哈茨木霉菌非水溶性葡聚糖酶的理化及生物学性质研究

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

目的研究源于哈茨木霉菌的非水溶性葡聚糖酶的理化性质,及其对口腔链球菌黏附和人工生物膜结构的影响。方法比较6种木霉菌产生非水溶性葡聚糖酶的能力,选出酶活性最高的菌株;确定该酶最适pH值和反应温度;RT-PCR扩增酶基因,连T载体,测序。通过黏附试验检测非水溶性葡聚糖酶抑制链球菌黏附的效果,并应用激光共聚焦显微镜观察其对人工生物膜结构的影响。结果哈茨木霉菌Th1产生的非水溶性葡聚糖酶活性最高,其反应最适pH值为5·5,温度40℃;基因序列与GenBank中的已知序列同源性达到92%。非水溶性葡聚糖酶能显著抑制链球菌的黏附;与未加酶组相比,加酶组生物膜高度降低,结构松散。结论哈茨木霉菌Th1产生的非水溶性葡聚糖酶能明显抑制细菌的黏附,影响生物膜的形成。

Objective 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. Methods 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 adherernce of Streptococcus sobrinus OMZ176, Streptococcus sobrinus 6715, Streptococcus mutans MT8148 were studied by adherence test. The optical sectionings of biofilms with or without mutanase supplementation were analyzed by confocal laser scanning microscopy （CLSM）. Results The highest .enzymatic activity was achieved by Trichoderma harzianum Thl, the maximum activity was at pH 5. 5 and at 40℃. 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. Conclusions The mutanase from Trichoderma harzianum Thl can inhibit the adherence of oral Streptococci and had an influence on the structure of oral biofilms.