MNP laboratory, Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakamangalam 629502, India.
Department of Microbiology, Periyar University, Salem, 636011, India.
Int J Biol Macromol. 2018 Oct 15;118(Pt A):195-208. doi: 10.1016/j.ijbiomac.2018.06.063. Epub 2018 Jun 14.
The present study was undertaken to evaluate the biosynthesis, molecular modeling and statistical optimization of xylanase production through Box-Behnken design by a mangrove associated actinobacterium Streptomyces variabilis (MAB3). Initially, the production of xylanase by the selected strain was carried through submerged fermentation using birchwood xylan as substrate. Further the xylanase production was statistically optimized through Box-Behnken design. It showed 5.30 fold increase of xylanase production by the isolate compared to 'one factor at a time approach' in the presence of the basal medium containing birchwood xylan (2.0% w/v) at pH 8.2, temperature 46.5 °C, inoculum size of 2% for 68 h. The analysis of variance (ANOVA) revealed high coefficient of determination (R = 0.9490) for the respective responses at significant level (P < 0.0001). The xylanase was purified by different purification steps and it resulted 5.30 fold increase with the yield of 21.27% at the final step using sephadex G-75 chromatography. The molecular weight of the purified xylanase was observed as 50 kDa on 10% SDS-PAGE. The homology 3D structure of the purified xylanase protein was predicted and this protein encodes with 420 amino acid residues. The maximum activity of purified xylanase was observed at pH 8, temperature 40 °C and the production medium supplemented with 1 mM Ca metal ion, 2.0% xylan and 1.5% NaCl. The kinetic parameters of the purified xylanase expressed the K and V values of 5.23 mg/ml and 152.07 μg/min/mg, respectively. Finally, the xylanolytic hydrolysis of pretreated agro-residues, especially the rice straw substituted medium yielded maximum (46.28 mg/g) level of reducing sugar and saccharification (63.18%), followed by bioethanol production (3.92 g/l) at 72 h of incubation. Based on the results, it could be confirmed that the selected isolate is a potent strain for xylanase production and also it can able to convert the pretreated agro-residues into economically important byproduct like bioethanol.
本研究通过红树林相关放线菌变栖链霉菌（MAB3）的 Box-Behnken 设计，评估了木聚糖酶的生物合成、分子建模和统计优化。最初，通过使用桦木木聚糖作为底物进行浸没发酵来筛选菌株生产木聚糖酶。然后，通过 Box-Behnken 设计对木聚糖酶的生产进行了统计优化。与“一次一个因素”方法相比，分离株的木聚糖酶产量提高了 5.30 倍，在含有桦木木聚糖（2.0%w/v）的基础培养基中，在 pH 8.2、温度 46.5°C、接种量为 2%的条件下培养 68 h。方差分析（ANOVA）显示，在显著水平（P<0.0001）下，各响应的高决定系数（R=0.9490）。木聚糖酶通过不同的纯化步骤进行纯化，在最后一步使用葡聚糖 G-75 层析时，产率提高了 5.30 倍，达到 21.27%。纯化的木聚糖酶的分子量在 10%SDS-PAGE 上观察到为 50 kDa。预测了纯化木聚糖酶蛋白的同源 3D 结构，该蛋白编码 420 个氨基酸残基。纯化木聚糖酶的最大活性在 pH 8、温度 40°C 和补充有 1 mM Ca 金属离子、2.0%木聚糖和 1.5%NaCl 的生产培养基中观察到。纯化木聚糖酶的动力学参数表示 K 和 V 值分别为 5.23 mg/ml 和 152.07μg/min/mg。最后，预处理农业残留物的木聚糖水解，特别是用稻秸替代培养基，产生了最大（46.28 mg/g）水平的还原糖和糖化（63.18%），随后在 72 h 的孵育期内产生了 3.92 g/l 的生物乙醇。根据这些结果，可以确认所选分离株是生产木聚糖酶的有效菌株，并且它还能够将预处理的农业残留物转化为具有经济重要性的副产物，如生物乙醇。
