Purification and properties of an α-(1 → 3)-glucanase (EC 3.2.1.84) from Trichoderma harzianum and its use for reduction of artificial dental plaque accumulation.

Abstract

Extracellular α-(1 → 3)-glucanase (mutanase, EC 3.2.1.84) produced by Trichoderma harzianum CCM F-340 was purified to homogeneity by ultrafiltration followed by ion exchange and hydrophobic interaction chromatography, and final chromatofocusing. The enzyme was recovered with an 18.4-fold increase in specific activity and a yield of 4.3%. Some properties of the α-(1 → 3)-glucanase were investigated. The molecular mass of the enzyme is 67 kDa, as estimated by SDS/PAGE, its isoelectric point 7.1, and the carbohydrate content 3%. The pH and temperature optima are 5.5 and 45°C, respectively. The enzyme is stable over a pH range of 4.5-6.0 and up to 45°C for 1 h. The Km and Vmax under standard assay conditions are 0.73 mg/ml and 11.39 x 10(-2) µmol/min/mg protein, respectively. The enzyme activity is stimulated by addition of Mg(2+) and Na(+), and significantly inhibited by Hg(2+). The α-(1 → 3)-glucanase preparation preferentially catalyzed the hydrolysis of various streptococcal mutans and fungal α-(1 → 3)-glucans. The 20-residue N-terminal sequence of the enzyme is identical with those of other α-(1 → 3)-glucanases from the genus Trichoderma, and highly similar to those from other fungi. The purified α-(1 → 3)-glucanase was effective in preventing artificial dental plaque formation. The easy purification from fermentation broth and high stability, and the effective inhibition of oral biofilm accumulation make this α-(1 → 3)-glucanase highly useful for industrial and medical application.

Application of chimeric glucanase comprising mutanase and dextranase for prevention of dental biofilm formation

“…We attempted to combine the open reading frames of the mut and dexA genes into one chimeric gene and examined the bi-functional effects of chimeric glucanase on the formation and decomposition of biofilm with the aim of finding a means of preventing dental caries. Mutanase has been isolated from various yeasts and bacteria and is known to inhibit formation of biofilm and decompose biofilm (10,14,15,17,18). Because we selected P. humicus NA1123 isolated from a traditional Japanese fermented food as the source of the mut genes, we considered it to be safer for an application in oral health promotion than other bacterial enzymes (30).…”

“…Mutanase has been isolated from various yeasts and bacteria and is known to inhibit formation of biofilm and decompose biofilm . Because we selected P. humicus NA1123 isolated from a traditional Japanese fermented food as the source of the mut genes, we considered it to be safer for an application in oral health promotion than other bacterial enzymes .…”

“…Although brushing can remove dental biofilm that is firmly adherent to tooth surfaces, biofilm removal from adjacent tooth surfaces and gingival margins is difficult (9). The use of topical mutanase or dextranase as an approach to control dental plaque has been explored in vitro and in vivo with variable results (10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Hayacibara et al (20) and Wiater et al (21,22) reported that the presence of either mutanase or dextranase alone had a modest effect on total amount of glucan formed, whereas both glucanases in combination reduced the total amount of glucan.…”

“…Although brushing can remove dental biofilm that is firmly adherent to tooth surfaces, biofilm removal from adjacent tooth surfaces and gingival margins is difficult . The use of topical mutanase or dextranase as an approach to control dental plaque has been explored in vitro and in vivo with variable results . Hayacibara et al .…”

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

“…Synthesis of mutanase (α-1,3-glucanase, EC 3.2.1.59) is induced in several fungi and bacteria when mutan (α-1,3-glucan) or oligosaccharides such as raffinose are present in the growth medium [52-54]. Mutanase production by Trichoderma harzianum is triggered by sugars or molecule with α-1,3 linkages, consistent with the ability of the enzyme to act on α-1,3 linkages in bacterial biofilms and extracellular polysaccharides such as α-mutan [55].…”