【摘要】：植物表面覆蓋的角質(zhì)層不易透水、透氣、耐酸堿、耐氧化,是植物細胞壁與周圍環(huán)境之間的物理和化學(xué)屏障,也一定程度影響了植物纖維的消化。角質(zhì)層主要組成成分為角質(zhì),可被角質(zhì)酶高效降解,且反應(yīng)溫和、無污染。本研究旨在篩選降解角質(zhì)能力較強的菌株,并對其產(chǎn)酶發(fā)酵條件和胞外粗酶酶學(xué)性質(zhì)進行探討,為降解植物角質(zhì)層,改善動物對植物纖維利用提供指導(dǎo)。高效角質(zhì)降解菌篩選:采用番茄角質(zhì)和角質(zhì)類似物聚己內(nèi)酯(PCL)-指示劑變色圈法從混合植物堆肥、動物糞便及病害果蔬表面分離角質(zhì)降解菌,篩選到8株降解角質(zhì)能力較強的菌株,分別命名為L3-2、L14、L16L、X3PB、X3PH、X4、X8P、X16。進一步比較各菌株在角質(zhì)誘導(dǎo)下發(fā)酵生產(chǎn)胞外酯酶活性大小,成功獲得2株角質(zhì)降解能力較突出的菌株L3-2和X8P,經(jīng)形態(tài)觀察和分子鑒定分別為Klebsiella pneumoniae L3-2和Acetobacter orientalis X8P。Klebsiella pneumoniae L3-2和Acetobacter orientalis X8P發(fā)酵條件優(yōu)化:結(jié)果表明,菌株L3-2在LB培養(yǎng)基中40℃、發(fā)酵3d產(chǎn)酶能力最強,1%玉米芯和0.5%牛肉膏明顯促進L3-2產(chǎn)酶(P㩳0.05)。菌株X8P在LB培養(yǎng)基中37℃、發(fā)酵4d產(chǎn)酶能力最強,1%橄欖油明顯促進X8P產(chǎn)酶。1%可溶性淀粉對兩菌株產(chǎn)酶有強抑制作用,而1%葡萄糖促進兩菌株產(chǎn)酶。菌株Klebsiella pneumoniae L3-2和Acetobacter orientalis X8P酯酶粗酶學(xué)性質(zhì)研究:結(jié)果表明,菌株L3-2胞外酯酶粗酶最適pH和溫度分別為pH 6.5和55℃,pH穩(wěn)定性不好。有機溶劑大都對其有抑制作用,甲醇(50%)和DMSO(50%)對其有促進作用(分別提高0.92倍和1.85倍)。甘油(50%和80%)對其有強激活作用(分別提高2.4倍和4.98倍)。Tween-20及Triton-100(1 mM和10 mM)可使L3-2胞外酯酶粗酶活提高2%-10%。金屬離子Na~+、Mg~(2+)、Mn~(2+)、Ca~(2+)、Ba~(2+)(1mM和10 mM)可使L3-2胞外酯酶活性提高2%-31%。菌株X8P胞外酯酶粗酶最適pH和溫度分別為pH 6.5和45℃,且有一定pH穩(wěn)定性,但在多數(shù)有機試劑中不穩(wěn)定,只在甘油中保留全部活性,在DMSO(50%)中活性保留66%。Tween-20(1 mM)、Tween-80(1 mM)和Triton-100(1 mM和10 mM)可使X8P酯酶粗酶活性提高3%-35%。金屬離子K~+、Mn~(2+)(1 mM和10 mM)可使X8P粗酶活性提高2%-20%(P㩳0.05)。
[Abstract]:The cuticle covered on the plant surface is not permeable, breathable, acid-alkali resistant and oxidized. It is a physical and chemical barrier between the cell wall of plant and the surrounding environment, and also affects the digestion of plant fiber to some extent. The keratinocytes are mainly composed of keratinocytes, which can be degraded efficiently by keratinases, and the reaction is mild and pollution-free. The purpose of this study was to screen the strains with strong ability to degrade keratinocytes, and to investigate the fermentation conditions and the enzymatic properties of extracellular crude enzymes, so as to provide guidance for the degradation of plant keratinocytes and the improvement of animal utilization of plant fibers. Screening of high efficient keratinizing bacteria: the keratinized bacteria were separated from the surface of mixed plant compost, animal feces and fruits and vegetables by (PCL) indicator method. Eight strains with strong keratolytic ability were selected and named L3-2L14L14 L16LBX3PHPX4X8PX16X16. The results showed that there were 8 strains with strong keratinolytic ability, and they were named L3-2L14, L16, L16 and L16, respectively. Two strains L3-2 and X8P were successfully obtained by comparing the extracellular esterase activity of each strain under keratinocyte induction fermentation. After morphological observation and molecular identification, the fermentation conditions of Klebsiella pneumoniae L3-2 and Acetobacter orientalis X8P.Klebsiella pneumoniae L3-2 and Acetobacter orientalis X8P were optimized, respectively. The results showed that the strain L3-2 was most capable of producing enzyme for 3 days after fermentation at 40 鈩，

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