Production and use of mutanase from Trichoderma harzianum for effective degradation of streptococcal mutans

Abstract

Basic cultural parameters affecting mutanase production by Trichoderma harzianum F-340 in shaken flasks and aerated fermenter cultures have been standardized. The best medium for enzyme production was Mandels medium A with initial pH 5.3, supplemented with 0.3% mutan and 0.05% peptone and inoculated with 20% of the 72-h mycelium as inoculum. It was shown that mycelial mass, used in the culture medium as a sole carbon source, induced mutanase synthesis and could be utilized as an inexpensive and easily available substitute for bacterial mutan. Application of optimized medium and cultural conditions enabled us to obtain a high mutanase yield (0.6-0.7 U/mL, 2.0-2.5 U/mg protein) in a short period of time (3-5 days), which was much higher than the best reported in literature. The enzyme in crude state was stable in the pH range of 4.5-6.0, and at temperatures of up to 40ºC; its maximum activity was recorded at 45ºC and at pH 5.5. The mutanase preparation obtained from the T. harzianum fungus was relatively stable under storage conditions, and showed a high hydrolytic potential in reaction with a mixed-linkage (α-1,3, α-1,6) water-insoluble mutan of streptococcal origin (hydrolysis yield reached a value of 69% in 24 h). Steady-state measurement of the enzymic reaction products during the hydrolysis revealed that mutanase exhibited an exo type of action on mutan. Thin-layer chromatographic analysis showed that glucose was the primary final product of mutan hydrolysis with mutanase. The potential application of mutanase in dentistry is discussed.

A novel thermostable α-1,3-glucanase from <i>Streptomyces thermodiastaticus</i> HF 3-3

“…a-1,3-glucanase exhibited a degrading activity against Streptococcal biofilms (Guggenheim and Haller, 1972;Imai et al, 1977;Inoue et al, 1988;Pleszczy¢ nska et al, 2010;Shimotsuura et al, 2008;Tsumori et al, 2011;Wiater et al, 2004Wiater et al, , 2005. However, the practical use of the enzyme still remains uncertain.…”

Comparative growth of trichoderma strains in different nutritional sources, using bioscreen c automated system

“…Peptone is a similar molecule frequently used for growth of fungi such as Mucor (32), Aspergillus (23), and Trichoderma (35). Studies involving Aspergillus flavus growth report that tryptone is as effective as peptone (22), and is cheaper.…”

Optimization of mutanase production by Trichoderma harzianum

“…Soybean peptone had the highest positive effect (15.1108) on mutanase production. It provided a source of nitrogen for fungal growth and enzyme production, which resulted in a higher yield than that reported for other organic nitrogen sources (Wiater et al, 2005b). An additional advantage of the use of this nitrogen source is the fact that soybean peptone is free of allergens and animal pathogens (Hemmer et al, 2011).…”

“…However, to our knowledge, there are few reports on detailed characteristics of production of fungal mutanases. In our previous studies, we described the effects of nutrients and culture conditions on mutanase production by various T. harzianum strains in flask cultures and batch fermentation using "one-variable-at-a-time" experiments (Wiater et al, 2005b). The "one-variable-at-a-time" approach is time consuming and does not account for the interactions among the medium components.…”