Mutanase Activity Research Articles

Mutanase Enzyme from Paracoccus mutanolyticus RSP02: Characterization and Application as a Biocontrol Agent.

Mutanases are enzymes that have the ability to cleave α-1,3 linkages in glucan polymer. In the present investigation, mutanase enzyme purified from the culture filtrate of Paracoccus mutanolyticus was evaluated for Streptococcal biofilm degradation and antimicrobial activity against pathogenic fungi along with enzyme kinetics, activation energies, pH and thermal stability. Biochemical and molecular characterization depicted that the enzyme showed optimum activity at pH 5.5 and at 50°C. It displayed Michaelis-Menten behaviour with a Km of 1.263 ± 0.03 (mg/ml), Vmax of 2.712 ± 0.15 U/mg protein. Thermal stability studies denoted that it required 55.46 and 135.43kJmol-1 of energy for activation and deactivation in the temperature range of 30-50°C and 50-70°C respectively. Mutanase activity was enhanced ~ 50 and 75% by Fe2+ and EDTA, respectively, while presence of Hg2+ and Mn2+ inhibit > 90% of its activity. This enzyme has a molecular mass of 138kDa and showed monomeric nature by Zymography. Scanning electron microscopy analysis of mutanase treated Streptococcal cells revealed cleavage of linkages among the cells and complete separation of cells, indicating its potential in dentistry as an anticaries agent in the prophylaxis and therapy of dental caries. In addition, antifungal activity of mutanase against Colletotrichum capsici MTCC 10147 and Cladosporium cladosporioide MTCC 7371 revealed that the enzyme has potential towards biological control of phytopathogens which could be used as an alternative bio-control agent against chemical pesticides in the future.

(1→3)-α-d-Glucan from Fruiting Body and Mycelium ofCerrena unicolor(Bull.) Murrill: Structural Characterization and Use as a Novel Inducer of Mutanase

Water-insoluble, alkali-soluble polysaccharide (marked as ASP) was extracted from the vegetative mycelium and fruiting body ofCerrena unicolorstrain. Monosaccharide examination of ASP demonstrated that the isolated biopolymer was composed mainly of glucose, xylose, and mannose monomers. The methylation investigation of studied polymers indicated that (1→3)-linkedα-D-Glcpis the major chain constituent (92.2% for glucans isolated from fruiting body and 90.1% from mycelium).1H NMR, FT-IR, and immunofluorescent labelling determinations confirmed that the polysaccharides isolated from both fruiting body and mycelium ofC. unicolorare (1→3)-α-d-glucans. The obtained (1→3)-α-d-glucans showed differences in viscosity and similar characteristics in optical rotations. (1→3)-α-d-Glucans extracted from mycelium and fruiting body ofC. unicolorwere also used as potential and specific inducers of mutanase synthesis byTrichoderma harzianum. The highest mutanase activity (0.38 U/mL) was obtained after induction of enzyme by (1→3)-α-d-glucan isolated from the mycelium ofC. unicolor, and this biopolymer has been suggested as a new alternative to streptococcal mutan for the mutanase induction inT. harzianum. (1→3)-α-d-Glucan-induced mutanase showed high hydrolysis potential in reaction with dextranase-pretreated mutan, where maximal degree of saccharification and solubilization of this bacterial homoglucan (83.1% and 78.4%, resp.) was reached in 3 h at 45°C.

English

The present paper describes optimization of fermentation conditions in shaken flasks and scale-up of fermentor production up to 115 L. The response surface methodology (RSM) has been successfully applied in standardization of mutanase production by Trichoderma harzianum CCM F-340. The model was very well fitted to the experimental data and explained more than 96% of the whole variation of the response (adjusted R2 = 0.962). In order to confirm the adequacy of the regression model based on the experimental data, validation cultures were grown in conditions created through optimization. The highest enzyme activity (0.747 U/mL) was reached in shaken flask cultures on Mandels’ medium in a volume of 140 mL modified in terms of carbon (cell wall preparation from the polypore fungus Laetiporus sulphureus 8.08 g/L) and nitrogen (soybean peptone 1.38 g/L) sources, under culture conditions 30°C, pH 5.3, agitation 270 rpm. The scale-up of the culture in the bioreactors with a working volume of 5 and 115 L resulted in a slight decrease in the mutanase activity (0.734 and 0.682 U/mL, respectively). The validation experiment showed a 70.6% increase in the production of mutanase compared with the culture before optimization. The results proved that the cultures could be scaled-up successfully from shaken flasks to the bioreactor scale. Our results indicate that in optimal conditions, T. harzianum could be a highly effective extracellular mutanase source. This report is the first to deal with optimization of mutanase biosynthesis using a mathematical model and scale-up of enzyme production in controlled fermentors with a view to facilitate application thereof in industry.   Key words: Mutanase, Trichoderma harzianum, response surface methodology (RSM), bioreactors, submerged culture.

Functional characterization of a Penicillium chrysogenum mutanase gene induced upon co-cultivation with Bacillus subtilis.

BackgroundMicrobial gene expression is strongly influenced by environmental growth conditions. Comparison of gene expression under different conditions is frequently used for functional analysis and to unravel regulatory networks, however, gene expression responses to co-cultivation with other microorganisms, a common occurrence in nature, is rarely studied under laboratory conditions. To explore cellular responses of the antibiotic-producing fungus Penicillium chrysogenum to prokaryotes, the present study investigates its transcriptional responses during co-cultivation with Bacillus subtilis.ResultsSteady-state glucose-limited chemostats of P. chrysogenum grown under penillicin-non-producing conditions were inoculated with B. subtilis. Physiological and transcriptional responses of P. chrysogenum in the resulting mixed culture were monitored over 72 h. Under these conditions, B. subtilis outcompeted P. chrysogenum, as reflected by a three-fold increase of the B. subtilis population size and a two-fold reduction of the P. chrysogenum biomass concentration. Genes involved in the penicillin pathway and in synthesis of the penicillin precursors and side-chain were unresponsive to the presence of B. subtilis. Moreover, Penicillium polyketide synthase and nonribosomal peptide synthase genes were either not expressed or down-regulated. Among the highly responsive genes, two putative α-1,3 endoglucanase (mutanase) genes viz Pc12g07500 and Pc12g13330 were upregulated by more than 15-fold and 8-fold, respectively. Measurement of enzyme activity in the supernatant of mixed culture confirmed that the co-cultivation with B. subtilis induced mutanase production. Mutanase activity was neither observed in pure cultures of P. chrysogenum or B. subtilis, nor during exposure of P. chrysogenum to B. subtilis culture supernatants or heat-inactivated B. subtilis cells. However, mutanase production was observed in cultures of P. chrysogenum exposed to filter-sterilized supernatants of mixed cultures of P. chrysogenum and B. subtilis. Heterologous expression of Pc12g07500 and Pc12g13330 genes in Saccharomyces cerevisiae confirmed that Pc12g07500 encoded an active α-1,3 endoglucanase.ConclusionTime-course transcriptional profiling of P. chrysogenum revealed differentially expressed genes during co-cultivation with B. subtilis. Penicillin production was not induced under these conditions. However, induction of a newly characterized P. chrysogenum gene encoding α-1,3 endoglucanase may enhance the efficacy of fungal antibiotics by degrading bacterial exopolysaccharides.

Structural Diversity of Streptococcal Mutans Synthesized under Different Culture and Environmental Conditions and Its Effect on Mutanase Synthesis

Streptococcal mutans synthesized under different conditions by growing cultures or by their glucosyltransferases were shown to exhibit a great structural and property diversity. Culturing and environmental factors causing structural differences in mutans were specified. All of the obtained biopolymers (76 samples) were water-insoluble and most of them (72) had a structure with a predominance of α-(1→3)-linked glucose (i.e., the content of α-(1→3)-linkages in the glucan was always higher than 50%, but did not exceed 76%). An exception were four glucans containing more than 50% of α-(1→6)-sequences. In these structurally unique mutans, the ratio of α-(1→3)- to α-(1→6)-bonds ranged from 0.75 to 0.97. Aside from one polymer, all others had a heavily branched structures and differed in the number of α-(1→3), α-(1→6), and α-(1→3,6) linkages and their mutual proportion. The induction of mutanase production in shaken flask cultures of Trichoderma harzianum by the structurally diverse mutans resulted in enzyme activities ranging from 0.144 to 1.051 U/mL. No statistical correlation was found between the total percentage content of α-(1→3)-linkages in the α-glucan and mutanase activity. Thus, despite biosynthetic differences causing structural variation in the mutans, it did not matter which mutan structures were used to induce mutanase production.

Comparative Studies on the Induction of Trichoderma harzianum Mutanase by α-(1→3)-Glucan-Rich Fruiting Bodies and Mycelia of Laetiporus sulphureus

Mutanase (α-(1→3)-glucanase) is a little-known inductive enzyme that is potentially useful in dentistry. Here, it was shown that the cell wall preparation (CWP) obtained from the fruiting body or vegetative mycelium of polypore fungus Laetiporus sulphureus is rich in α-(1→3)-glucan and can be successfully used for mutanase induction in Trichoderma harzianum. The content of this biopolymer in the CWP depended on the age of fruiting bodies and increased along with their maturation. In the case of CWP prepared from vegetative mycelia, the amount of α-(1→3)-glucan depended on the mycelium age and also on the kind of medium used for its cultivation. All CWPs prepared from the individually harvested fruiting body specimens induced high mutanase activity (0.53–0.82 U/mL) in T. harzianum after 3 days of cultivation. As for the CWPs obtained from the hyphal mycelia of L. sulpureus, the maximal enzyme productivity (0.34 U/mL after 3 days of incubation) was recorded for CWP prepared from the 3 week-old mycelium cultivated in Sabouraud medium. Statistically, a high positive correlation was found between the total percentage content of α-(1→3)-glucan in the CWP and the mutanase activity.

Mutanase from Paenibacillus sp. MP-1 produced inductively by fungal α-1,3-glucan and its potential for the degradation of mutan and Streptococcus mutans biofilm

Laetiporus sulphureus is a source of α-1,3-glucan that can substitute for the commercially-unavailable streptococcal mutan used to induce microbial mutanases. The water-insoluble fraction of its fruiting bodies from 0.15 to 0.2% (w/v) induced mutanase activity in Paenibacillus sp. MP-1 at 0.35 μ ml−1. The mutanase extensively hydrolyzed streptococcal mutan, giving 23% of saccharification, and 83% of solubilization of glucan after 6 h. It also degraded α-1,3-polymers of biofilms, formed in vitro by Streptococcus mutans, even after only 3 min of contact.

Cloning, heterologous gene expression and biochemical characterization of the α-1,3-glucanase from the filamentous fungus Penicillium purpurogenum

There has been much recent interest in α-1,3-glucanases (mutanases) as they have the potential to be used in the treatment of dental caries. Mutanases have been reported in a number of bacteria, yeast and fungi but remain a relatively uncharacterised family of enzymes. In this study we heterologously expressed the mutanase gene from the filamentous fungus Penicillium purpurogenum to enable further characterization of its enzymatic activity. The mutanase cDNA was cloned and expressed in the methylotrophic yeast Pichia pastoris. The molecular mass of the secreted protein was about 102 kDa. The recombinant enzyme hydrolyzed mutan with a specific activity of 3.9 U/mg of protein. The recombinant enzyme was specific for mutan and could not cleave a variety of other polysaccharides demonstrating a specificity for α-1,3-glucosidic linkages. The pH and temperature optima were pH 4.6 and 45 °C, respectively. Synthetic compounds were also tested as substrates to assess whether the P. purpurogenum mutanase has an exo- or endo-type mechanism of hydrolysis. The results suggest an endo-hydrolytic mode of action. The type of mechanism was confirmed since mutanase activity was not suppressed in the presence of inhibitors of exo-type enzymes.

Biochemical and Molecular Characterization of a Novel Type of Mutanase from Paenibacillus sp. Strain RM1: Identification of Its Mutan-Binding Domain, Essential for Degradation of Streptococcus mutans Biofilms

A novel type of mutanase (termed mutanase RM1) was isolated from Paenibacillus sp. strain RM1. The purified enzyme specifically hydrolyzed alpha-1,3-glucan (mutan) and effectively degraded biofilms formed by Streptococcus mutans, a major etiologic agent in the progression of dental caries, even following brief incubation. The nucleotide sequence of the gene for this protein contains a 3,873-bp open reading frame encoding 1,291 amino acids with a calculated molecular mass of 135 kDa. The protein contains two major domains, the N-terminal domain (277 residues) and the C-terminal domain (937 residues), separated by a characteristic sequence composed of proline and threonine repeats. The characterization of the recombinant proteins for each domain which were expressed in Escherichia coli demonstrated that the N-terminal domain had strong mutan-binding activity but no mutanase activity whereas the C-terminal domain was responsible for mutanase activity but had mutan-binding activity significantly lower than that of the intact protein. Importantly, the biofilm-degrading activity observed with the intact protein was not exhibited by either domain alone or in combination with the other. Therefore, these results indicate that the structural integrity of mutanase RM1 containing the N-terminal mutan-binding domain is required for the biofilm-degrading activity.

Paenibacillus strain MP-1: a new source of mutanase

A mutan-degrading bacterium, closely related to Paenibacillus curdlanolyticus, was isolated from soil. It produced 0.4 U mutanase ml(-1 )in 2 days in shake-flask cultures when bacterial mutan was the sole carbon source. Mutanase activity was optimal at pH 6.2 and 45 degrees C over 1 h and was stable between pH 5.8 and 12 at 4 degrees C for 24 h and up to 40 degrees C for 1 h. Mutan produced by Streptococcus mutans was rapidly hydrolyzed by this enzyme. The hydrolysis of mutan (1 g l(-1)) resulted in 17% saccharification over 2 h and, at the same time, glucan was entirely solubilized.

Enhancement of mutanase production inTrichoderma harzianumby mutagenesis

Conidia of Trichoderma harzianum F-340, an active producer of fungal mutanase, were mutagenized with physical and chemical mutagens used separately or in combination. After mutagenesis, the drop in conidia viability ranged from 0.004% to 71%. Among the applied mutagens, nitrosoguanidine gave the highest frequency of cultures with enhanced mutanase activity (98%). In total, 400 clones were isolated, and preliminarily evaluated for mutanase activity in flask microcultures. Eight most productive mutants were then quantified for mutanase production in shake flask cultures. The obtained results fully confirmed a great propensity of all the tested mutants to synthesize mutanase, the activity of which increased from 59 to 107% in relation to the parental T. harzianum culture. The best mutanase-overproducing mutant (T. harzianumn F-340-48), obtained with nitrosoguanidine, produced the enzyme activity of 1.36 U/ml (4.5 U/mg protein) after 4 days of incubation in shake flask culture. This productivity was almost twices higher than that achieved by the initial strain F-340, and, at present, is the best reported in the literature. The potential application of mutanase in dentistry is also discussed.

A study of the physicochemical and biological properties of mutanase from Trichoderma harzianum

To determine the physicochemical properties of the mutanase of Trichoderma harzianum isolated from China and to study the influence of mutanase on the adherence of oral Streptococci and the structure of oral biofilms. Six fungal strains belonging to Trichoderma were tested for mutanase production in the same cultural condition, the strain producing the highest mutanase activity was studied further and the pH and temperature optimum of the enzyme was determined. The RT-PCR method was used to obtain the gene coding for mutanase and the product was cloned to pMD18-T simple vector for sequencing. Inhibition effects of mutanase on the adherence of Streptococcus sobrinus OMZ176, Streptococcus sobrinus 6715, Streptococcus mutans MT8148 were studied by adherence test. The optical sectioning of biofilms with or without mutanase supplementation were analyzed by confocal laser scanning microscopy (CLSM). The highest enzymatic activity was achieved by Trichoderma harzianum Th1, the maximum activity was at pH 5.5 and at 40 degrees C. The nucleotide sequence was 92% homology with that of a known gene coding a mutanase (GenBank accession No. AJ243799). The adherence of Streptococcus sobrinus OMZ176, Streptococcus sobrinus 6715, Streptococcus mutans MT8148 was significantly inhibited by mutanase. Compared with control, the biofilms with mutanase supplementation had lower height and sparser structure. The mutanase from Trichoderma harzianum Th1 can inhibit the adherence of oral Streptococci and had an influence on the structure of oral biofilms.

Selection of method for obtaining an active mutanase preparation from Trichoderma harzianum

Crude mutanase preparations of Trichoderma harzianum were obtained from the culture supernatant by means of ammonium sulfate salting out, ultrafiltration, freeze-drying, concentration under reduced pressure, and fractional precipitation with organic solvents (methanol, ethanol, propanol, isopropanol, acetone). Ammonium sulfate was the worst precipitant, causing a fall in total mutanase activity by 47%. Other methods of enzyme recovery from the post-culture fluid yielded in most cases very good results in regard to specific and overall activities of the enzymatic preparations.