由 Paenibacillus macerans 生產耐熱型木聚醣酶

Abstract

本研究結果顯示 Paenibacillus macerans 以 500 mL Hinton 氏搖瓶培養的最佳溫度為 30℃，最佳誘導用培養基酸鹼值為 pH 7.0，而最佳誘導溫度為 45℃。所得的木聚醣酶，最好的酵素反應條件是 55℃ 與 pH 4.5。相對於葡萄糖、麥芽糖與木糖，於生長期間較適合的碳源是甘油。於酵素誘導期間，相對於誘導物蔗渣、黃豆粉、白楊木粉與山毛櫸木聚醣，麩皮具有最好的木聚醣酶酵素誘導能力。利用 Hinton 氏搖瓶培養 P. macerans，於 37℃ 培養 20 小時，添加 2% 麩皮誘導，同時升溫至 45℃，再繼續培養 34 小時後，酵素濃度為 15 IU/mL。以 5 公升發酵槽進行放大培養，於 37℃ 培養 9.5 小時，添加 2% 麩皮誘導，同時升溫至 45℃，再繼續培養 28 小時後，產生的酵素產量為 15 IU/mL，比搖瓶縮短了 16.5 小時的培養時間，便能得到相同的酵素濃度。將木聚醣酶搭配漆酶進行功能性試驗─利用紙漿進行生物漂白，能夠顯著地降低 3.7 單位卡巴值，並提升 ISO 白度 2%。利用糖苷水解酶 (glycoside hydrolase) 家族第 11 族的 4 個保守性序列 (conserved sequences) 與染色體步移技術 (genome-walking technique) 將來自耐熱細菌 P. macerans 的木聚醣酶基因調取出來，並選殖了 633 個核酸，此段序列對應到 211 個胺基酸殘基。當此段基因被轉殖並表現到大腸桿菌 Escherichia coli M15/pREP4 時，利用 anti-His taq 抗體偵測重組蛋白，發現重組菌株將表現出的木聚醣酶聚集到內涵體中，即使以 16℃、24 小時誘導，仍無法改善。將細菌以超音波震碎並離心後的上清液中，其所含的可溶性蛋白質，經過濃縮也測不出木聚醣酶活性。由 P. macerans 產生的木聚醣酶，同時具有熱穩定性與酸穩定性。可被應用在製漿造紙工業，進行生物漂白與從農、林、食品工業廢棄物來產生木寡醣 (xylooligosaccharides) 益生源。

Our reports showed that when cultured in 500 mL Hinton’s flask, the optimum temperature for Paenibacillus macerans is 30℃, the optimum pH of induction medium is pH 7.0, and the optimum induction temperature is 45℃. The xylanase of P. macerans was optimally active at 55℃ and pH 4.5. Compared with glucose, maltose, and xylose, the most suitable carbon source in growth stage was glycerol. Compared with bagasse, soya powder, white poplar powder, and beechwood xylan, the best carbon source we investigated to induce xylanase activity was wheat bran. While grown for 20 hours at 37℃, we added wheat bran to start induction phase, and rising temperature to 45℃ at the same time, continuing to culture for 34 hours, having the xylanase with the concentration of 15 IU/mL. To scale up, we cultured P. macerans in the 5 L bioreactor. While grown for 9.5 hours at 37℃, we added wheat bran, and rising temperature to 45℃, continuing to culture for 28 hours, having the xylanase activity with 15 IU/mL. Compared with flask culture, we could obtain the same amount of xylanase with shortened incubation time of 16.5 hours. Coordinated with laccase, we carried out functional assay─the bio-bleaching of pulp. The xylanase significantly reduced kappa number by 3.7 units, and increased the ISO brightness by 2%. We used 4 conserved sequences of glycoside hydrolase family 11 (GH11) and genome-walking technique to acquire xylanase nucleotide sequence in moderately thermophilic bacterium P. macerans. A xylanase gene of 633 bp was cloned that encodes a protein containing 211 amino acid residues. When the xylanase gene was cloned and expressed in Escherichia coli M15/pREP4, we used anti-His taq antibody to detect the recombinant protein, figuring out the recombinant strain produced almost all the xylanase in the inclusion body, even after long-time induction at low temperature. The soluble protein in the supernatant after sonication showed low xylanase activity, even concentrated. The xylanase of P. macerans is one of the rare xylanases that exhibits thermo- and acid stability, and thus, it is a suitable candidate for pre-bleaching of paper pulps and generating xylooligosaccharides from agro-residues for use as prebiotics.