New tyrosinases with putative action against contaminants of emerging concern.

Tyrosinases (EC 1.14.18.1) are type-3 copper metalloenzymes with strong oxidative capacities and low allosteric selectivity to phenolic and non-phenolic aromatic compounds, which have been used as biosensors and biocatalysts to mitigate the impacts of environmental contaminants over aquatic ecosystems. However, the widespread use of these polyphenol oxidases is limited by elevated production costs and restricted knowledge on their spectrum of action. Here, six tyrosinase homologs were identified and characterized from the genomes of four widespread freshwater ciliates using bioinformatics. Next, we performed a virtual screening to calculate binding energies between 3D models of these homologs and ~ 1000 contaminants of emerging concern (CECs), as an indirect approach to identify likely and unlikely targets for tyrosinases. Many fine chemicals, pharmaceuticals, personal care products, illicit drugs, natural toxins, and pesticides exhibited strong binding energies to these new tyrosinases, suggesting the spectrum of targets of these enzymes might be considerably broader than previously thought. Many ciliates, including those carrying tyrosinase genes, are fast-growing unicellular microeukaryotes that can be efficiently cultured, at large scales, under in vitro conditions, suggesting these organisms should be regarded as potential low-cost sources of new environmental biotechnological molecules.

Specific inhibitors of lysozyme and peptidases inhibit the growth of the rumen protozoan Entodinium caudatum without decreasing feed digestion or fermentation in vitro.

AIMS: Experiments were designed to determine the effects of different chemical inhibitors of lysozyme and peptidases on rumen protozoa and the associated prokaryotes, and in vitro fermentation using Entodinium caudatum as a model protozoan species. METHODS AND RESULTS: Imidazole (a lysozyme inhibitor), phenylmethylsulphonyl fluoride (PMSF, a serine peptidase inhibitor) and iodoacetamide (IOD, a cysteine peptidase inhibitor) were evaluated in vitro both individually and in two- and three-way combinations using E. caudatum monocultures with respect to their ability to inhibit the protozoan and their effect on feed digestion, fermentation and the microbiota. All the three inhibitors, both individually and in combination, decreased E. caudatum counts (P < 0·001), and IOD and its combinations with the other inhibitors significantly (P < 0·01) decreased ammonia concentration, with the two- and three-way combinations showing additive effective. Feed digestion was not affected, but fermentation and microbial diversity were affected mostly by PMSF, IOD and their combinatorial treatments potentially due to the overgrowth of Streptococcus luteciae accompanying with the disappearance of host ciliates. CONCLUSIONS: Entodinium caudatum depends on lysozyme and peptidase for digestion and utilization of the engulfed microbes and specific inhibition of these enzymes can inhibition E. caudatum without adversely affecting feed digestion or fermentation even though they changed the microbiota composition in the cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: The peptidase inhibitors may have the potential to be used in controlling rumen protozoa to improve ruminal nitrogen utilization efficiency.

Short communication: Gelatinization and enzymatic hydrolysis characteristics relevant to digestion and analysis of glycogen granules isolated from ruminal protozoa.

Glycogen is an α-glucan produced by rumen microbes from various feed carbohydrates. It may be digested ruminally or intestinally to provide nutrients. The physicochemical and enzymatic hydrolysis characteristics of microbial glycogen have not been described in detail, but do influence its conversion to absorbable nutrients in vivo, its nutritional comparability with plant starch sources, and its accurate analysis in vitro. The objectives of this study were to determine presence or absence of a gelatinization response and to describe enzymatic digestion characteristics of glycogen granules isolated from ruminal protozoa obtained from lactating dairy cows. Protozoal glycogen granules were determined to be 98.3% α-glucan. Granules displayed gelatinization, the breaking of hydrogen bonds between molecules or branches, at 65°C compared with purified wheat and corn starches, which initiated gelatinization at 50 and 65°C, respectively. Digestion of ungelatinized samples with amyloglucosidase for 2 h at 39°C showed approximately 3-fold greater hydrolysis to glucose for protozoal glycogen (25.2% of dry matter; DM) than for wheat (9.9% of DM) or corn (8.2% of DM) starches. Based on enzymatic digestion results, protozoal glycogen may be more readily digested than intact corn or wheat starches and should be gelatinized or the hydrogen bonds otherwise disrupted to allow more complete recovery in enzymatic analysis.

The utilization and digestion of cellulose by the rumen ciliate Diploplastron affine.

Rumen protozoa are known to contribute to fibre digestion, but the fibrolytic enzymes of the majority of ciliate species have been poorly recognised to date. The aims of the study were, first, to determine the influence of crystalline cellulose on the survival and population density of the ciliate Diploplastron affine when cultured in vitro, and second to identify and characterise the protozoal enzymes catalysing the hydrolysis of cellulose. It was found that crystalline cellulose, when added to a culture medium, increased the number of protozoa maintained in vitro. We observed that the bacteria-free ciliates fermented microcrystalline cellulose and produced 43.3 nmol volatile fatty acids/protozoon/h. A cell extract prepared from the bacteria-free ciliates degraded crystalline cellulose in the rate of 11.5 nmol released glucose/mg protein/min, whereas the degradation rates of carboxymethyl-cellulose (CMC), avicel and cellobiose were 343, 6.8 and 145 nmol released glucose/mg protein/min respectively. Two distinct peaks in the activity of relevant enzymes were identified following ion exchange chromatography of the protozoal cell extract and the presence of two different CMC-ases were confirmed by zymographic studies. CMC was mainly degraded to mono- and disaccharides but that some other oligosaccharides were also present. Cellobiose was the only product of avicel digestion.

The Sanguicolous Apostome Metacollinia luciensis Jankowski 1980 (Colliniidae, Apostomatia, Ciliophora) Is Not Closely Related to Other Sanguicolous Apostomes.

The apostome family Colliniidae includes species that are adapted to the hemocoel/blood of various invertebrates, particularly crustaceans. To explore the phylogeny of these sanguicolous apostomes, Metacollinia luciensis was collected in August 2015 at Roscoff from the amphipod host, Orchestia gammarellus. Ciliates were Protargol stained and DNA was extracted. The small subunit rRNA (SSUrRNA) and cytochrome c oxidase subunit I (cox1) genes were amplified. Molecular phylogenetic analyses of the SSUrRNA genes unambiguously grouped M. luciensis with other apostomes with robust bootstrap support, but separated it distinctly from the pseudocolliniid clade. While there are only cox1 sequences for a subset of these apostomes, M. luciensis was also distant from the pseudocolliniids and separated from them by species of the exuviotrophic apostome Hyalophysa. These results confirm the distinctness of the families Colliniidae and Pseudocolliniidae.

Antarctic marine ciliates under stress: superoxide dismutases from the psychrophilic Euplotes focardii are cold-active yet heat tolerant enzymes.

Oxidative stress is a particularly severe threat to Antarctic marine polar organisms because they are exposed to high dissolved oxygen and to intense UV radiation. This paper reports the features of three superoxide dismutases from the Antarctic psychrophilic ciliate Euplotes focardii that faces two environmental challenges, oxidative stress and low temperature. Two out of these are Cu,Zn superoxide dismutases (named Ef-SOD1a and Ef-SOD1b) and one belongs to the Mn-containing group (Ef-SOD2). Ef-SOD1s and Ef-SOD2 differ in their evolutionary history, expression and overall structural features. Ef-SOD1 genes are expressed at different levels, with Ef-SOD1b mRNA 20-fold higher at the ciliate optimal temperature of growth (4 °C). All Ef-SOD enzymes are active at 4 °C, consistent with the definition of cold-adapted enzymes. At the same time, they display temperatures of melting in the range 50-70 °C and retain residual activity after incubation at 65-75 °C. Supported by data of molecular dynamics simulation, we conclude that the E. focardii SODs combine cold activity, local molecular flexibility and thermo tolerance.

Inhibitory effect of melittin on endonuclease-like activity of centrin.

The xeroderma pigmentosum group C protein (XPC) and centrin2 are the primary initiators of global genome nucleotide excision repair (NER). Centrin, acts as a member of the EF-hand super family of calcium-binding proteins, playing roles in reconstitution of the vitro NER reaction. To understand the possible molecular and structural properties of the multiprotein process, the interactions of Euplotes octocarinatus centrin (EoCen), melittin, and DNA are described. EoCen shares a sequence identity of 66% with centrin2. Melittin possesses inverse direction hydrophobic triads-leucine-leucine-tryptophan (LLW) which are responsible for centrin binding. It is applied as a natural peptide to mimic centrin target peptide. As a result, it is proved that the integrated protein shows an endonuclease-like activity to DNA. Melittin is capable of interaction with both EoCen and DNA. More importantly, it is found that melittin displays an inhibitory effect on the endonuclease-like activity of centrin when it co-exists with EoCen and DNA in solution. Meanwhile, the DNA-melittin-EoCen ternary complex forms in the process. Quantitative analyses demonstrated by extensive biophysical assays reveal that binding of the peptide to DNA or centrin modulates the binding properties of it to another component. Furthermore, a possible positioning model of DNA and EoCen on melittin is proposed. This finding may constitute a model for that existing between centrin and its target peptide in NER process.

A single amino acid residue regulates the substrate affinity and specificity of indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase (IDO) is a heme-containing enzyme that catalyses the oxidative cleavage of L-Trp. The ciliate Blepharisma stoltei has four IDO genes (IDO-I, -II, -III and -IV), which seem to have evolved via two sequential gene duplication events. Each IDO enzyme has a distinct enzymatic property, where IDO-III has a high affinity for L-Trp, whereas the affinity of the other three isoforms for L-Trp is low. IDO-I also exhibits a significant catalytic activity with another indole compound: 5-hydroxy-l-tryptophan (5-HTP). IDO-I is considered to be an enzyme that is involved in the biosynthesis of the 5-HTP-derived mating pheromone, gamone 2. By analysing a series of chimeric enzymes based on extant and predicted ancestral enzymes, we identified Asn131 in IDO-I and Glu132 in IDO-III as the key residues responsible for their high affinity for each specific substrate. These two residues were aligned in an identical position as the substrate-determining residue (SDR). Thus, the substrate affinity and specificity are regulated mostly by a single amino acid residue in the Blepharisma IDO-I and IDO-III enzymes.

Novel Specificity of IDO Enzyme Involved in the Biosynthesis of Mating Pheromone in the Ciliate Blepharisma stoltei.

Mating pheromones (gamone 1 and gamone 2) in the ciliate Blepharisma are biologically active substances that trigger sexual reproduction (conjugation) under starvation conditions. Gamone 1 is a glycoprotein secreted by type I cells, and gamone 2 is a tryptophan (Trp)-derivative compound secreted by type II cells. Both gamones stimulate complementary mating type cells to promote each gamone production and induce pair formation. To elucidate the biosynthetic pathway of gamone 2, we investigated the enzymes involved in the pathway and the specificity of the enzymes. An RNA-seq analysis revealed that Blepharisma stoltei (Heterotrichea) possesses four indoleamine 2,3-dioxygenase (IDO) genes showing distinct expression patterns. Along with results from real-time PCR, these findings demonstrated that each IDO gene has different expression patterns that depend on the cellular conditions. Expression of IDO-I was correlated with the intensity of gamone 2 expression, and the recombinant IDO-I protein showed catalytic activity for 5-hydroxy-L-Trp (5-HTP) but very weak activity for L-Trp. Our results indicate that IDO-I is an enzyme evolutionary specialized to gamone 2 production in Blepharisma, and that the biosynthetic pathway for gamone 2 uses 5-HTP as an intermediate.

Phylogenomic analysis of integral diiron membrane histidine motif-containing enzymes in ciliates provides insights into their function and evolutionary relationships.

The Integral Membrane Histidine Motif-containing Enzymes (IMHME) are a class of binuclear non-heme iron proteins widely distributed among prokaryotes and eukaryotes. They are characterized by a conserved tripartite motif consisting of eight to ten histidine residues. Their known function is the activation of the dioxygen moiety to serve as efficient catalysts for reactions of hydroxylation, desaturation or reduction. To date most studies on IMHME were carried out in metazoan, phototrophic or parasitic organisms, whereas genome-wide analysis in heterotrophic free living protozoa, such as the Ciliophora phylum, has not been undertaken. In the seven fully sequenced genomes available we retrieved 118 putative sequences of the IMHME type, albeit with large differences in number among the ciliates: 11 sequences in Euplotes octocarinatus, 7 in Ichthyophthirius multifiliis, 13 in Oxytricha trifallax, 18 in Stylonychia lemnae, 25 in Tetrahymena thermophila, 31 in Paramecium tetraurelia and 13 in Pseudocohnilembus persalinus. The pool of putative sequences was classified in 16 orthologous groups from which 11 were related to fatty acid desaturase (FAD) and 5 to the fatty acid hydroxylase (FAH) superfamilies. Noteworthy, a large diversity on the number and type of FAD / FAH proteins were found among the ciliates, a feature that, in principle, may be attributed to peculiarities of the evolutionary process, such as gene expansion and reduction, but also to horizontal gene transfer, as we demonstrate in this work. We identified twelve putative enzymatic activities, from which four were newly assigned activities: sphingolipid Δ4-desaturase, ω3/Δ15 fatty acid desaturase, a large group of alkane 1-monooxygenases, and acylamide-delta-3(E)-desaturase, although unequivocal allocation would require additional experiments. We also combined the phylogenetics analysis with lipids analysis, thereby allowing the detection of two enzymatic activities not previously reported: a C-5 sterol desaturase in P. tetraurelia and a delta-9 fatty acid desaturase in Cohnilembus reniformis. The analysis revealed a significant lower number of FAD's sequences in the spirotrichea ciliates than in the oligohymenophorea, emphasizing the importance of fatty acids trophic transfer among aquatic organisms as a source of variation in metabolic activity, individual and population growth rates, and reproduction.

Evolutionary history of the chitin synthases of eukaryotes.

Chitin synthases are widespread among eukaryotes and known to have a complex evolutionary history in some of the groups. We have reconstructed the chitin synthase phylogeny using the most taxonomically comprehensive dataset currently available and have shown the presence of independently formed paralogous groups in oomycetes, ciliates, fungi, and all diatoms except raphid pennates. There were also two cases of horizontal gene transfer (HGT): transfer from fungus to early diatoms gave rise to diatom paralogous group, while transfer from raphid pennate diatom to Acantamoeba ancestor is, to our knowledge, restricted to a single gene in amoeba. Early evolution of chitin synthases is heavily obscured by paralogy, and further sequencing effort is necessary.

Enzymes Involved in Pyrophosphate and Calcium Metabolism as Targets for Anti-scuticociliate Chemotherapy.

Inorganic pyrophosphate (PPi) is a key metabolite in cellular bioenergetics under chronic stress conditions in prokaryotes, protists and plants. Inorganic pyrophosphatases (PPases) are essential enzymes controlling the cellular concentration of PPi and mediating intracellular pH and Ca(2+) homeostasis. We report the effects of the antimalarial drugs chloroquine (CQ) and artemisinin (ART) on the in vitro growth of Philasterides dicentrarchi, a scuticociliate parasite of turbot; we also evaluated the action of these drugs on soluble (sPPases) and vacuolar H+-PPases (H+-PPases). CQ and ART inhibited the in vitro growth of ciliates with IC50 values of respectively 74 ± 9 µM and 80 ± 8 µM. CQ inhibits the H+ translocation (with an IC50 of 13.4 ± 0.2 µM), while ART increased translocation of H+ and acidification. However, both drugs caused a decrease in gene expression of H+-PPases. CQ significantly inhibited the enzymatic activity of sPPases, decreasing the consumption of intracellular PPi. ART inhibited intracellular accumulation of Ca(2+) induced by ATP, indicating an effect on the Ca(2+) -ATPase. The results suggest that CQ and ART deregulate enzymes associated with PPi and Ca(2+) metabolism, altering the intracellular pH homeostasis vital for parasite survival and providing a target for the development of new drugs against scuticociliatosis.

Extracellular proteases are released by ciliates in defined seawater microcosms.

The biodegradation of proteins in seawater requires various proteases which are commonly thought to be mainly derived from heterotrophic bacteria. We, however, found that protists showed a high protease activity and continuously produced trypsin-type enzymes. The free-living marine heterotrophic ciliate Paranophrys marina together with an associated bacterium was isolated and used for microcosm incubation with different concentrations of killed bacteria as food for 10 days. The results showed that the co-existence of the ciliate with its associated bacterium produced a significant protease activity in both cell-associated and cell-free fractions while that in the associated bacterium only microcosm was negligible. The protease profiles are different between cell-associated and cell-free fractions, and a trypsin-type enzyme hydrolyzing Boc-Val-Leu-Lys-MCA was detected throughout the period in the presence of ciliates. This suggests that ciliates release proteases into the surrounding environment which could play a role in protein digestion outside cells. It has been previously suggested that bacteria are the major transformers in seawater. We here present additional data which indicates that protists, or at least ciliates with their specific enzymes, are a potential player in organic matter degradation in water columns.

Expression and characterization of cathepsin L-like cysteine protease from Philasterides dicentrarchi.

Philasterides dicentrarchi is a causative agent of scuticociliatosis in olive flounder Paralichthys olivaceus, aquaculture in Korea. In this study, a cDNA encoding a cathepsin L-like cysteine protease (PdCtL) of P. dicentrarchi (synonym Miamiensis avidus) was identified. To express the PdCtL recombinant protein in a heterologous system, 10 codons were redesigned to conform to the standard eukaryotic genetic code using polymerase chain reaction (PCR)-based site-directed mutagenesis. The recombinant P. dicentrarchi procathepsin L (proPdCtL) was expressed at high levels in E. coli Rosetta (DE3) pLysS with a pPET21a vector, and successfully refolded, purified, and activated into a functional and enzymatically active form. The optimal pH for protease activity was 5. Similar to other cysteine proteases, enzyme activity was inhibited by E64 and leupeptin. Immunogenicity of recombinant PdCtL was assessed by enzyme-linked immunosorbent assay, western blot, and specific anti-recombinant PdCtL antibodies were detected. Our results suggest that the biochemical characteristics of the recombinant ciliate proPdCtL protein are similar to those of the cathepsin L-like cysteine protease, that the PCR-based site-direct mutated ciliate gene was successfully expressed in a biochemically active form, and that the recombinant PdCtL acted as a specific epitope in olive flounder.

Ciliate Nassula sp. grazing on a microcystin-producing cyanobacterium (Planktothrix agardhii): impact on cell growth and in the microcystin fractions.

The proliferation of microcystins (MCs)-producing cyanobacteria (MCs) can have detrimental effects on the food chain in aquatic environments. Until recently, few studies had focused on the fate of MCs in exposed organisms, such as primary consumers of cyanobacteria. In this study, we investigate the impact of an MC-producing strain of the cyanobacterium Planktothrix agardhii on the growth and physiology of a Nassula sp. ciliate isolated from a non-toxic cyanobacterial bloom. We show that this Nassula sp. strain was able to consume and grow while feeding exclusively on an MC-producing cyanobacterium over a prolonged period of time (8 months). In short-term exposure experiments (8 days), ciliates consuming an MC-producing cyanobacterial strain displayed slower growth rate and higher levels of antioxidant enzymes than ciliates feeding on two non-MC-producing strains. Three high-performance methods (LC/MS, LC/MS-MS and ELISA) were used to quantify the free and bound MCs in the culture medium and in the cells. We show that ciliate grazing led to a marked decrease in free MCs (methanol extractable) in cells, the MCs were therefore no longer found in the surrounding culture medium. These findings suggest that MCs may have undergone redistribution (free vs bound MCs) or chemical degradation within the ciliates.

Biogenesis of telomerase ribonucleoproteins.

Telomerase adds simple-sequence repeats to the ends of linear chromosomes to counteract the loss of end sequence inherent in conventional DNA replication. Catalytic activity for repeat synthesis results from the cooperation of the telomerase reverse transcriptase protein (TERT) and the template-containing telomerase RNA (TER). TERs vary widely in sequence and structure but share a set of motifs required for TERT binding and catalytic activity. Species-specific TER motifs play essential roles in RNP biogenesis, stability, trafficking, and regulation. Remarkably, the biogenesis pathways that generate mature TER differ across eukaryotes. Furthermore, the cellular processes that direct the assembly of a biologically functional telomerase holoenzyme and its engagement with telomeres are evolutionarily varied and regulated. This review highlights the diversity of strategies for telomerase RNP biogenesis, RNP assembly, and telomere recruitment among ciliates, yeasts, and vertebrates and suggests common themes in these pathways and their regulation.

[The study of molecular mechanisms of action of natural amino acids and serotonin on adenylyl and guanylyl cyclases of the ciliates].

It has been previously shown that some amino acids and their derivatives are capable of regulating the activity of adenylyl cyclase (AC) and guanylate cyclase (GC) in free-living ciliates Dileptus anser and Tetrahymena. The aim of this work was to study the molecular mechanisms of action of methionine, tyrosine, alanine and neurohormone serotonin on the activity of enzymes-cyclases and the identification of their specific receptors in D. anser and T. pyriformis. Methionine and serotonin significantly increased the basal AC activity in both ciliates, and the AC effect of serotonin in T. pyriformis was carried out with the participation of Ca2+-dependent form of AC and heterotrimetic G proteins. AC stimulating effect of tyrosine and alanine was expressed weakly and only detected in D. anser. Serotonin is both ciliates and alanine in D. anser stimulated GC activity, whereas methionine and tyrosine had no effect on GC. Methionine and serotonin bind to surface receptors of the ciliates with high affinity. K(D) for [methyl-3H] methionine binding to D. anser and T. pyriformis were 7.5 and 35.6 nM, and for [3H] serotonin binding were 2.7 and 4.7 nM, respectively. Alanine and tyrosine bind to the ciliates with low affinity. Thus, ciliates D. anser and T. pyriformis have chemosignaling systems regulated by amino acids and their derivatives and including the enzymes with cyclase activity. There is an assumption that these systems are similar to hormonal signaling systems of higher eukaryotes and are their predecessors.

EF-1α and mitochondrial ATP synthase ß chain: alteration of their expression in encystment-induced Colpoda cucullus.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the total proteins contained in encystment-induced Colpoda cucullus showed that a 50-kDa protein (p50) disappeared, whereas the expression of a 49-kDa protein (p49) was enhanced in early phase of morphogenetic transformation into the resting cyst (i.e. 2-5 h after the onset of encystment induction). Puromycin or actinomycin D inhibited the alteration in the expression of p50 and p49 by the induction of encystment. These results suggest that the encystment-specific alteration in expression of these proteins is performed by a transcriptional regulation. Liquid chromatography tandem mass spectrometry analysis revealed that p50 is mitochondrial ATP synthase ß chains, and that p49 is elongation factor 1α.

Natural strategies for the spatial optimization of metabolism in synthetic biology.

Metabolism is a highly interconnected web of chemical reactions that power life. Though the stoichiometry of metabolism is well understood, the multidimensional aspects of metabolic regulation in time and space remain difficult to define, model and engineer. Complex metabolic conversions can be performed by multiple species working cooperatively and exchanging metabolites via structured networks of organisms and resources. Within cells, metabolism is spatially regulated via sequestration in subcellular compartments and through the assembly of multienzyme complexes. Metabolic engineering and synthetic biology have had success in engineering metabolism in the first and second dimensions, designing linear metabolic pathways and channeling metabolic flux. More recently, engineering of the third dimension has improved output of engineered pathways through isolation and organization of multicell and multienzyme complexes. This review highlights natural and synthetic examples of three-dimensional metabolism both inter- and intracellularly, offering tools and perspectives for biological design.

Ability of rumen protozoa Diploplastron affine to utilize ß-glucans.

The ability of the rumen ciliates to utilize ß-glucans other than cellulose and xylan is currently being recognized. The objective of the present study was to characterize the ability of the ciliate Diploplastron affine to digest some pachyman, laminarin, pustulan, curdlan and lichean. The protozoa were isolated from the rumen of sheep and either grown in vitro or inoculated into the rumen of ciliate-free sheep and maintained in natural conditions. In vitro culture studies showed that the enrichment of culture medium with the examined saccharides results in an increase in the number of ciliates in comparison to the control cultures. The increase was over 36 and 15 % when the growth medium was supplemented with pachyman (1,3-ß-glucan) and pustulan (1,6-ß-glucan), respectively. A positive correlation was also found between the population density of ciliates and the dose of saccharide supplemented to the growth medium. Enzyme studies were performed using the crude enzyme preparation obtained from ciliates treated with antibiotics. The ability of ciliates to digest the examined ß-glucans was tested by the quantification of reducing sugars released from the mentioned substrates during the incubation with crude enzyme preparation. The results showed that D. affine ciliates were able to digest both of them. The mean degradation rate varied between 6.7 and 28.2 µmol/L glucose per mg protein per h for pustulan and lichean, respectively, whereas the digestion velocity was the highest at 5.0-5.5 pH and 45-50°C.