Screening and Identification of High Cellulase-producing Bacteriafrom Soil at Mulberry Fields
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摘要: 为获得高产纤维素酶菌株，以桑园土壤为对象筛选纤维素酶产生菌。以羧甲基纤维素钠（CMC-Na）作唯一碳源，利用刚果红染色法从桑园土壤中筛选得到12株产纤维素酶菌株，其中菌株YZB46产酶效果最好，经酶学性质初步分析，YZB46在pH 6.0、40℃条件下发挥最佳内切葡聚糖酶活性，为17.08 U·mL-1，经传代，YZB46的产纤维素酶能力能稳定遗传。通过形态观察、革兰氏染色、生理生化特征及16S rDNA分析，发现菌株YZB46与芽孢杆菌属的Bacillus cereus同源性达99%，并将菌株YZB46鉴定为蜡样芽孢杆菌（Bacillus cereus）。Abstract: High cellulase-producing strains of microorganisms were screened and identified from soil at mulberry fields. Using CMC-Na as the sole carbon source, 12 target bacteria were isolated with Congo red dyeing. YZB46 was found to show the greatest cellulasesecretion among all. Preliminary analysis on thesecreted metabolitesindicated that the endo-cellulaseactivityreached 17.08 U·mL-1 at pH 6.0 and 40 ℃, and the enzymedisplayed a desirable genetic stability. Based on the results of morphological observation, gram-staining, physiological and biochemical characterizations, as well as 16S rDNA sequencing, the isolated YZB46 appeared to beclosely related to Bacillus cereus.
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Key words:
- cellulase /
- screening /
- identification
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图 2 YZB46菌株不同发酵时间的酶活力
Figure 2. Cellulase activities of metabolites after fermentation for varied durations
图 3 不同温度对YZB46的纤维素酶活力影响
Figure 3. Effect of temperature on cellulase activity of YZB46 metabolites
图 4 不同pH对YZB46的纤维素酶活力的影响
Figure 4. Effect of pH on cellulase activityof YZB46 metabolites
图 6 菌株YZB46的16srRNA系统发育树
Figure 6. Phylogenetic tree based on 16srRNA sequences of YZB46
表 1 菌株水解圈直径与菌落直径比值
Table 1. Ratio ofcolonyto transparent areadiameters
菌株号 水解圈直径D
/cm菌落直径d
/cmD/d YZB17 2.3 0.7 3.28 YZB24 2.6 1.1 2.36 YZB37 2.2 0.5 4.40 YZB46 2.6 0.4 6.50 YZB65 1.7 0.6 2.83 YZB83 2.2 0.8 2.75 YZB115 2.8 1.2 2.33 YZB129 0.6 0.2 3.00 YZB143 1.3 0.4 3.25 
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表 2 YZB46菌株不同传代次数的酶活力
Table 2. Cellulase activities inmetabolites of YZB46 in different generations
代次 原菌液 1 2 3 4 5 6 7 8 9 10 酶活力/
(U·mL-1)17.08 15.27 13.95 12.52 12.45 12.47 12.44 12.46 12.45 12.43 12.44
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表 3 菌株YZB46生理生化反应特征结果
Table 3. Physiological and biochemical characteristics of YZB46
特征 结果 吲哚试验 + 甲基红试验 - V-P测定 + 脲酶测定 + 脂酶(Tween80) - 接触酶 + 葡萄糖产酸 + D-甘露醇产酸 - 七叶苷水解 + 明胶水解 + 酪氨酸水解 + 淀粉水解 + 柠檬酸盐利用 + 硝酸盐还原 + L-阿拉伯糖产酸 - 葡萄糖产气 - 注：“+”表示阳性反应，“-”表示阴性反应。
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