客户采用我司Ni磁珠纯化His标签蛋白在《AMB Express》发表SCI论文

Recombinant protein expression and purification
Two BL21 strains with xylanase genes of PW-xyl9 or PW-xyl37 were selected for further characterization. The corresponding strains were incubated overnight in a shaker at 37 ℃ and 200 rpm; 4 mL of the overnight culture was then inoculated to 200 mL fresh LB medium in a 1 L shake flask for further culturing. Once the OD600 value of the two strains had reached 0.8, 200 mM IPTG was added, and the culture was cultivated at 20 ℃ for an additional 16 h. Cells were harvested and washed 3 × with PBS buffer, and were suspended in 20 mL PBS buffer with 1 mM phenylmethylsulfonyl fluoride (PMSF) which was protease inhibitor. The cells were disrupted by sonication for a total length of 15 min (Xiaomei, Kunshan, China), with setting parameters of 150 W, 3 s on, and 5 s off. The cell suspensions were centrifuged at 12,000 rpm at 4 ℃ for 20 min, and the supernatant was collected as crude enzyme. Magnetic beads (PuriMag, Xiamen, China) were used for protein purification, and the purified proteins obtained were concentrated in 10 kDa ultrafiltration tubes (Merck, Millipore, USA). The purity of proteins was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and their concentrations were measured with the Bradford method (Sangon Biotech, Shanghai, China).
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Characterization of efficient xylanases from industrial-scale pulp and paper wastewater treatment microbiota
Jia Wang, Jiawei Liang, Yonghong Li, Lingmin Tian & Yongjun Wei 
AMB Express volume 11, Article number: 19 (2021)
ABSTRACT: Xylanases are widely used enzymes in the food, textile, and paper industries. Most efficient xylanases have been identified from lignocellulose-degrading microbiota, such as the microbiota of the cow rumen and the termite hindgut. Xylanase genes from efficient pulp and paper wastewater treatment (PPWT) microbiota have been previously recovered by metagenomics, assigning most of the xylanase genes to the GH10 family. In this study, a total of 40 GH10 family xylanase genes derived from a certain PPWT microbiota were cloned and expressed in Escherichia coli BL21 (DE3). Among these xylanase genes, 14 showed xylanase activity on beechwood substrate. Two of these, PW-xyl9 and PW-xyl37, showed high activities, and were purified to evaluate their xylanase properties. Values of optimal pH and temperature for PW-xyl9 were pH 7 and 60 ℃, respectively, while those for PW-xyl37 were pH 7 and 55 ℃, respectively; their specific xylanase activities under optimal conditions were 470.1 U/mg protein and 113.7 U/mg protein, respectively. Furthermore, the Km values of PW-xyl9 and PW-xyl37 were determined as 8.02 and 18.8 g/L, respectively. The characterization of these two xylanases paves the way for potential application in future pulp and paper production and other industries, indicating that PPWT microbiota has been an undiscovered reservoir of efficient lignocellulase genes. This study demonstrates that a metagenomic approach has the potential to screen efficient xylanases of uncultured microorganisms from lignocellulose-degrading microbiota. In a similar way, other efficient lignocellulase genes might be identified from PPWT treatment microbiota in the future.

木聚糖酶是在食品，纺织和造纸工业中广泛使用的酶。已经从降解木质纤维素的微生物，例如牛瘤胃的微生物和白蚁后肠中鉴定出最有效的木聚糖酶。先前已通过宏基因组学从高效的制浆造纸废水微生物（PPWT）菌群中提取木聚糖酶基因，并将大多数木聚糖酶基因分配给GH10家族。在这项研究中，共克隆了40种来自某一种PPWT菌群的GH10家族木聚糖酶基因，并在大肠杆菌BL21（DE3）中表达。在这些木聚糖酶基因中，有14个在山毛榉底物上显示木聚糖酶活性。其中的两个，PW-xyl9和PW-xyl37，显示高活性，并被纯化以评估其木聚糖酶特性。 PW-xyl9的最佳pH和温度值分别为pH 7和60℃，而PW-xyl37的最佳pH和温度值分别为pH 7和55℃。在最佳条件下，它们的比木聚糖酶活性分别为470.1 U / mg蛋白和113.7 U / mg蛋白。此外，将PW-xyl9和PW-xyl37的Km值分别确定为8.02和18.8g / L。这两种木聚糖酶的表征为将来在制浆和造纸生产及其他行业中的潜在应用铺平了道路，这表明PPWT菌群一直是高效木质纤维素酶基因的未发现储藏库。这项研究表明，宏基因组学方法具有从降解木质纤维素的微生物中筛选未培养微生物的有效木聚糖酶的潜力。以类似的方式，将来可能会从PPWT处理菌群中鉴定出其他有效的木质纤维素酶基因。