Loperena et al. 2012; Singh and Singh 2012).

2.1 Amylases, Xylanases, Chitinases, and Lysozymes

Cold-adapted amylases have emerged as some of the leading biocatalysts with proven potential to be used in a wide array of industrial applications (Kuddus et al. 2011). Most of the cold-adapted amylases studied to date have been derived from prokaryotic organisms. There is only one report concerning these enzymes isolated from psychrophilic yeasts (De Mot and Verachtert 1987), that is, two amylases produced by C. antarctica (now P. antarctica) CBS 6678, which were purified to homogeneity using various chromatographic techniques and analyzed kinetically. Both of these enzymes, that is, α-amylase and glucoamylase, are monomeric glycoproteins hydrolyzing soluble starches at optimum temperatures of 62 and 57 °C, respectively. The optimum temperature of glucoamylase is in the range of mesophilic yeast glucoamylases (50–60 °C), while the optimum temperature of the other amylolytic enzyme is much higher than that of most of its yeast homologs (below 50 °C). Amylolytic preparations from C. antarctica exhibit high specificity for high-molecular-weight substrates, including raw starches. α-Amylase is also active toward cyclodextrins, hydrolyzing α-1,4-glycosidic bonds, while glucoamylase catalyzes the cleavage of α-1,6-glycosidic bonds in dextran, pullulan, and glycogen. Furthermore, De Mot and Verachtert (1987) emphasize the similarity of the amylolytic enzymes from C. antarctica CBS 6678 to their mold homologs in terms of glycosylation levels, substrate preferences, and sensitivity to inhibitors, and discuss the potential use of the strain producing these enzymes as a source of genetic material that could be cloned into mesophilic yeasts of the genus Saccharomyces to improve the fermentative properties of these microorganisms.

According to Kuddus et al. (2011), cold-active α-amylases have a wide range of potential applications. In the food industry, they may be used for the production of maltotetraose syrups, which are sweeteners and substitutes for sucrose that do not affect the initial taste and aroma of the product. These syrups are characterized by a high ability to bind water, thanks to which they ensure the proper level of humidity and texture in foodstuffs. Cold-active α-amylases may also be applied in the production of maltose and high-molecular-weight branched dextrins, which are used as fillers in the manufacture of bulk foods. In the cotton industry, they may be used for desizing raw fabric and processing denim textiles and in the detergent industry for cold washing. Other potential applications of cold-active α-amylases include the fruit industry (improved juice clarity), the baking industry (delayed bread staling), the paper industry (lower starch viscosity for appropriate coating of paper), the pharmaceutical industry (in dietary supplements to aid digestive processes), and in the treatment of food-processing wastewater (removal of starch waste). The application of psychrophilic enzymes in the above processes brings substantial benefits in terms of reduced energy consumption (and, by the same token, reduced costs) and environmental protection due to the fact that enzymes are biodegradable.

Many companies are interested in financing research on α-amylases despite the high costs and risk that it entails. Cooperation with commercial partners has led to identification of some new amylases from Antarctic microorganisms, which may have potential applications in industry. For instance, Bacillus licheniformis amylase was found to be highly active in the temperature range of 10–60 °C in a study by Borchert et al. (2004) sponsored by the company Novozymes. This discovery is the subject of US Patent no. 6673589, although most patents in this field concern processes rather than the substances isolated. Given the above, it should be mentioned that the results of research on amylases have not been commercialized to date.

Other identified cold-active eukaryotic glycosidases are xylanases from the yeasts Cryptococcus adeliensis (formerly Cryptococcus adeliae) and Cryptococcus albidus, which may be applied in low-temperature recycling of farm, industrial, and sewage waste (Shivaji and Prasad 2009). Furthermore, these enzymes are particularly useful in the hydrolysis of xylan, which is the main component of hemicelluloses in plant cell walls. The products of enzymatic degradation of this polysaccharide are used in biotechnological processes for, e.g., manufacture of fuels and organic solvents (Amoresano et al. 2000). The low-temperature activity of xylanases is also taken advantage of in the baking industry. The first tests on cold-adapted bacterial xylanases showed that their high activity at low temperatures and mechanism of action improve the quality of bread by increasing its volume. Some commercial preparations of psychrophilic bacterial xylanases are already available from the company Puratos (Grand-Bigard, Belgium) (Feller 2012).

Gomes et al. (2000) optimized culture conditions for Cryptococcus adeliensis with a view to efficient production of xylanase, reaching an activity of 400 nkat ml−1 during 168 h of culture at 4 °C. Among 13 tested sources of carbon, xylanolytic activity was induced to the highest degree by xylan, while the most preferred source of nitrogen was yeast extract. In the presence of other sources of carbon in the medium, the level of enzymatic activity was of constitutive nature. An unpurified preparation of xylanase from C. adeliensis revealed optimum activity at a pH of 5.0–5.5 and was stable in the pH range of 4–9 (21 h at 4 °C). Despite the fact that this protein exhibited optimum activity at a relatively high temperature (45–50 °C), it was very thermolabile with a half life of 78 min at 35 °C, while temperatures in the range of 40–50 °C led to a loss of 71–95 % of its initial activity within 5 min. The authors suggested that the low thermal stability of C. adeliensis xylanase may result from the increased structural flexibility of the molecule. Similar conclusions were advanced by Petrescu et al. (2000), who studied psychrophilic xylanase from the strain C. adeliensis ATCC 201412. The mature glycosylated enzyme consists of 338 amino acid residues and is 84 % identical to the amino acid sequence of its mesophilic homolog from C. albidus. Similar to the cold-adapted xylanase described above, the enzyme produced by the strain ATCC 201412 is thermolabile with a half life of 60 min at 30 °C, while the activity of its mesophilic homolog is not affected at this temperature. The kinetic adaptation of psychrophilic xylanase has also been confirmed in a study using differential scanning calorimetry (DSC), which revealed a difference between the melting points of these proteins (48 °C and 62 °C for psychro- and mesophilic xylanase, respectively). C. adeliensis and C. albidus xylanases differed in the degree of their activity in hydrolysis of xylan conducted at 5 °C (kcat ~ 14.8 and 4.9 s−1 for psychro- and mesophilic xylanase, respectively) as well as in the activation energy of the catalyzed reaction (Ea = 47.7 and 52.2 kJ mol−1 for C. adeliensis and C. albidus xylanase, respectively). The suggestion that the considerable thermolability of the cold-adapted xylanase results from increased molecule flexibility was partially confirmed by the analysis of a three-dimensional model of this enzyme. The authors observed some subtle differences between the structures of the psychro- and mesophilic proteins, such as a less tightly packed hydrophobic core, a lack of one salt bridge, and destabilization of α-helix macrodipoles in the psychrophilic enzyme. The spatial structure of the xylanase derived from the Antarctic yeast Cryptococcus albidosimilis (C. albidus TAE85) was resolved by a more accurate method, using mass spectometry (Amoresano et al. 2000). This enzyme was shown to be a protein composed of 338 amino acids, N- or O-glycosylated with GalGalNAc only at residue Asn254 in the polypeptide chain. It should be noted that this was the first report concerning the structural characteristics and glycosylation mechanism in a eukaryotic psychrophilic protein.

Furthermore, assay of the enzymatic activity of C. adeliensis grown in submerged culture revealed that this yeast produces endoglucanases, β-arabinofuranosidases, β-xylosidases, and β-glucosidases, which are less active than xylanases, as well as β-mannanases (Gomes et al. 2000). The authors suggest that C. adeliensis, which produces a wide range of hydrolytic enzymes useful for degradation of plant poly- and oligosaccharides, may be applied as a probiotic ingredient or therapeutic agent in feed.

To produce cold-active enzymes on a larger scale, researchers aim at expression of their genes in mesophilic microorganisms, whose culture is technologically simpler. An example here is the gene of a psychrophilic chitinase from the strain Glaciozyma antarctica PI12 with an optimum temperature of 15 °C, which was transferred to a mesophilic host, Komagataella pastoris (formerly Pichia pastoris). The latter gives a high yield of this enzyme in the presence of 1 % methanol at 25 °C (Ramli et al. 2011). This recombinant yeast enzyme was purified to homogeneity using immobilized metal affinity chromatography and characterized with a view to industrial applications. Based on bioinformatic analysis, it was classified as a class II chitinase, glycosyl hydrolase family 18. The regions forming the active site of G. antarctica PI12 chitinase also turned out to be highly conservative revealing strong identity to the catalytic domains of 5 eukaryotic homologous enzymes. The main advantages of this psychrophilic enzyme are its high activity at low temperatures (Topt 15 °C; at 5 °C the enzyme exhibits over 20 % of its maximum activity) and ability to efficiently hydrolyze chitin at acidic pH (pHopt 4.0). Just as the majority of typical cold-adapted enzymes, G. antarctica PI12 chitinase is characterized by low thermal stability (following 30 min incubation at 25 °C it lost 30 % of its initial activity). In turn, its activity rose by up to 20 % in the presence of 1 mM K+, Mn2+, and Co2+ ions. Examination of the substrate preferences of this enzyme revealed that its activity was highest in the hydrolysis of colloidal chitin, as compared to swollen chitin, carboxymethyl chitosan, and glycol chitosan. This was confirmed by kinetic experiments which showed that G. antarctica PI12 chitinase has higher Vmax and kcat values in low-temperature degradation of colloidal chitin than for other substrates. The authors proposed that due to its biochemical and kinetic properties, the enzyme should be an attractive tool for the production of chitooligosaccharides and in other biotechnological applications, such as biocontrol of microbial spoilage of refrigerated foods and mycoparasitic activity against phytopathogenic fungi in cold environments (Ramli et al. 2011, 2012).

Another interesting example is a lysozyme from the cold-adapted yeast Debaryomyces hansenii (muramidase, EC 3.2.1.17) (Wang et al. 2012). It cleaves the β-(1,4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, the major bacterial cell wall polymer, and acts as a nonspecific innate immunity molecule against the invasion of bacterial pathogens.

Culture conditions for this enzyme were optimized using statistical design methods, the Plackett–Burman design (PBD) and the development and validation of a quadratic model. As a result, the maximum lysozyme yield was about 3.5-fold higher than the baseline value that greatly enhances the application potential of this enzyme. In general, cold-adapted lysozymes are successfully used as food preservatives for fish and meat products as well as fruits and vegetables (Wang et al. 2012).

2.2 Pectinolytic Enzymes

Psychrophilic yeasts are a good source of pectinolytic enzymes. Many yeast species produce them, usually several types of pectinases at the same time, often also in the form of isoenzymes. These proteins may be applied in the fruit-processing industry, which would be especially interested in cold-active pectinolytic enzymes for reducing juice viscosity at ambient temperature and for improving the clarity of the final product (Alimardani-Theuil et al. 2011).

All known psychrophilic yeasts with pectinolytic activity (also called PPYs, pectinolytic–psychrophilic yeasts) belong to the genera Cystofilobasidium, Cryptococcus, and Mrakia. In contrast to psychrophilic bacteria, which predominantly synthesize alkaline pectinases, yeasts mostly produce enzymes with optimum activity at acidic pH.

The first yeast strain described in the literature in terms of pectinolytic enzyme production was Cystofilobasidium capitatum PPY-1 (Nakagawa et al. 2002). When grown on a pectin or polygalacturonate substrate as a source of carbon at 5 °C and below, this yeast synthesized a wide range of pectinolytic enzymes: pectin methylesterases (PME), polygalacturonases (PG), and pectin lyases (PNL), but it did not produce pectate lyases (PAL). The activity of these enzymes in culture supernatants, determined for hydrolysis of pectins and polygalacturonate at 20 °C, amounted to 85, 205, and 1.7 U mg−1 protein, respectively. The authors also tested the ability of C. capitatum PPY-1 to degrade pectin substances at 5 °C and obtained activity levels of 267, 101, and 0.01 U mg−1 protein for PME, PG, and PNL, respectively. One of these enzymes, that is, PPY-1 PG, was purified to homogeneity using various chromatographic methods and exhibited an activity of 458 U mg−1 protein under optimum reaction conditions (Topt 45 °C, pHopt 4.4). At 0 °C, its activity was reduced to about 20 % of its maximum activity, which confirms its adaptation to cold (Nakagawa et al. 2005a). The enzyme was found to be stable in the pH range of 2.5–7.0 and up to about 50 °C. It was shown that the enzyme most effectively hydrolyzes polygalacturonate (degree of esterification (DE) 0 %) and pectins moderately esterified with methanol (DE 60 %). Interestingly, based on identification of polygalacturonate hydrolysis products at the initial stages of enzymatic reaction, the authors suggest that PPY-1 PG exhibits mechanisms of action typical of both endo- and exopolygalacturonases. Nakagawa et al. (2005a) emphasize that the special properties of C. capitatum PPY-1 PG make this protein useful for industrial applications and especially for low-temperature pectin degradation in the food industry. PPY-1 PG is quite unique as its N-terminal sequence of 20 amino acids does not show high homology with any sequences of other PG deposited in databases. The highest degree of homology was found for Saccharomyces cerevisiae PG (52 %).

Another C. capitatum PPY-1 enzyme that has been purified and characterized in terms of cold adaptation was pectin lyase (PNL) (Nakagawa et al. 2005b). An efficient purification procedure was developed for this enzyme using traditional chromatographic techniques. As a result, a homogeneous protein was obtained with a high specific activity (1,230 U mg−1 protein). Based on SDS-PAGE, its molecular mass was estimated at 42 kDa and a 25-amino acid N-terminal sequence was determined. PPY-1 PNL exhibits maximum activity in pectin hydrolysis at 40 °C, while in the range of 0–20 °C, it retains about 5–50 % of its maximum activity, similar to its homolog from Penicillium paxilli. Moreover, in pectin hydrolysis, the purified enzyme is characterized by high optimum pH (8.0). Thus, it may be considered a cold-adapted alkaline enzyme. Pectin lyase shows a preference for hydrolysis of highly methoxylated pectins and reduces the viscosity of pectin solutions to that of water after 1.5 h reaction at 5 °C.

Nakagawa et al. (2005b) suggest that both enzymes, that is, C. capitatum PPY-1 PG and PNL, which depolymerize pectin substances (but differ in substrate specificity and optimum pH) may be used in the form of a mixture in the fruit and vegetable industry to reduce juice and fruit/vegetable pulp viscosity, enhance clarification, and aid depectinization.

Other psychrophilic yeast strains with pectinolytic activity were isolated by Birgisson et al. (2003) from frozen soil, plant leaves, and branches in southwestern Iceland. Based on rRNA sequencing, they classified the yeasts as C. capitatum, Cryptococcus macerans (anamorph of Cystofilobasidium macerans), Cystofilobasidium lari-marini (now C. capitatum) and Cryptococcus aquaticus (now Mrakiella aquatica). They showed that the activity of pectinolytic enzymes largely depends on culture conditions, that is, source of carbon, pH, and temperature. Biosynthesis was the most efficient in the presence of pectin as a source of carbon but was repressed on pure glucose. The optimum temperature for the production of polygalacturonases by Cystofilobasidium was 14 °C, while in the case of Cryptococcus, it was by 5 °C lower. The optimum pH of culture medium also varied among the strains: it was 3.2 for C. lari-marini (about 60 U ml−1), 2.6 for C. macerans (about 50 U ml−1), and 3.9 for C. capitatum (about 37 U ml−1). The authors conducted preliminary analysis of the pectinolytic enzymes contained in the culture supernatant and found that polygalacturonases were produced most efficiently by Cystofilobasidium strains, reaching maximum hydrolytic activity at 40 °C and pH 5.0. The same type of enzymes isolated from Cryptococcus was characterized by Topt = 50 °C and pHopt = 4.0. They appear to be cold adapted, as they exhibit relatively high activity in a low-temperature range (0–20 °C): Cystofilobasidium PG retains about 20–40 % of its maximum activity, while its Cryptococcus homologs retain only 10–30 %. Just as most other psychrophilic enzymes, they are characterized by low thermal stability and undergo partial inactivation at temperatures above 30 °C. Birgisson et al. (2003) emphasize marked kinetic adaptations of yeast polygalacturonases to a cold environment and indicate their usefulness for low-temperature processes as well as ease of selective inactivation after completing the process. Another advantage attributed to the yeast strains is their ability to biosynthesize polygalacturonase isoenzymes, which makes it possible to select proteins with optimum biochemical properties for industrial applications.

Evaluation of the enzymatic activity of psychrophilic yeasts in submerged cultures revealed that they can synthesize other pectinolytic enzymes, albeit not as active as polygalacturonases: Cystofilobasidium strains produce pectin lyase, C. aquaticus pectin esterase, and C. macerans strains produce pectin lyase, pectate lyase, and pectin esterase (Birgisson et al. 2003).

Pectinolytic activity was also observed in yeast strains isolated from forest soil in Abashiri (Hokkaido, Japan) by Nakagawa et al. (2004). Of particular, importance was discovery of a new species of psychrophilic yeast with pectinolytic activity, that is, Mrakia frigida. On a substrate with 1 % w/v pectin as a source of carbon, at 5 °C, this strain synthesizes pectin methylesterase and polygalacturonase with a yield of 16.0 and 7.6 mU mg−1 protein, respectively.

Margesin et al. (2005) studied two strains of M. frigida, one of which was isolated from Alpine glacier cryoconite (A15), while the other originated from a sediment sample containing mud, spring water, and moss from the Gyda peninsula in northern Siberia (AG25). They analyzed the biochemical properties of pectate lyases (PL) and found that their production in both isolates depended on medium composition, pH, and amount of oxygen dissolved in the medium. PL synthesis by both strains was the most efficient in a medium-containing yeast extract at neutral pH, while optimum temperatures were 5 and 1 °C for A15 and AG25, respectively. Both enzymes exhibited highest activity at 30 °C and were highly active in the temperature range of 0–20 °C (about 20–70 % of the maximum activity). Above the optimum temperature, the activity of the lyases was significantly repressed (after 15 min of incubation at 30 °C they lost about 40 % of their maximum activity, while at 50 °C, they underwent complete denaturation). The cold adaptation of these enzymes is also shown by lower values of Ea and other thermodynamic constants (ΔH*, ΔG*, ΔS*) as compared to their mesophilic homologs. Another interesting biochemical property of M. frigida PL is their strong alkaline preference (pH 8.5 and 9.0 for A15 and AG25, respectively) and stability (1 h activity in a pH range of 7–10). The enzymes are completely denatured in the presence of EDTA (10 mmol l−1), SDS (1 %), KMnO4 (10 mmol l−1), and l-cysteine (10 mmol l−1); only Ca2+ ions enhance the rate of the reaction they catalyze. According to the authors, the functional advantages of pectate lyases from psychrophilic yeasts include their much greater relative activity at low temperatures as compared to the widely used mesophilic enzymes (by a factor of 4–7) and their alkalinity (Margesin et al. 2005).

2.3 Other Glycosidases

Considerably less research has been devoted to cold-active yeast enzymes hydrolyzing di- and oligosaccharides than to enzymes degrading polysaccharides, even though such glycosidases would also prove valuable for industrial applications, especially for food processing and synthesis of probiotic oligosaccharides (Sheik Asraf and Gunasekaran 2010).

From the technological standpoint, of greatest interest are cold-active β-galactosidases, which might be applied in low-temperature hydrolysis of lactose, which is one of the main components of milk and whey (Loveland et al. 1994; Sheridan and Brenchley 2000). Manufacture of lactose-free milk and milk products is of particular importance for consumers with intolerance of this disaccharide, who often suffer from serious gastrointestinal problems. Cold-active β-galactosidases have also been shown to possess transglycosylation activity whereby lactose hydrolysis takes place with simultaneous transfer of monosaccharides to higher oligosaccharides to form tri- and tetrasaccharides (Karasova-Lipovova et al. 2003; Benesova et al. 2005). Such galacto-oligosaccharides (either produced directly in milk or from whey as an additive in dairy products) can be used as ingredients in probiotic foodstuffs to enhance the growth of bifidobacteria in the large intestine, or as low-calorie sweeteners due to their resistance to metabolism in the small intestine (Huston 2008).

The majority of commercial β-galactosidase preparations contain mesophilic enzymes obtained from yeasts and filamentous fungi. The process of removing lactose from milk or sweet whey using mesophilic β-galactosidases from Kluyveromyces lactis or Kluyveromyces fragilis (now Kluyveromyces marxianus) (Ladero et al. 2000) is conducted for a short time (several hours) at pH 6.0–7.0 and 30–40 °C to prevent infection. Such conditions result in a relatively low degree of lactose degradation. Thus, psychrophilic β-galactosidases would be a perfect alternative to these enzymes, because they exhibit sufficiently high activity at below 20 °C and are not inactivated by sodium or calcium ions or by galactose. The application of such β-galactosidases would reduce the risk of infection with microflora and considerably decrease the energy consumption of the process (Białkowska et al. 2009). Only a few cold-active β-galactosidases have been described to date. They are mostly derived from bacteria of the genera Arthrobacter, Flavobacterium, Pseudoalteromonas, Rahnella, Planococcus, and Carnobacterium (Hoyoux et al. 2001; Turkiewicz et al. 2003a; Wierzbicka-Woś et al. 2011), while two are from yeasts of the genus Guehomyces. The first yeast β-galactosidase was isolated from the psychrophilic strain Guehomyces pullulans R1 by Nakagawa et al. (2006) in selective tests conducted on agar plates containing X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). It was found to be active both in culture supernatants and in biomass. In lactose hydrolysis, the extracellular fraction of the enzyme was characterized by a relatively high optimum temperature for a cold-active enzyme (50 °C), but at 0 °C, it retained as much as 20 % of its maximum activity. Crude enzymatic preparations revealed the highest activity at pH 4.0, with stability in the pH range of 3–7. It is emphasized that the acidic nature of G. pullulans R1 β-galactosidase is its unique feature as most of its known psychrophilic homologs hydrolyze lactose most efficiently under near-alkaline pH conditions. Thus, it is suggested that this yeast enzyme may be used for industrial processes conducted in acidic environments. An example of such a process is manufacture of galacto-oligosaccharide syrups from sour whey, where β-galactosidases from filamentous fungi (active in the pH range of 2.5–4.5) are currently applied. β-Galactosidases operating at acidic pH can also reduce the pollution impact and increase the technical usefulness of whey (a product of the cheese industry) by producing glucose- and galactose-rich syrups that can be used as sweeteners in a variety of foodstuffs and substrates that are easily fermented by alcohol-producing microorganisms (Gerday et al. 2005). G. pullulans β-galactosidase was found useful for low-temperature hydrolysis of lactose in milk as 10 U ml−1 of this enzyme hydrolyzed about 80 % of the disaccharide over 96 h at 10 °C (Nakagawa et al. 2006).

The other yeast β-galactosidase is derived from the psychrotolerant strain G. pullulans 17-1, isolated from sea sediment in Antarctica (Song et al. 2010). The biosynthesis of this enzyme, induced by the presence of lactose in the growth medium, is most efficient at pH 4.5. The optimum temperature and pH of the crude protein preparation for hydrolysis of o-nitrophenyl-β-D-galactopyranoside (ONPG) are 50 °C and 4.0, respectively. According to the authors, the cold adaptation of G. pullulans 17-1 β-galactosidase is not particularly pronounced if one compares its optimum temperature with those of mesophilic β-galactosidases from the yeasts K. lactis ATCC 8583 and K. fragilis (Topt = 37 and 30 °C, respectively). However, a major advantage of this enzyme is its extracellular secretion and high hydrolytic activity (25.3 U ml−1) in culture supernatants on a medium scale (2 L bioreactor). Song et al. (2010) reported that this represents the highest activity (in hydrolysis of a synthetic substrate) among all β-galactosidases produced by wild yeast strains obtained to date, even as compared to the activity of β-galactosidases from mesophilic yeasts: under optimum conditions, K. lactis synthesizes an enzyme characterized by an activity of 0.9 U ml−1, while the enzyme produced by K. fragilis 34440 has an activity of 9.2 U ml−1.

Turkiewicz et al. (2005) showed Leucosporidium antarcticum (now Glaciozyma antarctica; Turchetti et al. 2011) to produce cold-active α-glucosidase and β-fructofuranosidase. The former was found to be active in both culture supernatants and biomass, while the latter was not secreted and was strictly limited to the cell. Unfortunately, following release from the cell, invertase underwent prompt and irreversible inactivation. Although the rapidly progressing inactivation was inhibited in the presence of EDTA (2 mM) and Mn2+ (1 mM), the protective action of these compounds declined with time of incubation, due to which it was impossible to obtain even partially purified enzymatic preparations. The high in vitro sensitivity of G. antarctica to inactivation also led to limited pH stability of the enzyme, which retained maximum activity during 30-min incubation at 4 °C within a very narrow acidic pH range (4.3–4.8). For comparison, α-glucosidase from the same strain, secreted to the growth medium, was stable over the same time within a 3-fold wider neutral pH range (6.0–7.5). These enzymes preferred very different pH of the reaction environment and exhibited different optimum temperatures. Both their low thermal stability and high catalytic activity at 0 °C (over 20 % of the maximum activity for invertase) show that G. antarctica glycosidases are indeed cold-active.

2.4 Proteases

Other cold-adapted yeast hydrolases with a range of applications as wide as that of glycosidases are two exoproteases: an aspartyl protease isolated from the Antarctic strain Cryptococcus humicola (formerly Candida humicola) and a serine protease from the endemic yeast G. antarctica. There are relatively few data on extracellular yeast proteases, even in respect of mesophilic yeasts, as it is thought that these organisms are not able to synthesize significant amounts of these proteins. It seems that this view largely results from the fact that the predominant yeast species both in research and industrial applications is S. cerevisiae. Only a few strains of this yeast exhibit extracellular proteolytic activity and that to a small degree (Sturley and Young 1988).

Psychrophilic proteases have a wide range of industrial and technological applications, e.g., they are used as additives in detergents and foodstuffs, as bioremediators, in biotransformations, and in molecular biology. These enzymes are also used as an alternative to rennet to reduce production costs by accelerating the maturation of slow-ripening cheeses in a low-temperature and low-moisture environment. In seafood processing, cold-active proteases aid in removing scales and skin from fish as well as in extracting carotenoproteins from shellfish. They are also used to impart tenderness to refrigerated meat products and to improve their flavor. Furthermore, proteases active at room temperature may be used as an ingredient in medicinal gels for wound and scar healing and topical treatment of skin infections (Huston 2008).

The discovery of an extracellular protease produced by C. humicola by Ray et al. (1992) confirms the fact that aspartyl (rarely cysteine) enzymes predominate among yeasts inhabiting even very distant biotopes. They are often called acidic proteases as they reveal maximum activity in environments with relatively low-pH levels (3.0–4.5). The Antarctic exoprotease was characterized by an even lower optimum pH in hydrolysis of hemoglobin (pH 1–1.2) and additionally exhibited kinetic adaptations to low temperature: it was active in the temperature range of 0–45 °C with a distinct optimum at 37 °C and retained 12–15 % of its maximum activity at 0 °C. It should be stressed that so far this has been the only aspartyl protease isolated from Antarctic yeasts. The majority of aspartyl protease are derived from mesophilic microorganisms, such as Rhodotorula glutinis (Kamada et al. 1972), Cryptococcus albidus var. aerius (Federici 1982), Candida albicans (Rüchel 1981), Candida parapsilosis (Rüchel et al. 1986), Candida tropicalis (Togni et al. 1991), Candida olea (Nelson and Young 1987), Saccharomycopsis lipolytica (Yamada and Ogrydziak 1983), Aureobasidium pullulans (Chi et al. 2007), Sporidiobolus ruineniae (Kim 2009), Pichia farinosa CO-2 (Kim 2010), Metschnikowia pulcherrima IWBT Y1123, and Candida apicola IWBT Y1384 (Reid et al. 2012).

Another interesting proteolytic enzyme isolated from psychrophilic yeasts is G. antarctica extracellular serine protease (Turkiewicz et al. 2003b). This class of enzymes, degrading protein and synthetic substrates in neutral or alkaline environments, includes only a few extracellular yeast proteases, mostly produced by mesophilic Yarrowia lipolytica (Tobe et al. 1976) and Aureobasidium pullulans (Ogrydziak 1993). Interestingly, G. antarctica is the first psychrophilic subtilase belonging to the proteinase K subfamily (subtilisin family) to be described. An efficient purification procedure was developed for this enzyme using traditional chromatographic methods. The resulting homogeneous protein had a very high specific activity (85 U mg−1 protein). It was shown that the enzyme is stable under conditions of storage and purification without protectants and is not activated by bivalent metal ions. It should be stressed that G. antarctica subtilase exhibits a very high specific activity in hydrolysis of protein substrate (hemoglobin denatured with urea), which is much higher than that of many previously isolated microbial or animal psychrophilic proteases. Similar to most other microbial serine proteases, G. antarctica 171 subtilase has wide substrate specificity, with the greatest affinity to substrates characteristic of chymotrypsin and chymotrypsin-like enzymes. In this respect, it differs significantly from Y. lipolytica AEP, which is of typical trypsin-like nature (Ogrydziak 1993). At 30 °C, the enzyme exhibits an approximately 140-fold higher specificity constant (kcat/Km) for the hydrolysis of BzTyrOEt (N-Benzoyl-l-tyrosine ethyl ester) than for the hydrolysis of BzArgOEt (N-Benzoyl-l-arginine ethyl ester) (37.4 and 0.27 s−1 mM−1, respectively). Furthermore, G. antarctica 171 subtilase displays a significant shift in optimum temperatures toward the low range (25 °C), which is by more than 35 °C lower than the optimum temperature of the best characterized mesophilic subtilisin Carlsberg (Topt = 65 °C; Kulakova et al. 1999). Another unique property of G. antarctica 171 subtilase is its high activity at 0 °C (over 20 % of its maximum activity). Interestingly, it remains active at temperatures as low as −10 °C. The enzyme displays high amidase activity (pHopt in the range of 8.0–8.5) against N-SucAAPFpNA (N-Succinyl-AAPF-p-nitroanilide) and N-SucAAPLpNA (N-Succinyl-AAPL-p-nitroanilide). Similar to most other naturally occurring psychrophilic enzymes, this protease is characterized by high sensitivity to the denaturing action of temperature (half life at 30 °C is 2.5 h). Adaptation to low temperatures is attributed by the authors to the presence of additional loops in the molecule, e.g., a 5-amino acid loop near the N-terminus. Although the gene encoding this 35.5-kDa protein was not isolated, the location of a total of 108 amino acid residues was identified and the N-terminal sequence was determined (58 amino acids; the sequence of 38 was determined by the Edman method, and another 23 by proteomic analysis). Based on the sequences of homologous proteases, the sequences of four peptides defined in proteomic analysis of trypsin-digested G. antarctica subtilase were aligned (Turkiewicz, unpublished results), among which are sequences of the catalytic triad of the enzyme Ser224…His69…Asp45 (the two first residues were labeled according to residue numbering in proteinase K), which are highly conservative.

Recently, Zaliha et al. (2012) patented a bifunctional enzyme derived from the strain L. antarcticum (now G. antarctica) PI12, which exhibited both lipolytic and proteolytic activity (see Chap.16).

2.5 Phytases and Other Esterases

Of industrial importance is the cold-active phytase isolated from the Antarctic yeast Cryptococcus laurentii AL27 (myo-inositol hexakisphosphate phosphohydrolase, EC 3.1.3.8) (Pavlova et al. 2008). It is a phosphatase that catalyzes phytate hydrolysis to phosphorylated myo-inositol derivatives and inorganic phosphate and the first known yeast-derived Antarctic phytase. This enzyme is used as a dietary ingredient that plays a significant role in feeding monogastric animals. Its main task is to reduce inorganic phosphorus in animal fodder to prevent excessive concentrations of this element in animal waste. Furthermore, this phosphatase increases assimilation of some dietary elements (Ca, Cu, Zn, Mn, and Fe), proteins, and amino acids. Over the past several years, special attention has been given to the products of phytase activity, that is, myo-inositol products with varying degrees of phosphorylation, due to the potential application in the pharmaceutical industry (Pavlova et al. 2008).

The strain C. laurentii AL27 was chosen as a result of a 2-step selection procedure as the best phytase producer among 124 strains of Antarctic yeasts. The first step involved strain cultivation on calcium- and phytate-containing solid medium, and the second step consisted of culturing the isolates selected in the first step in a liquid medium containing glucose as a source of carbon (20 g l−1). C. laurentii AL27, grown at 24 °C, was characterized by the highest intracellular phytase activity (23.8 U g−1 biomass) on a medium containing sucrose (40 g l−1) and KH2PO4 (at a concentration supplying phosphorus at 5 mg l−1). Pavlova et al. (2008) indicate that Antarctic phytase plays a useful role by efficiently degrading phytates directly in the stomachs of monogastric animals (37–40 °C, pH 2–3). The major advantages of the C. laurentii enzyme include its relatively low optimum temperature (40 °C as compared to 45–60 °C for most other phytases), an optimum pH of 4.8, and stability over a wide pH range (2.0–5.0; at pH 2.0 the protein preserves about 50 % of its maximum activity).

An interesting cold-active yeast enzyme was described by Zimmer et al. (2006), who isolated an esterase from Rhodotorula mucilaginosa. The yeast was grown on (2R,3R)-(−)-di-O-benzoyl-tartrate, which it stoichiometrically hydrolyzed to monoesters. The monoesters were subsequently broken into benzoates and tartrates and metabolized. The enzyme was obtained from the culture supernatant, purified, and determined to be a monomeric glycosylated 86 kDa benzoyl esterase. Its optimum pH was 7.5, and the optimum temperature was 45 °C. The psychrophilic properties of this esterase were proven by its high activity at 0 °C, which amounted to 20 % of its activity at 30 °C. This shows that the enzyme may be used in low-temperature settings, e.g., in manufacturing volatile aromas and fragrances. R. mucilaginosa esterase hydrolyzed short-chain p-nitrophenyl-alkyl esters as well as some benzoyl esters, including benzoyl-methyl ester, phenyl-benzoyl ester, ethylene–glycol-dibenzoyl ester, 1,5-anhydro-d-fructose-tribenzoyl ester, and cocaine. The enzyme is potentially useful for hydrolysis of 1,5-anhydro-d-fructose-tribenzoyl ester, which could be used as a reagent for specific insertion or release of protecting groups in sugars (benzoylated carbohydrates are precursors in the synthesis of, e.g., beta-l-nucleoside analogs with antiviral activity). Thus, R. mucilaginosa esterase seems to be a useful low-temperature reagent in the production of benzoylated compounds for the pharmaceutical, cosmetic, and fine chemical industries (Zimmer et al. 2006).

Another cold-active esterase is produced by Y. lipolytica CL180 (rYli180). It preferentially hydrolyzes the S-enantiomer of racemic ofloxacin ester (Kim et al. 2007). The gene of this protein was cloned into Escherichia coli, and the product of its expression, with a mass of 53 kDa, was purified to homogeneity by means of immobilized metal affinity chromatography. Based on the determination of its biochemical properties, it was shown that this esterase preferentially hydrolyzes p-nitrophenyl esters of fatty acids with short-chain lengths (≤10 carbon atoms) and is a cold-active protein (Topt = 35 °C; 40–60 % of maximum activity in the range of 0–20 °C). According to the authors, these features make it a very attractive enzyme for potential application as a biocatalyst, especially in the manufacture of thermolabile chemicals.

Sabri et al. (2001) described an acyl-CoA thioesterase produced by the psychrophilic Antarctic yeast Rhodotorula aurantiaca. Its application potential is not as yet known, albeit it plays an important role in the metabolism of myristoyl-CoA, and, indirectly, also in the regulation of yeast growth at different temperatures.

3 Conclusions and Future Prospects

Literature data show cold-active enzymes to have several advantages over their mesophilic and thermophilic homologs. Despite some difficulties linked to their relatively high cost, low activity and stability under typical process conditions, and the low degree of biodiversity of the psychrophilic microbes known to date, cold-active enzymes may successfully replace traditional ones in a range of industrial applications. Some relatively new recombinant DNA technologies, including site-directed mutagenesis and metatranscriptomics, enable the production of an increasing number of recombinant enzymes. Thanks to this, it is possible to obtain cheaper cold-tolerant enzymes with novel or modified activity. It seems advisable to pursue commercially viable production of such enzymes in heterologous hosts and their modification by chemical or protein engineering methods with a view to obtaining higher activity levels and greater robustness. Some strains could be genetically improved to make them more suitable for the expression of cold-adapted enzymes, which would have significant positive implications for their use in various fields of biotechnology and industry.

While research into useful cold-active yeast enzymes is largely limited to hydrolases, it seems that other classes of enzymes could be equally relevant for industrial applications. For instance, oxidoreductases are playing an increasingly important role in biotransformations conducted in organic solvents (microaqueous systems) (Straathof et al. 2002; Burton 2003; Solano et al. 2012). Enzymes that could be particularly suited to such applications include those produced by yeasts from cold biotopes with low water activity, such as, e.g., yeasts inhabiting some Antarctic environments. The only cold-active oxidoreductase from psychrophilic yeasts that has been discovered to date is D-galacturonate reductase from Cryptococcus diffluens (Hamada et al. 2011).