木聚糖酶产生菌的筛选与发酵条件的初步优化
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摘要
木聚糖(xylanase EC 3．2．1．8)是植物体内存在的半纤维素的主要成分，是自然界中仅次于纤维素的最为丰富的多糖。木聚糖酶是戊聚糖酶的一种，可以将木聚糖降解成低聚木糖和木糖的复合酶系，主要应用于造纸、饲料、医药、能源、纺织等行业，具有极大的商业价值和广阔的应用前景。
本研究从四川青城山、九寨沟、拉萨、山东临祈等地采集林木长期自然覆盖下的土壤。用RBB-xylan选择培养基进行初筛，根据不同菌株所形成的透明圈的大小，确定需要复筛的菌株，经产酶基本培养基复筛后，筛选到4株产酶稳定且活力较高的菌株，选择其中产木聚糖酶较高的一株X-18，作为进一步发酵条件优化的实验菌株。该菌细胞形态为杆状，革兰氏染色为阳性，有芽孢，分类鉴定的结果表明，菌株X-18属于芽孢杆菌属(Bacillus sp．)细菌，因此，该菌命名为Bacillus sp．No X-18。进行单因素试验，结果显示甘露糖、蔗糖为最佳产酶的碳源，其木聚糖酶活力分别为17．24 IU／mL、16．68 IU／mL。最佳氮源为蛋白胨，其木聚糖酶活力为18．5 IU／mL：双因素试验，结果显示木聚糖和阿拉伯糖的碳源组合菌株酶活最高，为19．86 IU／mL,蛋白胨和草酸铵的组合最有利于产酶，酶活为19．71 IU／mL；培养基成分的综合优化试验，结果显示最佳培养基是：1．5％蔗糖、1％阿拉伯糖、0．37％NH_4Cl、0．37％酵母膏、0．5％K_2HPO_4、0．02％MgSO_4·7H_2O，pH 8．0，其木聚糖酶活力可达74．21IU／mL。最佳培养条件为：起始PH8．0，50ml装液量，32℃，发酵时间72h。
对Bacillus sp．No X-18木聚糖酶的部分酶学特性进行了研究，结果表明，菌株X-18酶活最适温度为50℃，其热稳定性较好，在50℃保温1小时仍达到100％的酶活力。该酶pH值范围较广，在pH 5．0至pH8．0范围内均能保持较高的活性，最适pH值为5．0，与其相关报道的研究相比，该木聚糖酶酸碱性较好。
Xylan is the main component of hemicellulose in the plant, and it is the second only to cellulose in natural amylose. Xylanase is a kind of pentosanase which can degrade the xylan to XyLo-oligosaccharides and xylose. It's mainly applied in paper making, foodstuff, medicine energy sources, and textile industry and so on. Both commerce value and application foreground are considerable. All bacteria in our study were collected from soil covered by lots of wood and leaves for long time in different area,the districts contain Qing Cheng mountain, Jjiuzhaigou valley in Si chuan Province, Lhasa, Linqi city in Shandong Province and else. The bacteria were first screened by RBB-xylan culture medium according to whether appear hydrolyzed holecircle or not. And the bacteria with clarity circle were cultivated in ferment medium for the next screening. We obtained four excellent xylanase enzyme producing strains through the anterior process. Finally we choose the high enzyme activity strain No. X-18 to carry out ferment condition optimization experiment. The Gram stain and spore stain are both Positive Rod toward the bacteria. The result of classification and characterization indicate the No. X-18 belongs to Bacillus sp. The result of single factor experiment show the most appropriate carbon sources are mannose and cane sugar corresponding to the enzyme activity is 17.24 IU/mL 16.68 IU/mL respectively, the most appropriate nitrogen sources is peptone corresponding to the enzyme activity is 18.5 IU/mLon the other hand, the result of double factors reveal the most high activity 19.86 IU/mL is made up of the compound of xylan and arabinose refer to the carbon source, When use the compound of peptone and ammonium oxalate as nitrogen source the enzyme is considerable up to 19.71 IU/mL.The optimal medium composition as 1.5% sucrose, 1% arabinose,0.37% NH_4Cl, 0.37% yeast extract, 0.5% K_2HPO_4, 0.02%MgSO_4·7H_2O, pH 8.0 after a series of the single factor experiment, double factors experiment and medium component optimization experiment. Furthermore, the optimal culture condition described below: 50mL initial PH 8.0 ferment medium in 250ml flask at 32℃for 72h. The enzyme activity can reach 74.21IU/mL at the optimal condition.We investigate part zymological property about the Bacillus sp.No X-18 xylanase, find the optimum temperature is50℃,And its heat stability is fine, it keep 100% activity after incubating at 50℃for 1 hour. Its pH range also is wide.the activity can keep high from pH5.0to pH8.0,and the optimum pH is 5.0,tolerance of the enzyme is much beffer than it reported already.
本研究从四川青城山、九寨沟、拉萨、山东临祈等地采集林木长期自然覆盖下的土壤。用RBB-xylan选择培养基进行初筛，根据不同菌株所形成的透明圈的大小，确定需要复筛的菌株，经产酶基本培养基复筛后，筛选到4株产酶稳定且活力较高的菌株，选择其中产木聚糖酶较高的一株X-18，作为进一步发酵条件优化的实验菌株。该菌细胞形态为杆状，革兰氏染色为阳性，有芽孢，分类鉴定的结果表明，菌株X-18属于芽孢杆菌属(Bacillus sp．)细菌，因此，该菌命名为Bacillus sp．No X-18。进行单因素试验，结果显示甘露糖、蔗糖为最佳产酶的碳源，其木聚糖酶活力分别为17．24 IU／mL、16．68 IU／mL。最佳氮源为蛋白胨，其木聚糖酶活力为18．5 IU／mL：双因素试验，结果显示木聚糖和阿拉伯糖的碳源组合菌株酶活最高，为19．86 IU／mL,蛋白胨和草酸铵的组合最有利于产酶，酶活为19．71 IU／mL；培养基成分的综合优化试验，结果显示最佳培养基是：1．5％蔗糖、1％阿拉伯糖、0．37％NH_4Cl、0．37％酵母膏、0．5％K_2HPO_4、0．02％MgSO_4·7H_2O，pH 8．0，其木聚糖酶活力可达74．21IU／mL。最佳培养条件为：起始PH8．0，50ml装液量，32℃，发酵时间72h。
对Bacillus sp．No X-18木聚糖酶的部分酶学特性进行了研究，结果表明，菌株X-18酶活最适温度为50℃，其热稳定性较好，在50℃保温1小时仍达到100％的酶活力。该酶pH值范围较广，在pH 5．0至pH8．0范围内均能保持较高的活性，最适pH值为5．0，与其相关报道的研究相比，该木聚糖酶酸碱性较好。
Xylan is the main component of hemicellulose in the plant, and it is the second only to cellulose in natural amylose. Xylanase is a kind of pentosanase which can degrade the xylan to XyLo-oligosaccharides and xylose. It's mainly applied in paper making, foodstuff, medicine energy sources, and textile industry and so on. Both commerce value and application foreground are considerable. All bacteria in our study were collected from soil covered by lots of wood and leaves for long time in different area,the districts contain Qing Cheng mountain, Jjiuzhaigou valley in Si chuan Province, Lhasa, Linqi city in Shandong Province and else. The bacteria were first screened by RBB-xylan culture medium according to whether appear hydrolyzed holecircle or not. And the bacteria with clarity circle were cultivated in ferment medium for the next screening. We obtained four excellent xylanase enzyme producing strains through the anterior process. Finally we choose the high enzyme activity strain No. X-18 to carry out ferment condition optimization experiment. The Gram stain and spore stain are both Positive Rod toward the bacteria. The result of classification and characterization indicate the No. X-18 belongs to Bacillus sp. The result of single factor experiment show the most appropriate carbon sources are mannose and cane sugar corresponding to the enzyme activity is 17.24 IU/mL 16.68 IU/mL respectively, the most appropriate nitrogen sources is peptone corresponding to the enzyme activity is 18.5 IU/mLon the other hand, the result of double factors reveal the most high activity 19.86 IU/mL is made up of the compound of xylan and arabinose refer to the carbon source, When use the compound of peptone and ammonium oxalate as nitrogen source the enzyme is considerable up to 19.71 IU/mL.The optimal medium composition as 1.5% sucrose, 1% arabinose,0.37% NH_4Cl, 0.37% yeast extract, 0.5% K_2HPO_4, 0.02%MgSO_4·7H_2O, pH 8.0 after a series of the single factor experiment, double factors experiment and medium component optimization experiment. Furthermore, the optimal culture condition described below: 50mL initial PH 8.0 ferment medium in 250ml flask at 32℃for 72h. The enzyme activity can reach 74.21IU/mL at the optimal condition.We investigate part zymological property about the Bacillus sp.No X-18 xylanase, find the optimum temperature is50℃,And its heat stability is fine, it keep 100% activity after incubating at 50℃for 1 hour. Its pH range also is wide.the activity can keep high from pH5.0to pH8.0,and the optimum pH is 5.0,tolerance of the enzyme is much beffer than it reported already.
引文
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[2]许君．细菌碱性木聚糖酶菌种筛选、产酶条件与酶学性质研究[硕士论文]．郑州：河南 农业大学，2004年．
[3] Huang DF、Lm M. Geme Engineering of Agricultural Microorgnism, Beijing:Science Press,2001.
[4] Fang LY, Zou XT, Xu ZR. Molecular Biology and Gene Engineering of XylanaseGene. China Feed, 2002, 13(7): 11-13.
[5]蔡敬民，张洁，于宙等．芽孢杆菌木聚糖酶的发酵条件研究．工业微生物，1996，26(2)： 17-19．
[6] 周世宁，陆勇军，杜扬．木聚糖降解菌的筛选和木聚糖酶性质的研究．中山大学学报 (自然科学版)，1996，35(1)：91-96．
[7]许君，任艳丽，陈红歌等．碱性木聚糖酶产生菌的筛选与产酶条件．生物技术，2004， 14(3)：52-54．
[8]崔月明，木聚糖酶产生菌的选育、酶学性质及xynA基因的克隆表达[硕士论文]．广西， 广西大学．2005．
[9]徐明，邵佩兰．木聚糖酶产生菌的土壤筛选．宁夏农学院学报，1999，20(3)：61-64．
[10]孙迅，朱陶，朱启忠等．产胞外木聚糖酶放线菌的分离与筛选．微生物学杂志，1998， 18(4)：29-32．
[11]刘琳．木聚糖酶菌株的筛选及p-1，4-木聚糖酶基因的克隆[硕士论文]．武汉：湖北大学．
[12]黄运红，龙中儿，李素珍等．木聚糖酶高产微生物的筛选和鉴定．江西师范大学学报 (自然科学版)．2002，26(3)：245-248．
[13]陈从瑾，黄克瀛，周虎．木聚糖酶产生菌的筛选及其酶学性质初步研究．中南林业科 技大学学报．2007，27(2)：70-74．
[14] Poutanen, K, et al,Enzyme Biomass conversion,ACS Symp.ser.460,America Chemical society,Washington,DC.USA,1991,426.
[15]张红莲，姚斌，范云六．木聚糖酶的分子生物学及其应用．生物技术通报，2002(3)： 23-30．
[16] Kulkami N,Shendye A and Rao M.Molecular and biotechnological aspects of xylanase.FEMS Microbiol Rev,1999,23:411-456.
[17]秦玉花．高产木聚糖酶芽孢杆菌的分离筛选及其酶性质研究[硕士论文]．雅安：四川 农业大学，2006．
[18] Okazaki. W. Akiba. T,Horikoshi,K.et.al. Purification and characterization of xylanase from Flaws. Journal of Fermentation Technology,1985,62,361-369.
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