黑曲霉产木聚糖酶液体发酵工艺的研究
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摘要
木聚糖是自然界存在的可再生的一种植物成分。木聚糖酶能降解木聚糖成木糖或木寡糖。木聚糖酶在饲料、食品、纺织、造纸等行业有着广泛的用途，是现代酶学研究的热点之一。本实验在固体发酵的基础上，进行了适合液体发酵菌株的筛选和工艺的探索，为木聚糖酶液体发酵生产打下基础。通过以木聚糖酶固体发酵高产菌株黑曲霉F27(A.niger F27)为出发菌株，经钴—60诱变，筛选得到适合于液体发酵的菌株L，在5％的麸皮筛选培养基上酶活为1966u／ml。在摇瓶水平上进行了培养基筛选实验，筛选到适合于木聚糖酶发酵主要碳源—玉米芯、主要氮源—蛋白胨、无机盐离子—Mg~(2+)、氨基酸—His，还进行了表面活性剂Tween—80的添加量等因素试验。在得到的发酵培养基的基础上，对发酵条件进行优选，发现在pH值5.5、转速220rpm、500ml摇瓶装量为50ml、温度28～30℃下酶活可达到8226u／ml。在摇瓶发酵工艺优化的基础上进行了发酵罐放大试验，并对发酵罐发酵工艺进行了优化，在玉米芯6％、葡萄糖0.5％、蛋白胨1％和尿素0.3％等的改良培养基上，培养条件为：14h内温度为30℃，14h后28℃，消前pH5.5，罐压0.06mpa～0.08mpa通气1：1.0～1.5搅拌100～150rpm基础上，得到酶活为7200u／ml。
Xylan is a component of plants that can be recycled in nature. Xylanase is the enzyme family that dehydrates xylan into xylose or oligoxylose. Xlyanase has extensive applications in the industries of feed, food, fabric and papermaking, and has become one of the hot spots of modern enzymology. Fermentation technics optimization and screening of the strain suitable for liquid fermentation were carried out on the basis of solid fermentation. The strain L suitable for liquid fermentation was selected by mutation of Co-60 with the A.niger 27,the xylanase high production strain as starting strain. The xylanase activity of the strain L on the selective medium of 5% wheat bran was 1966u/ml. Optimization of the medium was carried out on the level of flask, corncob, tryptone, Mg2+ and Hisdidane were selected for the production of xylanase. Effects of other factors, such as Tween-80 on the production of xylanase were also ascertained. After the orthonagol design, the optimal medium for the production of xylanase on the level of flask was established. The fermentation condition was also optimized and the xylanase activity reached 8226u/ml. The optimal temperature, pH, rotate speed and charge volume for xylanase production were 28-30℃, pH5.5, 220rpm and 50/500ml, respectively. The optimization of fermentation technics on the level of the jar was also carried out. The optimal medium for xylanase production was composed of 6%corncob, l%treptone and 0.3%urea. The optimal temperature, pH, vessel pressure, aeration rate and rotate speed were 30℃ in the first 14hr and 28℃ after 14hr, pH5.5 before sterilization, 0.06mpa~0.08mpa,l: 1.0-1.5 and 100~150rpm, respectively. The xylanase activity reached 7200u/ml after optimization.
Xylan is a component of plants that can be recycled in nature. Xylanase is the enzyme family that dehydrates xylan into xylose or oligoxylose. Xlyanase has extensive applications in the industries of feed, food, fabric and papermaking, and has become one of the hot spots of modern enzymology. Fermentation technics optimization and screening of the strain suitable for liquid fermentation were carried out on the basis of solid fermentation. The strain L suitable for liquid fermentation was selected by mutation of Co-60 with the A.niger 27,the xylanase high production strain as starting strain. The xylanase activity of the strain L on the selective medium of 5% wheat bran was 1966u/ml. Optimization of the medium was carried out on the level of flask, corncob, tryptone, Mg2+ and Hisdidane were selected for the production of xylanase. Effects of other factors, such as Tween-80 on the production of xylanase were also ascertained. After the orthonagol design, the optimal medium for the production of xylanase on the level of flask was established. The fermentation condition was also optimized and the xylanase activity reached 8226u/ml. The optimal temperature, pH, rotate speed and charge volume for xylanase production were 28-30℃, pH5.5, 220rpm and 50/500ml, respectively. The optimization of fermentation technics on the level of the jar was also carried out. The optimal medium for xylanase production was composed of 6%corncob, l%treptone and 0.3%urea. The optimal temperature, pH, vessel pressure, aeration rate and rotate speed were 30℃ in the first 14hr and 28℃ after 14hr, pH5.5 before sterilization, 0.06mpa~0.08mpa,l: 1.0-1.5 and 100~150rpm, respectively. The xylanase activity reached 7200u/ml after optimization.
引文
1．毕瑞明，孙迅．黑曲霉产木聚糖酶发酵条件的研究。工业微生物．2000，30(1)，53-55
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3．陈惠忠 高培基．产木聚糠酶菌株的选育及其液体发酵条件。微生物学报．1990，30(5)351-357
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6．邓远北．应用概率统计。科学出版社：北京，2001
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8．范代娣．细胞培养与蛋白质工程。化学工业出版社：北京，2000
9．方洛云，邹晓庭，许梓蒙．木聚糖酶基因的分子生物学与基因工程。畜禽业 NO2，2002．
10．郭勇．生物制药技术。中国轻工业出版社：北京，2000
11．洪枫，余世袁．调控pH值提高木聚糖酶活力的研究。南京林业大学学报．1999，23(5)．-23-26
12．胡沂淮 邵蔚蓝．木聚糖酶。生命的化学，NO3，J22，281—285
13．侯炳炎．木聚糖酶及其应用。动物科学和动物医学，2002，19：4
14．韩希强．均匀设计和回归软件的应用。江西医药信息，1999，78-82
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19．李孝辉．木聚糖酶在食品和饲料工业中的应用，粮油和饲料工业，1999，NO12，30—32
20．刘相梅，祁蒙．木聚糖酶基因克隆、表达与分泌及定点诱变研究进展。生物工程进展．2001，21(2)．-28-31
21．李蕤，程海燕．产碱性木聚糖酶芽孢杆菌HS的筛选及发酵条件研究。工业微生物．2002，32(2)．-30-33
22．刘超纲，勇强．纤维素和木聚糖复合历导合成木聚糖酶的研究。林产化学与工业．2001，21(1)．-69-73
23．李卫芬，孙建义．木聚糖酶的特性研究。浙江大学学报，农业与生命科学版．2001，27(1)．-103-106
24．欧凯，王书翰．产木聚糖酶优良菌株的选育。纤维素科学与技术．1999，7(4)．-29-33
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27．石军，陈安国．木聚糖酶生产与应用研究进展。饲料工业．2001，22(9)．40-43
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29．吴淑芳，尤纪雪．木聚糖酶处理改善麦草浆漂白特性的研究。西南造纸．2000，29(6)，10-11
30．吴克，刘斌．木霉T6木聚糖酶液态发酵生产研究。食品与发酵工业．2001，27(3)，9-12
31．王海，石波．不同诱导物对Streptomyces olivaceovirdis E-86产高活性木聚糖酶的研究。食品与发酵工业．2002，28(6)，23-26
32．熊宗贵．发酵工艺原理。中国医药出版社：北京，1995
33．徐勇，陈牧．木聚糖酶水解制取低聚木糖的研究。林产化学与工业．2002，22(2)．-57-60
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36．杨革，李亚．细菌耐碱性木聚糖酶产生菌的选育及其酶学性质的初步研究。南京农业大学学报，1999，22(1)，111-113
37．俞文和．抗生素工艺学。辽宁出版社：沈阳，1987
38．杨瑞金，许时婴．具有高木二糖形成活力的木聚糖酶生产菌株的筛选与产酶培养基的优化。工业微生物．2001，31(1)．-23-26
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40．周德庆．工业发酵培养基的特点及其原料组分。工业微生物 1992，22(3)：19-28
41．朱静，严自正．微生物产生的木聚糖酶的功能和应用。生物工程学报，12(4)，375-378，1996
42．张红莲，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)．23-26，30
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2．白云铃 许正宏，孙微，陶文沂．细菌木聚糖酶高产菌株的筛选及产酶条件。微生物学报，2000，40(4)，440—443
3．陈惠忠 高培基．产木聚糠酶菌株的选育及其液体发酵条件。微生物学报．1990，30(5)351-357
4．陈红歌，朱静，梁改芹，严自正，张树政．酸性木聚糖酶产生菌的筛选及产酶条件。微生物学报，1999，Vol 39，No 4
5．佟晓华，张强．高产木聚糖酶菌株的诱变选育。农业与技术．1997，(6)．-28-29
6．邓远北．应用概率统计。科学出版社：北京，2001
7．冯文英，李振岩，常清荣，粱改芹，朱静，严自正．木聚糖酶的制备及其在麦草浆漂白中的应用。中国造纸．2002，21(2)．8-13
8．范代娣．细胞培养与蛋白质工程。化学工业出版社：北京，2000
9．方洛云，邹晓庭，许梓蒙．木聚糖酶基因的分子生物学与基因工程。畜禽业 NO2，2002．
10．郭勇．生物制药技术。中国轻工业出版社：北京，2000
11．洪枫，余世袁．调控pH值提高木聚糖酶活力的研究。南京林业大学学报．1999，23(5)．-23-26
12．胡沂淮 邵蔚蓝．木聚糖酶。生命的化学，NO3，J22，281—285
13．侯炳炎．木聚糖酶及其应用。动物科学和动物医学，2002，19：4
14．韩希强．均匀设计和回归软件的应用。江西医药信息，1999，78-82
15．怀文辉 何秀萍，郭文杰，张博润。微生物木聚糖降解酶的研究进展和应用前景。微生物学通报，2000，VOL27，J2，137—140
16．李永泉．微波诱变选育木聚糖酶高产菌。微波学报．2001，17(1)，-50-53
17．李阜棣，喻子牛，何绍江主编，农业微生物试验指导，农业出版社：北京，1996
18．刘瑞田 曲音波。木聚糖酶分子的结构区域，生物工程进展，1998，NO6，VOL18，26—29
19．李孝辉．木聚糖酶在食品和饲料工业中的应用，粮油和饲料工业，1999，NO12，30—32
20．刘相梅，祁蒙．木聚糖酶基因克隆、表达与分泌及定点诱变研究进展。生物工程进展．2001，21(2)．-28-31
21．李蕤，程海燕．产碱性木聚糖酶芽孢杆菌HS的筛选及发酵条件研究。工业微生物．2002，32(2)．-30-33
22．刘超纲，勇强．纤维素和木聚糖复合历导合成木聚糖酶的研究。林产化学与工业．2001，21(1)．-69-73
23．李卫芬，孙建义．木聚糖酶的特性研究。浙江大学学报，农业与生命科学版．2001，27(1)．-103-106
24．欧凯，王书翰．产木聚糖酶优良菌株的选育。纤维素科学与技术．1999，7(4)．-29-33
25．曲音波，高培基．制浆造纸用木聚糖酶的研究进展。生物工程进展．1998，18(6)．36-40
26．孙迅，王宜磊．木聚糖酶高产菌株Bacillus pumilus H-101的筛选及产酶条件的研究。微生物学杂志．1997，17(2)．-17-22
27．石军，陈安国．木聚糖酶生产与应用研究进展。饲料工业．2001，22(9)．40-43
28．天津轻工业学院．工业发酵分析。中国轻工出版社：北京，1991
29．吴淑芳，尤纪雪．木聚糖酶处理改善麦草浆漂白特性的研究。西南造纸．2000，29(6)，10-11
30．吴克，刘斌．木霉T6木聚糖酶液态发酵生产研究。食品与发酵工业．2001，27(3)，9-12
31．王海，石波．不同诱导物对Streptomyces olivaceovirdis E-86产高活性木聚糖酶的研究。食品与发酵工业．2002，28(6)，23-26
32．熊宗贵．发酵工艺原理。中国医药出版社：北京，1995
33．徐勇，陈牧．木聚糖酶水解制取低聚木糖的研究。林产化学与工业．2002，22(2)．-57-60
34．禹慧明，林勇．木聚糖酶高产菌株选育。工业微生物．2002，32(1)，43-44，53
35．杨瑞鹏，赵学慧．木聚糖酶高产菌株筛选与鉴定。华中农业大学学报．1990，9(3)，311-314
36．杨革，李亚．细菌耐碱性木聚糖酶产生菌的选育及其酶学性质的初步研究。南京农业大学学报，1999，22(1)，111-113
37．俞文和．抗生素工艺学。辽宁出版社：沈阳，1987
38．杨瑞金，许时婴．具有高木二糖形成活力的木聚糖酶生产菌株的筛选与产酶培养基的优化。工业微生物．2001，31(1)．-23-26
39．周德庆主编．微生物学实验手册，上海科技出版社，1983
40．周德庆．工业发酵培养基的特点及其原料组分。工业微生物 1992，22(3)：19-28
41．朱静，严自正．微生物产生的木聚糖酶的功能和应用。生物工程学报，12(4)，375-378，1996
42．张红莲，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)．23-26，30
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