VTT Biotechnology, P.O. Box 1000, FIN-02044 VTT, Finland.
Enzyme Microb Technol. 2013 Apr 10;52(4-5):272-8. doi: 10.1016/j.enzmictec.2013.01.005. Epub 2013 Jan 17.
Isolates from gardening waste compost and 38 culture collection microbes were grown on agar plates at pH 4.0 with the cutinase model substrate polycaprolactone as a carbon source. The strains showing polycaprolactone hydrolysis were cultivated in liquid at acidic pH and the cultivations were monitored by assaying the p-nitrophenyl butyrate esterase activities. Culture supernatants of four strains were analyzed for the hydrolysis of tritiated apple cutin at different pHs. Highest amounts of radioactive hydrolysis products were detected at pHs below 5. The hydrolysis of apple cutin by the culture supernatants at acidic pH was further confirmed by GC-MS analysis of the hydrolysis products. On the basis of screening, the acidic cutinase from Aspergillus niger CBS 513.88 was chosen for heterogeneous production in Pichia pastoris and for analysis of the effects of pH on activity and stability. The recombinant enzyme showed activity over a broad range of pHs with maximal activity between pH 5.0 and 6.5. Activity could be detected still at pH 3.5.
从园艺废物堆肥和 38 个培养物微生物中分离出的菌株在 pH 4.0 的琼脂平板上生长，以作为碳源的角蛋白酶模型底物聚己内酯进行培养。显示聚己内酯水解的菌株在酸性 pH 下进行液体培养，并通过测定对硝基苯丁酸酯酶活性来监测培养。对四种菌株的培养液在不同 pH 值下对氚标记苹果角质的水解进行分析。在 pH 值低于 5 的情况下检测到最高量的放射性水解产物。通过对水解产物的 GC-MS 分析进一步证实了在酸性 pH 值下培养物上清液对苹果角质的水解作用。基于筛选，选择黑曲霉 CBS 513.88 的酸性角质酶进行毕赤酵母中的异源生产，并分析 pH 值对活性和稳定性的影响。重组酶在广泛的 pH 值范围内表现出活性，最适 pH 值在 5.0 到 6.5 之间。在 pH 值为 3.5 时仍能检测到活性。
