The Basic And Application Study Of Mutanase From Trichoderma Harzianum Th1

Caries is a chronic infectious disease of the dental hard tissues mainly due to bacteria. Mutans streptococci including S.mutans and S.sobrinus have been confirmed to be highly cariogenic pathogens in humans. The colonization of Mutans streptococci and the formation of dental plaque result in the development of caries. The acidogenic, aciduric properties and the ability of synthesizing extracellular glucans play an important role in the formation of dental caries. Using the specific hydrolase which can hydrolyze the polysaccharides matrix of dental plaque have been considered as a effective method of preventing the colonization of major cariogenic bacteria in teeth surface and preventing of the following dental caries. But the research about this method of caries prevention is limited in China.In this study Trichoderma harianum Th1 (isolated from the soil in ShanDong Province) was found to secret mutanase. Then the mutanase of Trichoderma harianum Thl was partial purified, the properties were studied and the gene was cloned and sequenced too. The effect of mutanase on mutans streptococci growth, adherence and the formation of biofilm was assessed in vitro. With the confocal laser scanning microscopy the influence of mutanase on the structure of mixed-species biofilm was detected and the susceptibility of biofilm supplemented with mutanase to Chlorhexidine was studied. Furthermore, the effect of mutanase on dental caries development was measured in a rat model.Part OneThe aim of part one is to isolate and characterize the mutanase. Six fungal strains belonging to Trichoderma were tested for mutanase production in the enzyme-inducing media. The optimal enzymatic ctivity was achived by Trichoderma harianum Th1, a strain isolated from the soil in ShanDong Province. This strain produce the highest mutanase activity(0.071U/ml) in a shorter time (72h). The purification achived was 1.8-fold by means ofammonium sulfate salting out. This enzyme from Trichoderma harianum Th1 was an endo-alpha-l,3-glucanase. The pH optimum of the enzyme was 5.5 and the temperature optimum was 40.The RT-PCR method was used to obtain the gene coding for mutanase and the 1.8Kb product was cloned to pMD18-T simple vector for sequencing. Comparison to homologue using BLAST showed the nucleotide sequence was 94% homology with that of a known gene coding a mutanase and the amino acid sequence was 96% homology .It was deduced that the difference between two genes was due to the different strain from the different country. The mutan of some amino acid may result in the change of some of the enzyme properties. The structure of Trichoderma harianum Thl mutanase was predicted with the molecular biological softs.Part TwoThe potential anti-bacterial action of mutanase was investigated in BHI media, the results showed the mutanase did not prevent S.mutans MT8148 growth in liquid media. Inhibitory of mutanase on adherence of S.mutans MT8148, S.sobrinus 6715 and S.sobrinus OMZ176 was studied. It was found that mutanase showed significant inhibitory effect on bacterial adherence to glass surface when the sucrose exist and the inhibitory effect had no significant difference among different concentrations. Mutansae also had inhibited significantly the biofilm formation of S.sobrinus 6715 and S.sobrinus OMZ176, but it did not inhibit the biofilm formation of S.mutans MT8148. The different adherent mechanism of S.mutans and S.sobrinus may explainthis phenomenon.Biofilms were fed with or without mutanase supplementation. The structure and viability distribution of the biofilms were examined using confocal laser scanning microscopy (CLSM). Compared with non-supplemented biofilms, mutanase-supplemented biofilms had sparser structure and lower height. In non-supplemented biofilms, the lower vitality values were found in layers adjacent to the substrate, the middle layers were higher, and then lower at the outermost surface. Different vitality distribution was appeared in the mutanase-supplemented biofilms:the vitality values of inner, middle and outer layers in mutanase-supplemented biofilms were almost approximate. Antimicrobial susceptibilities of biofilms with or without mutanase treatment were also evaluated too. Chlorhexidine treatment in mutanase-supplemented biofilms gave greater reduction in viable counts than in non-supplemented biofilms. The viable count of non-supplemented biofilms was reduced significantly when incubated with mutanase before exposure to Chlorhexidine. Part ThreeIn order to test the anti-cariogenic activity of mutanase from Trichoderma harianum Thl, the gnotobiotic rats model were used. S.mutans MT8148, S.sobrinus 6715 and the mixture of two were inoculated separately to rats. The test groups were locally sprayed with mutanase in oral cavity, meanwhile the destill water was given to the controls. Significant differences occur between the test groups and controls. The weights and the coefficient of viscera had no significant difference between the test groups and controls. It suggested that mutanase had no toxicity to the body when it was sprayed in oral cavity, but it should be studied further if the dose or frequency of the use of mutanase was changed.