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客戶(hù)采用我司Ni磁珠純化His標(biāo)簽蛋白在《AMB Express》發(fā)表SCI論文

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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原文更多內(nèi)容請(qǐng)參考如下：

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，紡織和造紙工業(yè)中廣泛使用的酶。已經(jīng)從降解木質(zhì)纖維素的微生物，例如牛瘤胃的微生物和白蟻后腸中鑒定出最有效的木聚糖酶。先前已通過(guò)宏基因組學(xué)從高效的制漿造紙廢水微生物（PPWT）菌群中提取木聚糖酶基因，并將大多數(shù)木聚糖酶基因分配給GH10家族。在這項(xiàng)研究中，共克隆了40種來(lái)自某一種PPWT菌群的GH10家族木聚糖酶基因，并在大腸桿菌BL21（DE3）中表達(dá)。在這些木聚糖酶基因中，有14個(gè)在山毛櫸底物上顯示木聚糖酶活性。其中的兩個(gè)，PW-xyl9和PW-xyl37，顯示高活性，并被純化以評(píng)估其木聚糖酶特性。 PW-xyl9的最佳pH和溫度值分別為pH 7和60℃，而PW-xyl37的最佳pH和溫度值分別為pH 7和55℃。在最佳條件下，它們的比木聚糖酶活性分別為470.1 U / mg蛋白和113.7 U / mg蛋白。此外，將PW-xyl9和PW-xyl37的Km值分別確定為8.02和18.8g / L。這兩種木聚糖酶的表征為將來(lái)在制漿和造紙生產(chǎn)及其他行業(yè)中的潛在應(yīng)用鋪平了道路，這表明PPWT菌群一直是高效木質(zhì)纖維素酶基因的未發(fā)現(xiàn)儲(chǔ)藏庫(kù)。這項(xiàng)研究表明，宏基因組學(xué)方法具有從降解木質(zhì)纖維素的微生物中篩選未培養(yǎng)微生物的有效木聚糖酶的潛力。以類(lèi)似的方式，將來(lái)可能會(huì)從PPWT處理菌群中鑒定出其他有效的木質(zhì)纖維素酶基因。