Chen Feifei, Ye Jianren, Sista Kameshwar Ayyappa Kumar, Wu Xuelian, Ren Jiahong, Qin Wensheng, Li De-Wei
Jiangsu Key Laboratory for Prevention and Management of Invasive Species, Co-innovation Center for Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing, China.
Department of Biology, Lakehead University, Thunder Bay, ON, Canada.
Front Microbiol. 2020 Jan 22;10:3137. doi: 10.3389/fmicb.2019.03137. eCollection 2019.
The efficient industrial conversion of plant-derived cellulose to simple sugars and other value-added chemicals requires various highly stable and reactive enzymes. Industrial processes especially synchronous saccharification and fermentation (SSF)-based production of cellulosic bio-ethanol require enzymes that are active at lower temperatures. In this study, we have identified, characterized, and expressed the cold-adaptive endo-1,4-β-glucanase (BpEG) isolated from the JK-SH007. The analysis of the predicted amino acid sequence indicated that BpEG belongs to GH family 8. The without the signal peptide was cloned into the expression vector pET32a and significantly expressed in BL21 (DE3) competent cells. The SDS-PAGE and Western blot analysis of BpEG revealed that the recombinant BpEG was approximately 60 kDa. Purified recombinant BpEG exhibited hydrolytic activity against carboxymethyl cellulose (CMC) and phosphoric acid swollen cellulose (PASC), but not crystalline cellulose and xylan substrates. High performance, anion exchange, chromatography-pulsed amperometric detector (HPAEC-PAD) analysis of the enzymatic products obtained from depolymerization of 1,4-β-linked biopolymers of different lengths revealed an interesting cutting mechanism employed by endoglucanases. The recombinant BpEG exhibited 6.0 of optimum pH and 35°C of optimum temperature, when cultured with CMC substrate. The BpEG enzyme exhibited stable activity between pH 5.0 and 9.0 at 35°C. Interestingly, BpEG retained about 42% of its enzymatic activity at 10°C compared to its optimal temperature. This new cold-adaptive cellulase could potentially achieve synchronous saccharification and fermentation (SSF) making BpEG a promising candidate in the fields of biofuel, biorefining, food and pharmaceutical industries.
将植物来源的纤维素高效工业转化为单糖和其他增值化学品需要各种高度稳定且具有活性的酶。工业过程，尤其是基于同步糖化发酵（SSF）的纤维素生物乙醇生产，需要在较低温度下具有活性的酶。在本研究中，我们鉴定、表征并表达了从JK-SH007中分离出的冷适应性内切-1,4-β-葡聚糖酶（BpEG）。对预测氨基酸序列的分析表明，BpEG属于糖基水解酶家族8。将去除信号肽后的序列克隆到表达载体pET32a中，并在BL21（DE3）感受态细胞中显著表达。对BpEG的SDS-PAGE和蛋白质免疫印迹分析表明，重组BpEG约为60 kDa。纯化后的重组BpEG对羧甲基纤维素（CMC）和磷酸膨胀纤维素（PASC）具有水解活性，但对结晶纤维素和木聚糖底物无活性。对不同长度的1,4-β-连接生物聚合物解聚得到的酶促产物进行高效阴离子交换色谱-脉冲安培检测（HPAEC-PAD）分析，揭示了内切葡聚糖酶采用的一种有趣的切割机制。当以CMC为底物培养时，重组BpEG的最适pH为6.0，最适温度为35°C。BpEG酶在35°C下于pH 5.0至9.0之间表现出稳定的活性。有趣的是，与最适温度相比，BpEG在10°C时仍保留约42%的酶活性。这种新型冷适应性纤维素酶有可能实现同步糖化发酵（SSF），使BpEG成为生物燃料、生物精炼、食品和制药行业中有前景的候选酶。
