Ramani Gurusamy, Meera Balasubramanian, Vanitha Chinnathambi, Rajendhran Jeyaprakash, Gunasekaran Paramasamy
Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai, 625021, India.
J Ind Microbiol Biotechnol. 2015 Apr;42(4):553-65. doi: 10.1007/s10295-014-1549-6. Epub 2015 Jan 28.
A partial peptide sequence of β-glucosidase isoform (Bgl4) of Penicillium funiculosum NCL1 was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The cDNA (bgl4) encoding Bgl4 protein was cloned from P. funiculosum NCL1 RNA by consensus RT-PCR. The bgl4 gene encoded 857 amino acids that contained catalytic domains specific for glycoside hydrolase family 3. The cDNA was over-expressed in Pichia pastoris KM71H and the recombinant protein (rBgl4) was purified with the specific activity of 1,354.3 U/mg. The rBgl4 was a glycoprotein with the molecular weight of ~130 kDa and showed optimal activity at pH 5.0 and 60 °C. The enzyme was thermo-tolerant up to 60 °C for 60 min. The rBgl4 was highly active on aryl substrates with β-glucosidic, β-xylosidic linkages and moderately active on cellobiose and salicin. It showed remarkably high substrate conversion rate of 3,332 and 2,083 μmol/min/mg with the substrates p-nitrophenyl β-glucoside and cellobiose respectively. In addition, the rBgl4 showed tolerance to glucose concentration up to 400 mM. It exhibited twofold increase in glucose yield when supplemented with crude cellulase of Trichoderma reesei Rut-C30 in cellulose hydrolysis. These results suggested that rBgl4 is a thermo- and glucose-tolerant β-glucosidase and is a potential supplement for commercial cellulase in cellulose hydrolysis and thereby assures profitability in bioethanol production.
通过基质辅助激光解吸/电离飞行时间质谱法鉴定了绳状青霉NCL1的β-葡萄糖苷酶同工型（Bgl4）的部分肽序列。通过一致性逆转录聚合酶链式反应（RT-PCR）从绳状青霉NCL1 RNA中克隆了编码Bgl4蛋白的cDNA（bgl4）。bgl4基因编码857个氨基酸，其中包含糖苷水解酶家族3特有的催化结构域。该cDNA在巴斯德毕赤酵母KM71H中过表达，重组蛋白（rBgl4）经纯化后比活性为1,354.3 U/mg。rBgl4是一种分子量约为130 kDa的糖蛋白，在pH 5.0和60°C时表现出最佳活性。该酶在60°C下耐热60分钟。rBgl4对具有β-葡萄糖苷键、β-木糖苷键的芳基底物具有高活性，对纤维二糖和水杨苷具有中等活性。它对底物对硝基苯基β-葡萄糖苷和纤维二糖的底物转化率分别高达3,332和2,083 μmol/min/mg。此外，rBgl4对高达400 mM的葡萄糖浓度具有耐受性。在纤维素水解中添加里氏木霉Rut-C30的粗纤维素酶时，其葡萄糖产量提高了两倍。这些结果表明，rBgl4是一种耐热且耐葡萄糖的β-葡萄糖苷酶，是纤维素水解中商业纤维素酶的潜在补充剂，从而确保了生物乙醇生产的盈利能力。
