2.2. Alkaline peroxide pretreatment
Milled rice hulls were slurried in water (15%, w/v) containing H2O2 (0, 3.75, 7.5, or 10.0%, v/v) and adjusted to pH 11.5 using NaOH and shaken in an incubator at 250 rpm at 25 or 35 ◦C for 6 or 24 h. The pH of the pretreated rice hulls was adjusted to 5.0 using concentrated HC1 before enzymatic saccharification.
2.3. Enzyme assays
Carboxymethyl cellulase (CMCase), _-glucosidase, xylanase, _-xylosidase, _-l-arabinofuranosidase, and ferulic acid esterase activities were assayed by the procedures described previously .
2.4. Enzymatic saccharification
The enzymatic saccharification of the alkaline peroxide pretreated rice hulls was performed by shaking gently (100 rpm) at 45 ◦C after adjusting the pH to 5.0 with HC1 and adding enzymes at each enzyme dose of 0.04 ml/g hulls for 72 h, unless otherwise stated. Samples (1 ml) were withdrawn and stored at −20 ◦C before analysis.
2.5. Bacterial strain and preparation of inoculum
Recombinant E. coli strain FBR5 was maintained in glycerol vials at−20 ◦C for use as a working stock . It was plated on Luria broth (LB; 10 g tryptone, 5 g yeast extract, and 5 g NaCl per l) containing 4.0 g xylose and 20 mg tetracycline solidified with 15 g agar per l. Plates were incubated at 35 ◦C. Cells from a single well-isolated colony were inoculated into a 125 ml flask containing 100 ml of LB with 20 g xylose and 20 mg tetracycline per l. Cultures were incubated for 24h at 35◦C and 100 rpm and used as seed culture for fermentation experiments.
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2.2. 鹼性過氧化物預處理被碾碎的米船身是slurried 在水(15% 中, w/v) 包含H2.O2 (0, 3.75, 7.5, 或10.0%,
v/v) 並且對酸鹼度11.5 調整了使用NaOH 和震動在孵養器在250 轉每分鐘在25 或35?C 對6 或24 h 。被預處理的米船身的酸鹼度被調整了到5.0 使用被集中的HC1 在enzymatic 糖化之前。2.3. 酵素分析用試樣Carboxymethyl cellulase (CMCase), _ - glucosidase, xylanase, _ - xylosidase, _ - l-arabinofuranosidase, 和ferulic 酸esterase 活動由規程檢驗了早先被描述[ 10 ] 。2.4. Enzymatic 糖化鹼性過氧化物被預處理的米船身的enzymatic 糖化由柔和地震動進行了(100 轉每分鐘) 在45?C 在調整酸鹼度到5.0 與HC1 和增加酵素以後在0.04 ml/g 船身各酵素藥量為72 h, 除非另外說明。樣品(1 機器語言) 被撤出了和被存放了在?20?C 在分析之前。2.5. 接種物再組合E. 桿菌張力的細菌張力和準備FBR5 被維護了在丙三醇小瓶at?20?C 至於使用作為運作的股票[ 14 ] 。它被鍍了在Luria 湯(磅; 10 g tryptone 、5 g 酵母抽提物, 和5 g NaCl 每l) 包含4.0 g 木糖和20 毫克四圜素變硬了與15 瓊脂每l. 板材被孵化在35?C 的g 。細胞從唯一很好被隔絕的殖民地被接種了入一個125 機器語言燒瓶包含100 機器語言磅以20 g 木糖並且20 毫克四圜素每l. 文化被孵化了為24h 在35?C 和100 轉每分鐘和使用了當種子文化為發酵實驗