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 Burkholderia pyrrocinia JK-SH007. The analysis of the predicted amino acid sequence indicated that BpEG belongs to GH family 8. The BpEG without the signal peptide was cloned into the expression vector pET32a and significantly expressed in Escherichia coli 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.

中文翻译：

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伯克霍尔德氏菌JK-SH007的一种新型的冷适应性内源1,4-β-葡聚糖酶：基因表达，酶特性和作用方式的表征。

从植物纤维素到单糖和其他增值化学品的有效工业转化需要各种高度稳定和反应性的酶。工业过程，尤其是基于同步糖化和发酵（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。在这项研究中，我们已经鉴定，表征并表达了从伯克霍尔德菌（Burkholderia pyrrocinia）JK-SH007分离的冷适应性内切1,4-β-葡聚糖酶（BpEG）。对预测的氨基酸序列的分析表明，BpEG属于GH家族8。将没有信号肽的BpEG克隆到表达载体pET32a中，并在大肠杆菌BL21（DE3）感受态细胞中显着表达。BpEG的SDS-PAGE和Western blot分析表明重组BpEG约为60 kDa。纯化的重组BpEG对羧甲基纤维素（CMC）和磷酸溶胀的纤维素（PASC）表现出水解活性，但对结晶纤维素和木聚糖底物没有水解活性。高性能，阴离子交换，色谱脉冲安培检测器（HPAEC-PAD）对从不同长度的1,4-β连接的生物聚合物解聚获得的酶产物的分析显示，内切葡聚糖酶采用了一种有趣的切割机理。与CMC底物一起培养时，重组BpEG的最佳pH值为6.0，最佳温度为35°C。在35°C下，BpEG酶在pH 5.0和9.0之间表现出稳定的活性。有趣的是 与最佳温度相比，BpEG在10°C时保留了约42％的酶活性。这种新型的冷适应性纤维素酶可以潜在地实现同步糖化和发酵（SSF），从而使BpEG成为生物燃料，生物精炼，食品和制药行业领域中有希望的候选者。