Department of Biochemistry, State University of Maringá, Av. Colombo, 5790, Maringá, PR, 87020-900, Brazil.
Department of Technology, State University of Maringá, Av. Ângelo Moreira da Fonseca, 1800, Umuarama, PR, 87506-370, Brazil.
Appl Biochem Biotechnol. 2020 Sep;192(1):257-282. doi: 10.1007/s12010-020-03307-9. Epub 2020 May 6.
A new cellulase producer strain of Penicillium digitatum (RV 06) was previously obtained from rotten maize grains. This work aim was to optimize the production and characterize this microorganism produced cellulase. A CMCase maximum production (1.6 U/mL) was obtained in stationary liquid culture, with an initial pH of 5.0, at 25 °C, with 1% lactose as carbon source, and cultured for 5 days. The produced enzyme was purified by ammonium sulfate precipitation and exclusion chromatography. The purified enzyme optimal temperature and pH were 60 °C and 5.2, respectively. The experimental T of thermal inactivation was 63.68 °C, and full activity was recovered after incubation of 7 h at 50 °C. The purified 74 kDa CMCase presented K for CMC of 11.2 mg/mL, V of 0.13 μmol/min, k of 52 s, and k/K of 4.7 (mg/mL) s. The purified enzyme had a high specificity for CMC and p-nitrophenyl cellobioside and released glucose and cellobiose as final products of the CMC hydrolysis. The enzyme trypsin digestion produced peptides whose masses were obtained by MALDI-TOF/TOF mass spectrometry, which was also used to obtain two peptide sequences. These peptide sequences and the mass peak profile retrieved a CBHI within the annotated genome of P. digitatum PD1. Sequence alignments and phylogenetic analysis confirmed this enzyme as a CBHI of the glycoside hydrolase family 7. The P. digitatum PD1 protein in silico structural model revealed a coil and β-conformation predominance, which was confirmed by circular dichroism of the P. digitatum RV 06 purified enzyme.
从腐烂的玉米颗粒中，先前获得了产纤维素酶的青霉新菌株（RV 06）。本工作旨在优化该微生物产生纤维素酶的生产，并对其进行特性分析。在静止液体培养中，初始 pH 值为 5.0、25°C、1%乳糖作为碳源时，可获得最大 CMCase 产量（1.6 U/mL），培养 5 天。通过硫酸铵沉淀和排阻层析对产生的酶进行纯化。纯化酶的最适温度和 pH 值分别为 60°C 和 5.2。实验测定的热失活动力学 T 值为 63.68°C，在 50°C 孵育 7 小时后可完全恢复活性。纯化的 74 kDa CMCase 的 CMC K 值为 11.2 mg/mL，V 值为 0.13 μmol/min，k 值为 52 s，k/K 值为 4.7（mg/mL）s。纯化酶对 CMC 和对硝基苯基纤维二糖苷具有高特异性，释放葡萄糖和纤维二糖作为 CMC 水解的最终产物。酶胰蛋白酶消化产生的肽，其质量通过 MALDI-TOF/TOF 质谱获得，还用于获得两个肽序列。这些肽序列和质量峰谱检索到青霉 PD1 注释基因组中的 CBHI。序列比对和系统发育分析证实该酶为糖苷水解酶家族 7 的 CBHI。青霉 RV 06 纯化酶的圆二色性证实了青霉 PD1 蛋白的计算结构模型中螺旋和β-构象占主导地位。
