CN110004070B - 一株产木聚糖酶黑曲霉基因工程菌及其构建方法与应用 - Google Patents
一株产木聚糖酶黑曲霉基因工程菌及其构建方法与应用 Download PDFInfo
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Abstract
本发明公开了一株产木聚糖的黑曲霉基因工程菌,该菌株中Msb2基因失活;所述Msb2基因的核苷酸序列如SEQ ID NO.1所示,所述Msb2基因失活后的核苷酸序列如SEQ ID NO.2所示。本发明还公开了上述基因工程菌的构建方法。本发明进一步地公开了基因工程菌在发酵生产木聚糖酶中的应用。本发明通过构建Msb2基因的黑曲霉基因工程菌,使黑曲霉在发酵产木聚糖酶过程中生物膜产量减少,并且黏附性变弱,提高了木聚糖酶的产量,缩短了发酵周期。
Description
技术领域
本发明属于生物基因工程领域,具体涉及一株产木聚糖的黑曲霉基因工程菌及其构建方法与应用。
背景技术
植物纤维是自然界中储存量最大、最丰富,也是最廉价的一类可再生资源,其中半纤维素约占15%~30%。作为半纤维素的主要组成成分,木聚糖广泛分布于高等植物的细胞壁中,是自然界中存储量仅次于纤维素的多糖。木聚糖酶是一类可以将木聚糖降解成低聚糖和木糖的水解酶类,主要由β-1,4-内切木聚糖酶和β-木糖苷酶组成,其功能分别是切断木聚糖主链中的木糖苷键以及将寡聚糖进一步降解为单体木糖。
同时木聚糖酶作为一种工业酶制剂,在诸多领域具有广泛应用。在植物性饲料中,添加木聚糖酶可以降解饲料中的抗营养因子非淀粉多糖,从而提高禽畜对营养物质的利用率。在食品行业,木聚糖酶可以将半纤维素含量较高的农业废弃物降解成为低聚木糖。在生物能源转化领域,木聚糖酶可与纤维素酶系协同作用,将植物纤维降解为戊糖和己糖,并以其作为发酵底物,进一步通过微生物生产乙醇。在生物制浆漂白领域,木聚糖酶可以作为生物漂白助剂应用于造纸工业。
目前,木聚糖酶主要依靠微生物发酵法获得,现已知能够合成木聚糖酶的微生物主要涉及真菌、链霉菌、细菌和酵母等。曲霉属菌株是生产木聚糖酶的优良菌株,成为国内外木聚糖酶生产研究的重点。
菌丝体固定在谷梗或其他一些惰性材料上,或者采用海藻酸钠等载体材料包埋的方法,重复使用,既降低了发酵液粘度,利于氧气及营养物质的传递,提高了细胞利用率和生物反应速率,同时也便于产物的分离提取等后处理加工。近年来,丝状真菌的固定化发酵生产得到了越来越多的关注。
发明内容
发明目的:为了解决现有技术中黑曲霉发酵生产木聚糖酶时产量低、发酵周期长的问题。
本发明还要解决的技术问题是,提供上述黑曲霉基因工程菌的构建方法。
本发明最后要解决的技术问题是提供上述黑曲霉基因工程菌在发酵制备木聚糖酶中的应用。
发明内容:
一株产木聚糖的黑曲霉菌,其分类命名为黑曲霉菌(Aspergillus niger),菌株号为SJ1,已保藏于中国典型培养物保藏中心,保藏时间为2019年02月28日,保藏编号为CCTCCNO:M2019115,保藏地址为中国·武汉·武汉大学,邮编430072。该菌株通过稀释土壤,以基础木聚糖筛选培养基筛选所得,培养基配方为木聚糖10g/L,KH2PO4、KH2PO4、MgSO4、NH4NO3、NaCl、MnS04、ZnSO4、FeSO4、琼脂用量均2%。后经菌种鉴定为黑曲霉,***进化树如图1所示。
上述产木聚糖的黑曲霉菌在制备木聚糖中的应用在本发明的保护范围之内。
一株产木聚糖的黑曲霉基因工程菌,该菌株中Msb2基因失活。所述的失活是指该菌株中Msb2基因不能表达得到具有活性的蛋白质,所述的失活可以是在Msb2基因中***一段核苷酸序列,或者缺失黑曲霉中的Msb2基因的部分序列,或者用其他核苷酸序列替换Msb2基因的全序列。
其中,所述Msb2基因的核苷酸序列如SEQ ID NO.1所示,所述Msb2基因失活后的核苷酸序列如SEQ ID NO.2所示。Msb2基因为黑曲霉CBS513.88信号黏蛋白Msb2(Gene ID:4986934)同源蛋白,90%相似度。通过同源重组的方法将Msb2全长2955bp替换成潮霉素抗性表达元件2740bp。
其中,所述黑曲霉为黑曲霉SJ1,保藏于中国典型培养物保藏中心,保藏号为CCTCCNO:M201911。
上述产木聚糖的黑曲霉基因工程菌的构建方法,包括如下步骤:
(1)提取黑曲霉SJ1基因组;
(2)构建基因敲除片段△Msb2,所述基因敲除片段的核苷酸序列如SEQ ID NO.3所示;
(3)制备黑曲霉原生质体;
(4)将基因敲除片段导入原生质体中进行同源重组,得到Msb2基因失活黑曲霉基因工程菌。
上述产木聚糖的黑曲霉基因工程菌在制备木聚糖酶中的应用在本发明的保护范围之内。
具体的应用方法如下:以产木聚糖的黑曲霉基因工程菌为发酵菌剂,多孔纤维材料为固定化载体,发酵制备木聚糖酶,所述的固定化载体为聚氨酯。
所述的固定化载体通过如下方法进行预处理:
将固定化载体用0.5~2M的NaOH浸泡30min~2h,用水冲洗至pH为中性,在0.5~2MHCl中浸泡30min-2h,用水冲洗至pH为中性,烘干,得到经过预处理的固定化载体;
作为优选:将固定化载体用1M的NaOH浸泡1h,用水冲洗至pH为中性,在1M HCl中浸泡1h,用水冲洗至pH为中性,烘干,得到经过预处理的固定化载体。
所述发酵的条件如下:
发酵培养基:木聚糖10~70g/L、NaNO3 2~6g/L、KH2PO4 0.3~2g/L、MgSO4·7H2O0.1~0.8g/L、吐温-80 0.1~0.8g/L,以麸皮浸出液配制;优选木聚糖30g/L、NaNO34.2g/L、KH2PO4 1.0g/L、MgSO4·7H2O 0.5g/L、吐温-80 0.5g/L;
所述的麸皮浸出液按照如下方法制备:称取15~60g/L的麸皮,加入800mL水,煮沸30min-2h后,过滤得滤液,用水再定容至1L;优选称取40g/L的麸皮,加入800mL水,煮沸1h后,过滤得滤液,用水再定容至1L。
发酵条件如下:28~32℃、150~300r/min培养3~6d,优选30℃、220r/min培养4d。
有益效果:
本发明通过对生物膜形成相关Msb2基因进行敲除,减少生物膜的形成,进而提高木聚糖酶的产量,提高菌体对底物的利用能力,缩短发酵周期。本发明以聚氨酯为固定化载体实现了摇瓶发酵产木聚糖酶,酶活稳定,酶活由1515.34U/mL提高至1633.27U/mL,产量提高7.8%,周期由4d缩短至3.5d。本发明使用培养基为纯清液培养基,发酵结束得发酵液上清即为木聚糖酶液,可直接用于后续实验。
附图说明
图1为黑曲霉原始菌的***进化树。
图2为黑曲霉SJ1基因组的电泳图,图1中,1、2泳道为黑曲霉SJ1基因组,M为10000bp DNA marker。
图3为PAN7-1质粒图谱;
图4为Msb2基因上游同源臂、下同源臂及抗性基因片段的电泳图,M为2000bp DNAmarker,泳道1为Msb2上游同源臂,泳道2为抗性基因片段,泳道3为Msb2下游同源臂。
图5为基因△Msb2敲除片段的电泳图,泳道1为△Msb2敲除片段,M为5000bp DNAmarker;
图6为结晶紫染色图;
图7为结晶紫染色OD值差异图;
图8为原始黑曲霉和黑曲霉基因工程菌的发酵结果;
图9为黑曲霉原始菌和Msb2敲除菌株固定化发酵4d后,载体菌量附着图。
具体实施方式
实施例1:提取黑曲霉SJ1基因组。
将黑曲霉孢子接入YPD培养基,30℃、220rpm培养12h。12000rpm、4℃下离心5min,弃上清液。用PBS(8g/L NaCl,0.2g/L KCl,1.44g/L Na2HPO4,0.24g/L KH2PO4,pH 7.4)洗两遍。然后立即离心(4℃、12000rpm、2min),弃上清。将菌体在液氮研磨成粉状后,根据TaKaRa MiniBEST Plant Genomic DNA Extraction Kit试剂盒说明书进行操作。通过琼脂糖凝胶电泳测定黑曲霉基因组浓度如图2所示。
实施例2:构建基因△Msb2敲除片段。
(1)设计引物:PCR获得Msb2基因上游同源臂和下游同源臂,所述Msb2基因上游同源臂的核苷酸序列如SEQ ID NO.4所示,所述Msb2基因下游同源臂的核苷酸序列如SEQ IDNO.5所示,以pAN7-1质粒为模板PCR扩增出抗性表达元件,pAN7-1质粒图谱如图3,对Msb2上游同源臂、下游同源臂和抗性片段进行胶回收纯化,使用0.8%(0.8g/100mL)琼脂糖凝胶对纯化的Msb2上游同源臂、下游同源臂、抗性元件进行电泳检测,结果如图4。然后以游同源臂、下游同源臂、抗性元件采用overlap pcr的方法构建出Msb2的敲除片段△Msb2,对△Msb2敲除片段胶回收纯化,琼脂糖凝胶电泳及测序验证。△Msb2敲除片段结果如图5。引物如下表:
表1 △Msb2敲除片段构建引物
PCR体系如下:
胶回收获得Msb2上下同源臂,抗性片段和△Msb2敲除片段:
使用takara试剂盒(TaKaRa MiniBEST Agarose Gel DNA Extraction KitVer.4.0)试剂盒纯化。
实施例3:黑曲霉原生质体的制备与转化。
1.将黑曲霉接种到3个PDA平板,长满孢子,待用。
2.加适量刮孢缓冲液(在0.9% NaCl中加入0.02%的tween80,115℃灭菌20min。)到平板中,用涂布棒将孢子刮下,得到孢子液,血球计数板计数。
3.接种107个孢子到50ml YPD培养基,30°220rpm培养10h。
4.配制酶液,(Yatalase,lysing enzyme,崩溃酶,蜗牛酶各0.1g/10ml,葡聚糖酶50μl/10ml,纤维素酶400μl/10ml),再用20ml无菌注射器过滤除菌到灭菌的三角瓶中。
5.孢子萌发完毕后,用Miracloth过滤,留下菌丝,称重,取2g菌丝至酶液中。30°200rpm下酶解3h。
6.酶解完成后,用Miracloth过滤,取滤液,4°5000rpm离心10min。去上清,加入1ml1M山梨醇,用枪吹吸混匀,再加入15ml山梨醇,离心,去上清。然后再重复一次。去掉上清,加入1ml缓冲液(100ml含有KCl 4.47g,CaCl2 0.735g,MOP 0.2093g,KOH调pH至6.0),用枪吹吸混匀,然后放4°冰箱待用。
9.吸取△Msb2敲除片段到1.5ml灭菌离心管中
10.之前做好的原生质体用1ml移液枪轻柔吹吸几次混匀(如果是现做的则无需混匀或只需上下颠倒几次就行)
11.吸100μl的原生质体到1.5ml离心管中,与10ul△Msb2敲除片段混合,再加入50μl的缓冲液A(100ml含有PEG8000 25g,CaCl2 1.47g,KCl 4.47g,10mM Tris pH7.5),混匀,置于冰上并计时20min。
12.20min后,加入1ml缓冲液A,上下颠倒几次,放置在室温并计时20min。
20min后,吸取200,300,500μl涂布于潮霉素浓度为150mmol/L的PDA培养基,正置培养。
实施例4:菌落PCR验证。
1.将黑曲霉接种到3个PDA平板,长满孢子,待用。
2.加适量刮孢缓冲液到平板中,用涂布棒将孢子刮下,得到孢子液,血球计数板计数。
3.取2ul孢子加入缓冲液(100ml含有0.01M Tris,10Mm EDTA,1M KCl,PH9.5)中,95℃水浴10分钟,取出冰浴5分钟,为模板,以验证引物△Msbcheck1(序列:CGCTCTGTCCATTCATTTCAGC)和△Msbcheck2(序列:ATGGCAACATCAACAACGCT)引物进行PCR验证,pcr得到片段进行测序验证,得到敲除Msb2的敲除菌株。
实施例5:结晶紫染色实验
将原始黑曲霉和敲除菌分别接种到PDA平板,待长满孢子,加3mL刮孢缓冲液到平板中,用涂布棒将孢子刮下,转移至灭菌的5mL离心管中,用刮孢缓冲液定容到2mL,得到孢子液。用血球计数板定量并稀释至106个/mL,随后继续稀释至105,104,103。
在24孔板中预先加入1ml的曲霉基本培养基,然后将不同浓度的孢子液取2uL接种到培养基中。30℃下静置培养48h,使黑曲霉在孔板底部成膜。之后倒掉培养基,用PBS冲洗2遍后,加入0.1%的结晶紫染色15min。之后倒掉结晶紫,用PBS冲洗2遍后,加入冰醋酸在震荡仪中放置30min,使结晶紫脱色。然后进行观察以及酶标仪检测OD570。结晶紫染色孔板结果如图6所示,OD值差异图如图7所示。
实施例6:发酵验证。
1,载体为聚氨酯材料,预处理方法:将载体用1M的Na(OH)浸泡1h,用纯水洗净后,在1M HCl中浸泡1h,之后用纯水冲洗至pH为中性,再放入65℃烘箱中烘干至恒重。将剪成相同大小的载体适量加入培养基中,与培养基一同115℃灭菌20min,冷却待用。
2,麸皮浸出液:称取40g/L的麸皮,加入800mL水,煮沸1h后,用4层纱布过滤得滤液,用水再定容至1L。
3,培养基配方:斜面培养基:马铃薯葡萄糖琼脂(PDA)培养基。种子培养基蔗糖10g/L,以麸皮浸出液配制。发酵培养基(g/L):木聚糖30、NaNO3 4.2、KH2PO4 1.0、MgSO4·7H2O 0.5、吐温-80 0.5,以麸皮浸出液配制。以上培养基在121℃、105Pa条件灭菌15min,待用。
4.将保藏菌种接种于PDA斜面培养基中,置于30℃恒温培养箱培养3d后,接入装有100mL种子培养基的500mL摇瓶中,30℃、220r/min培养24h。
5.将培养好的种子以5%(体积分数)接种至发酵培养基,30℃、220r/min培养4d。
6.粗酶液制备:发酵液用冷冻离心机5000rpm离心10min,即的粗酶液。
7.木聚糖酶活测定:取适当稀释的粗酶液50μL,加入0.45mL的0.05mol/L的柠檬酸缓冲液(pH 4.8)及1mL、1.2%(质量分数)的木聚糖底物,在50℃条件下反应30min。木糖生成量的测定参照文献进行。酶活定义(U):在50℃条件下反应30min,每分钟降解底物生成1μmol木糖所需的酶量。
8.以上述方法,将黑曲霉SJ1和Msb2敲除菌株接种到加0.1g载体的100mL种子培养基培养24h后转接载体至发酵培养基,30℃、220r/min培养4d,不同时间取发酵液检测酶活,酶活数据如图8所示。将固定化发酵4d后的载体取出,用PBS冲洗3遍洗掉吸附的菌丝。得到附着菌丝的载体,如图9所示,原始菌的菌量较多,敲除菌的菌量适中。
序列表
<110> 南京工业大学
<120> 一株产木聚糖酶黑曲霉基因工程菌及其构建方法与应用
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2955
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgatcgcgc ccacgacttc attgttggcc gcgttcctgt ccttgggcgg cctcgagctc 60
gtcgctgccc gggaggcagg acagatccgg gacttgggag cgcgtgaggt tagctccgcc 120
gttcctggtt cctcccagcc cgttacgcct gtcgagttga caaccaggtc cgaaacgagt 180
acgcatgatt cagcgccgac ttcggacaat gaacaaacaa gcccgatcgt tgacccttca 240
cctgctgtta tcgttgttcc tgtaaccatt tccgtggacg ccaatggtgt tacgcatacc 300
attgtgggca cggcacccaa cgttcccggt gcttcaacga caactagctc cacccttgag 360
cgtgcagaga gtaccccggc ggcagctgct gctacttcga cggcgagcgc gactacatcg 420
gacaaggatt caactaccag tgactcggaa aagcagaaat ctacatccga atccgagaaa 480
cacacttccg cgtctgaatc gtcagcgaaa ccagaggcag cttcttcgtc agcagcagca 540
acgacggcag agacagcttc ttcgtcggcc gcagcggcag cagcagcagc tactacatct 600
agccagagtt cgttggctga ctcctgggag agcttcattt caagtatctt ggggtcctct 660
accagcacca gcgcaacctc cacggccagc cctaccgcat cgccagccga gtccagctct 720
gcaacccccg ctcccgcttc aagctctgct ggcgtcattg tagatgtcac ctcgagcaag 780
agttctacaa gcgatagctc gacgacttcg gagagctcta cggctaagga ttccaccact 840
cagagctctt ccagcccagc atcctcgacc cctgcttcca ctacggagtc ttcggccgag 900
gcttcgcaat cggaatctgc cacttctagc tcgaaggcag agacttcgag tcaacttatc 960
actagccaga gcgcatctgc ccagagttct tccgctcagg tccctctggc tgagtctact1020
tcgaagtccg agtcttcaac ccagagctcc tcgagtcagg ttacatctgt tcaggccacc1080
tcatcaggcg cagcctctca gactactggg tcagcatcct cgagcctcat cccgtctgca1140
tctgaagatg tgacctcttc ttcttccacc ccctcatcat caggaggtgt cctcggcttc1200
ctctcggacc tcttccctac taccaccact tccagcggat ctgcctctgc tactagcaag1260
agtgcaagcg agtctgccga ctccagtgct ttccctctga gcctccctac catctccgtt1320
agtgttcctg tggttacgcc tgcctcatct tctggatcgg tcaatattgt ttctgcccta1380
cttggcacat cctcaggcgc ttccttggaa ccttcggcca ttagtctggg tatctcggcc1440
acttccacgg ctcttatccc cggagcttcc agcggaaccc agagctcctc agcgggagcc1500
tcgactccga tcatcagcgg tggcattgtc atcatgccca cttcgactga atctgcttct1560
gcttccgtct caggatctgc ctcgacgggt ctttttggaa ccacttcaga atcagcatct1620
tcattggctt cgggatccgc ctctggatct gcgggcgtta ttcctacatc ggtgtctggg1680
tctgtatctg gatctgcagg actgttccct acgtcggcgt ctggatccgt ttcgggatct1740
gcaggtgtta ttcctacatc ggctcctggc tctgcatctg gatctgtgtc gggatctgct1800
tctggatccg tttcaggctc tgccattggc accagctctg catccaagtt tacctccatt1860
tctggccctg cctcttcgat tcctttgatc acttccacca aggagtcctc gggtctggta1920
tctgcgacgc cttcctatgc cccgtccact accagcaagg aaacgcccac gcctatcgca1980
caacccacca cgacgaccac ctcggccgct gagacgactg tcgcccccaa accgaccacg2040
accacctccg attctgacaa ctacctgccg tcaagcattc tcgtggttga gcccacaact2100
actacgtcac tgagcacggg cacagctacc cattcgagta ccagcactgc tgctgctcag2160
cttcctggat cgatctctcc ttctggtggt cttcccgagc agccggccaa cgataccttg2220
gtccaaatcg gattcaatgg caaactgcgc tggagctttg tggccacaac gcccttgtct2280
tcctcccaga tcttcatgta tgttcccgag ggtatgggct acgcgttgca gctcaatgac2340
tctgacgtgg tcatgtacga cctccaaccc tacgacaact cggcaagcac tggctacatt2400
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cacaacccta tttcgaggct ctacgagcag cccaacgaga cggtcaagac attgatggcc2520
atgatcgacc cttccatccc tcttctggtg ggcgactcgg gttcttccag tggcagctcg2580
tccaacagcg gtggatcggg tagcagcagc agcaacggcg atggcagtga cggtggctac2640
gaccaaagcg atgctggtac cagctcatct ggaaccacca aggctagtgc cgttggtata2700
ggatgtggtg ctgtggccgg tgctgcagca tatggtgctg gtatgttctg ggttgcgcgt2760
cggtaccgga agaagcgcca gttgcaccag cgctccccct cgagcgtcga ccagatgagc2820
gaaggccgcg gcgctgggtc cgtcttcggc gcaggtggcc gtctctcccg caacagccag2880
aacagccggg gcactggccg cacccagatg atcagcgcgc cggtgatggc cgagaactcg2940
ctcggctgga actaa 2955
<210> 2
<211> 2798
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gctattgtta cccaaccatt cgaatcaatt aaaatccgcc gcctccacca tttgtagaaa 60
aatgtgacga actcgtgagc tctgtacagt gaccggtgac tctttctggc atgcggagag 120
acggacggac gcagagagaa gggctgagta ataagccact ggccagacag ctctggcggc 180
tctgaggtgc agtggatgat tattaatccg ggaccggccg cccctccgcc ccgaagtgga 240
aaggctggtg tgcccctcgt tgaccaagaa tctattgcat catcggagaa tatggagctt 300
catcgaatca ccggcagtaa gcgaaggaga atgtgaagcc aggggtgtat agccgtcggc 360
gaaatagcat gccattaacc taggtacaga agtccaattg cttccgatct ggtaaaagat 420
tcacgagata gtaccttctc cgaagtaggt agagcgagta cccggcgcgt aagctcccta 480
attggcccat ccggcatctg tagggcgtcc aaatatcgtg cctctcctgc tttgcccggt 540
gtatgaaacc ggaaaggccg ctcaggagct ggccagcggc gcagaccggg aacacaagct 600
ggcagtcgac ccatccggtg ctctgcactc gacctgctga ggtccctcag tccctggtag 660
gcagctttgc cccgtctgtc cgcccggtgt gtcggcgggg ttgacaaggt cgttgcgtca 720
gtccaacatt tgttgccata ttttcctgct ctccccacca gctgctcttt tcttttctct 780
ttcttttccc atcttcagta tattcatctt cccatccaag aacctttatt tcccctaagt 840
aagtactttg ctacatccat actccatcct tcccatccct tattcctttg aacctttcag 900
ttcgagcttt cccacttcat cgcagcttga ctaacagcta ccccgcttga gcagacatca 960
ccatgcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg1020
tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag1080
gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt1140
atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg1200
aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag1260
acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga1320
tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg1380
gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact1440
ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga1500
tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca1560
acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt1620
tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta1680
tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc1740
tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca1800
atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg1860
ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg1920
tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat1980
agagtagatg ccgaccgcgg gatccactta acgttactga aatcatcaaa cagcttgacg2040
aatctggata taagatcgtt ggtgtcgatg tcagctccgg agttgagaca aatggtgttc2100
aggatctcga taagatacgt tcatttgtcc aagcagcaaa gagtgccttc tagtgattta2160
atagctccat gtcaacaaga ataaaacgcg ttttcgggtt tacctcttcc agatacagct2220
catctgcaat gcattaatgc attgactgca acctagtaac gccttncagg ctccggcgaa2280
gagaagaata gcttagcaga gctattttca ttttcgggag acgagatcaa gcagatcaac2340
ggtcgtcaag agacctacga gactgaggaa tccgctcttg gctccacgcg actatatatt2400
tgtctctaat tgtactttga catgctcctc ttctttactc tgatagcttg actatgaaaa2460
ttccgtcacc agcncctggg ttcgcaaaga taattgcatg tttcttcctt gaactctcaa2520
gcctacagga cacacattca tcgtaggtat aaacctcgaa atcanttcct actaagatgg2580
tatacaatag taaccatgca tggttgccta gtgaatgctc cgtaacaccc aatacgccgg2640
ccgaaacttt tttacaactc tcctatgagt cgtttaccca gaatgcacag gtacacttgt2700
ttagaggtaa tccttctttc tagaagtcct cgtgtactgt gtaagcgccc actccacatc2760
tccactcgag cccatcagta tttctcctat aaaccatg 2798
<210> 3
<211> 6740
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ttctccgtat actattgatc gtttggcatc tagtgtacct tttgatgggg ggttcgacca 60
ggacggcatg cgtacatgcc ttctccattg ggacgttcct ccggctagac acgcacatac 120
tgtccgttca cactatccat tcattcattc ggccattagt aaatacactc atacatatta 180
gccgggatgt aatgacttgt agtgcatgag gtgtaggcag agagtccaga accggtgaca 240
cgcaacgaca gcaacgaaag caactccagt atcaaatagc aaagacctag aacatgctca 300
accccaggtt gttgcgggca tttgtcgtca gtgagtgagc aaatgtgcaa ttgggctaat 360
gaggatgcga ggtgaagata cggcaaccaa ggtacaatac cgtacggcaa gaggtgggac 420
aaagtggaag ggggcccacc tctacccggt gcgtgtagtt tatggccttg tcatggggtg 480
gttggcattg tagccacggc cttttccttt gcttttcccg cgtgcttcct gccttttccc 540
ccctctgctt tggtgtgcct cgtttttccc ctgccgtgcc tttttccctt ccttccctcc 600
tcccccccct tttttccagt ggtaccaaac cacaactccc acagcccaac ccggaaatcc 660
aagtggtggt ggtaagagct aagtaaagta aagtaaagtc aaaaagtaaa agccgggccc 720
caaaataatc aaatctacca aaaaaaggcg atgacacgga ccggcaaacc aactaagcca 780
aactaatcca aaccgccagc cagaagtaat ctccccttcc ctctcactct cctttcattc 840
attttagtgc tgttgttatt attactgttc ctaattacta ccaccatttt tttgattttg 900
atttttcccg tccttccttt ggtattatta ttattattct tctgcttctt cactttgcat 960
cgcctatctt tcttttcatt ctttactatc atcctttccc tcttcctcct ctctcttcct1020
ctctctttcc ccccctcttc gccccgtttt tgcttcgcca gcgccggtgc ttcgttgacg1080
tagcccacct gcgaacacct tttctacgtc aacgagccgt tgggacttgc caattcatcc1140
tcagcctttc tctccttgca tcccatcccc ctcccacggt cgcgcgccct cttcaggtac1200
taccaccacc tccaaccgcc gttcaccata acaacccacc cccctcctcc actaccatta1260
ccacctccac ctcctccctc cctctccacg gccgtttttc tcacttaaca tccctttctt1320
agatcttgtc gctcgctcct taccgcctct acattcgtta ttgttttgtt tgctggtagt1380
ttttcgtgag gggttactgt tggtttctcg tcgaccacac cccctgccgc tctttgaacc1440
cgctttgttt ataccgaact cccgccgttg gtgcgcgcac atccatccgt cagctgtcag1500
tgcactatta gagcttgtcg ctttcgatac ttttgattcg gagatttttt tggtgatata1560
gttttgtgat atctgcaaac tttctggttg tttgttcgac tcaatatacc aggaagctta1620
ccgaccgttg tcatcgcatc cccagccgtc gaccgattcg ggatatcgcc atcgctcgcg1680
ccatccttcg attgagatag gttcgcttca agtggttcaa atcgtggaga ggtggatata1740
cgaaaacgcc gggaatagga catctatata gcccgggggt tagactattc gaatttccaa1800
ccaccgaccc catgcatcag caatcttaac gactggcttt gatccttgac gtttgaatac1860
tccgtgccta gggatatttg gttttggagg ttcccatctg agccttcgct ctgtccattc1920
atttcagcat tgttacggtc ttttcgacca cctgaccact ctttatcgcc tgctattgtt1980
acccaaccat tcgaatcaat taaaatccgc cgcctccacc atttgtagaa aaatgtgacg2040
aactcgtgag ctctgtacag tgaccggtga ctctttctgg catgcggaga gacggacgga2100
cgcagagaga agggctgagt aataagccac tggccagaca gctctggcgg ctctgaggtg2160
cagtggatga ttattaatcc gggaccggcc gcccctccgc cccgaagtgg aaaggctggt2220
gtgcccctcg ttgaccaaga atctattgca tcatcggaga atatggagct tcatcgaatc2280
accggcagta agcgaaggag aatgtgaagc caggggtgta tagccgtcgg cgaaatagca2340
tgccattaac ctaggtacag aagtccaatt gcttccgatc tggtaaaaga ttcacgagat2400
agtaccttct ccgaagtagg tagagcgagt acccggcgcg taagctccct aattggccca2460
tccggcatct gtagggcgtc caaatatcgt gcctctcctg ctttgcccgg tgtatgaaac2520
cggaaaggcc gctcaggagc tggccagcgg cgcagaccgg gaacacaagc tggcagtcga2580
cccatccggt gctctgcact cgacctgctg aggtccctca gtccctggta ggcagctttg2640
ccccgtctgt ccgcccggtg tgtcggcggg gttgacaagg tcgttgcgtc agtccaacat2700
ttgttgccat attttcctgc tctccccacc agctgctctt ttcttttctc tttcttttcc2760
catcttcagt atattcatct tcccatccaa gaacctttat ttcccctaag taagtacttt2820
gctacatcca tactccatcc ttcccatccc ttattccttt gaacctttca gttcgagctt2880
tcccacttca tcgcagcttg actaacagct accccgcttg agcagacatc accatgcctg2940
aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc3000
tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg3060
gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc3120
ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg3180
agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg3240
aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg3300
ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca3360
ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg3420
tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg3480
ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc3540
tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt3600
cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc3660
agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt3720
atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg3780
atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg3840
ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac3900
tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagagtagat3960
gccgaccgcg ggatccactt aacgttactg aaatcatcaa acagcttgac gaatctggat4020
ataagatcgt tggtgtcgat gtcagctccg gagttgagac aaatggtgtt caggatctcg4080
ataagatacg ttcatttgtc caagcagcaa agagtgcctt ctagtgattt aatagctcca4140
tgtcaacaag aataaaacgc gttttcgggt ttacctcttc cagatacagc tcatctgcaa4200
tgcattaatg cattgactgc aacctagtaa cgccttncag gctccggcga agagaagaat4260
agcttagcag agctattttc attttcggga gacgagatca agcagatcaa cggtcgtcaa4320
gagacctacg agactgagga atccgctctt ggctccacgc gactatatat ttgtctctaa4380
ttgtactttg acatgctcct cttctttact ctgatagctt gactatgaaa attccgtcac4440
cagcncctgg gttcgcaaag ataattgcat gtttcttcct tgaactctca agcctacagg4500
acacacattc atcgtaggta taaacctcga aatcanttcc tactaagatg gtatacaata4560
gtaaccatgc atggttgcct agtgaatgct ccgtaacacc caatacgccg gccgaaactt4620
ttttacaact ctcctatgag tcgtttaccc agaatgcaca ggtacacttg tttagaggta4680
atccttcttt ctagaagtcc tcgtgtactg tgtaagcgcc cactccacat ctccactcga4740
gcccatcagt atttctccta taaaccatgg atcgtgtact ttgttcaata ctagacaggg4800
ccaaaatata agccgaagcc atgacgcaac gctctagaga ggttgttggg ggcgttgggc4860
ccctaatctg tgtggaatac ataccaagcg ttgttgatgt tgccatttgt tcccaactgt4920
attcacattt tatcttgata tgccatcgcg tcctcatttc cctggcattt gtttgattat4980
atcctttttc tttttctttt ctactcttct tattccccac acgttttgtg ccattttcca5040
ctaccttttc atccattcca ttcttactct ccttatggtg acacagaaaa agacctgcca5100
ccgcaatcac ttttttactc ttcttctatc ccattttcta tatcttcttt ccatcctctc5160
catatgacct atcccatctt ccctcagcca tggatctacc tatccctcct gactgaactc5220
accccatccc ctacgctttc ttaatgattg ggtggtaggt atctgctgtg acgagtgaac5280
gccgtgctat actctcctac cccgcgtttt gttttctacc tctttgtccg tcctttttta5340
tctcgctatg tgtacagatg tatatttctc ttttcttatt tttcaagtag atcgaaatgc5400
aagcatgcaa agctagctat ttagcttagc ccagctagtt ggattaaatc tgactcggtt5460
gggttgggct gaatgaaaat gttcaaatgt ggttgattct tttgtagaga tgtaggagta5520
gttctctgat tgttttgatg tgtgaaggta tttttgaacc gggacctagg tagggattgt5580
ttctgttgat aggcaaatgc ggaggaccga tgagtagcag gtaatgtgtt tgatctggtc5640
cttgaatgat gtggttcgtg gtaatgatga tgactgatga tgtttggctg aaaggagagc5700
agaaaaacgc taagagacaa ttattatgta cagtacaaga atgcagcgga gagagaggcg5760
aatgataaga aagtcagaag aaccatttcg tccttccatc cgtccatccc tccatcgcca5820
atgacataag aaaaccccgg gcaagagaaa aagaagagtt aaaatgaaga gatactggaa5880
cgaaagtgta ggaagggatc aatcaatcac tcaatcactc attcttctct ttcgaattcg5940
acccgaaacc caacaacgcc aacggacccc tctcgccgcg ctgctcacgc tcctgcatgc6000
gctgctcctt cgctcgacga cgttcctcct tatacttctc ccaggagacg ttcgcatcgt6060
agcccatgac ctcgccatta ttcttctcag cctggaattt aaggaagtgg cttagagagg6120
tctcctcgtc atgaggcgtc ctcacggcct gaggtaacgg acggacggca tcttcaccgg6180
catacgcacg gagtccggta tggaaggccc attcgacggc ctcttcgacg ggcttgtcga6240
ataggtaggg gaggaaaggc acgacggaga gaccgagcta gcaagtagta atttgcatgt6300
gttagcggaa taattctata ccgcaagcgg agaaacagaa ctcaccccga taggcgccca6360
agtacggaaa aagacactcc gggaattctt caacatgcgc ccagagtact tgaccaccga6420
gtggatggta aaggccggaa gacccatact ggcgataccc tggaacacgg cgcgctgggc6480
catgacggtg cggtagtctt cagctagggg gacgtgagtc gtgggccatg gcatcagcgt6540
atcgctgctg ctactgcctg ttaacgagcc ggcgatgttg ccggttacca tgcccttggc6600
gacttgctcg tgggtaaggt cgctggcatc tttgtaggcc tcgcagggag gcgctaggac6660
gcggcgattg cggaggtagg ctttgtagcc ttcgtgggcg acatcgccga ttagataggt6720
ccaggaaatg ccgtaggcgg 6740
<210> 4
<211> 1797
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gagagtccag aaccggtgac acgcaacgac agcaacgaaa gcaactccag tatcaaatag 60
caaagaccta gaacatgctc aaccccaggt tgttgcgggc atttgtcgtc agtgagtgag 120
caaatgtgca attgggctaa tgaggatgcg aggtgaagat acggcaacca aggtacaata 180
ccgtacggca agaggtggga caaagtggaa gggggcccac ctctacccgg tgcgtgtagt 240
ttatggcctt gtcatggggt ggttggcatt gtagccacgg ccttttcctt tgcttttccc 300
gcgtgcttcc tgccttttcc cccctctgct ttggtgtgcc tcgtttttcc cctgccgtgc 360
ctttttccct tccttccctc ctcccccccc ttttttccag tggtaccaaa ccacaactcc 420
cacagcccaa cccggaaatc caagtggtgg tggtaagagc taagtaaagt aaagtaaagt 480
caaaaagtaa aagccgggcc ccaaaataat caaatctacc aaaaaaaggc gatgacacgg 540
accggcaaac caactaagcc aaactaatcc aaaccgccag ccagaagtaa tctccccttc 600
cctctcactc tcctttcatt cattttagtg ctgttgttat tattactgtt cctaattact 660
accaccattt ttttgatttt gatttttccc gtccttcctt tggtattatt attattattc 720
ttctgcttct tcactttgca tcgcctatct ttcttttcat tctttactat catcctttcc 780
ctcttcctcc tctctcttcc tctctctttc cccccctctt cgccccgttt ttgcttcgcc 840
agcgccggtg cttcgttgac gtagcccacc tgcgaacacc ttttctacgt caacgagccg 900
ttgggacttg ccaattcatc ctcagccttt ctctccttgc atcccatccc cctcccacgg 960
tcgcgcgccc tcttcaggta ctaccaccac ctccaaccgc cgttcaccat aacaacccac1020
ccccctcctc cactaccatt accacctcca cctcctccct ccctctccac ggccgttttt1080
ctcacttaac atccctttct tagatcttgt cgctcgctcc ttaccgcctc tacattcgtt1140
attgttttgt ttgctggtag tttttcgtga ggggttactg ttggtttctc gtcgaccaca1200
ccccctgccg ctctttgaac ccgctttgtt tataccgaac tcccgccgtt ggtgcgcgca1260
catccatccg tcagctgtca gtgcactatt agagcttgtc gctttcgata cttttgattc1320
ggagattttt ttggtgatat agttttgtga tatctgcaaa ctttctggtt gtttgttcga1380
ctcaatatac caggaagctt accgaccgtt gtcatcgcat ccccagccgt cgaccgattc1440
gggatatcgc catcgctcgc gccatccttc gattgagata ggttcgcttc aagtggttca1500
aatcgtggag aggtggatat acgaaaacgc cgggaatagg acatctatat agcccggggg1560
ttagactatt cgaatttcca accaccgacc ccatgcatca gcaatcttaa cgactggctt1620
tgatccttga cgtttgaata ctccgtgcct agggatattt ggttttggag gttcccatct1680
gagccttcgc tctgtccatt catttcagca ttgttacggt cttttcgacc acctgaccac1740
tctttatcgc ctgctattgt tacccaacca ttcgaatcaa ttaaaatccg ccgcctc 1797
<210> 5
<211> 1930
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gcccactcca catctccact cgagcccatc agtatttctc ctataaacca tggatcgtgt 60
actttgttca atactagaca gggccaaaat ataagccgaa gccatgacgc aacgctctag 120
agaggttgtt gggggcgttg ggcccctaat ctgtgtggaa tacataccaa gcgttgttga 180
tgttgccatt tgttcccaac tgtattcaca ttttatcttg atatgccatc gcgtcctcat 240
ttccctggca tttgtttgat tatatccttt ttctttttct tttctactct tcttattccc 300
cacacgtttt gtgccatttt ccactacctt ttcatccatt ccattcttac tctccttatg 360
gtgacacaga aaaagacctg ccaccgcaat cactttttta ctcttcttct atcccatttt 420
ctatatcttc tttccatcct ctccatatga cctatcccat cttccctcag ccatggatct 480
acctatccct cctgactgaa ctcaccccat cccctacgct ttcttaatga ttgggtggta 540
ggtatctgct gtgacgagtg aacgccgtgc tatactctcc taccccgcgt tttgttttct 600
acctctttgt ccgtcctttt ttatctcgct atgtgtacag atgtatattt ctcttttctt 660
atttttcaag tagatcgaaa tgcaagcatg caaagctagc tatttagctt agcccagcta 720
gttggattaa atctgactcg gttgggttgg gctgaatgaa aatgttcaaa tgtggttgat 780
tcttttgtag agatgtagga gtagttctct gattgttttg atgtgtgaag gtatttttga 840
accgggacct aggtagggat tgtttctgtt gataggcaaa tgcggaggac cgatgagtag 900
caggtaatgt gtttgatctg gtccttgaat gatgtggttc gtggtaatga tgatgactga 960
tgatgtttgg ctgaaaggag agcagaaaaa cgctaagaga caattattat gtacagtaca1020
agaatgcagc ggagagagag gcgaatgata agaaagtcag aagaaccatt tcgtccttcc1080
atccgtccat ccctccatcg ccaatgacat aagaaaaccc cgggcaagag aaaaagaaga1140
gttaaaatga agagatactg gaacgaaagt gtaggaaggg atcaatcaat cactcaatca1200
ctcattcttc tctttcgaat tcgacccgaa acccaacaac gccaacggac ccctctcgcc1260
gcgctgctca cgctcctgca tgcgctgctc cttcgctcga cgacgttcct ccttatactt1320
ctcccaggag acgttcgcat cgtagcccat gacctcgcca ttattcttct cagcctggaa1380
tttaaggaag tggcttagag aggtctcctc gtcatgaggc gtcctcacgg cctgaggtaa1440
cggacggacg gcatcttcac cggcatacgc acggagtccg gtatggaagg cccattcgac1500
ggcctcttcg acgggcttgt cgaataggta ggggaggaaa ggcacgacgg agagaccgag1560
ctagcaagta gtaatttgca tgtgttagcg gaataattct ataccgcaag cggagaaaca1620
gaactcaccc cgataggcgc ccaagtacgg aaaaagacac tccgggaatt cttcaacatg1680
cgcccagagt acttgaccac cgagtggatg gtaaaggccg gaagacccat actggcgata1740
ccctggaaca cggcgcgctg ggccatgacg gtgcggtagt cttcagctag ggggacgtga1800
gtcgtgggcc atggcatcag cgtatcgctg ctgctactgc ctgttaacga gccggcgatg1860
ttgccggtta ccatgccctt ggcgacttgc tcgtgggtaa ggtcgctggc atctttgtag1920
gcctcgcagg 1930
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gagagtccag aaccggtgac 20
<210> 7
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gaggcggcgg attttaattg attcgaatgg ttgggtaaca atagc 45
<210> 8
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gctattgtta cccaaccatt cgaatcaatt aaaatccgcc gcctc 45
<210> 9
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
catggtttat aggagaaata ctgatgggct cgagtggaga tgtggagtgg gc 52
<210> 10
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gcccactcca catctccact cgagcccatc agtatttctc ctataaacca tg 52
<210> 11
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
cctgcgaggc ctacaaagat 20
<210> 12
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
cgctctgtcc attcatttca gc 22
<210> 13
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
atggcaacat caacaacgct 20
Claims (6)
1.一株产木聚糖酶的黑曲霉基因工程菌,其特征在于,该菌株是Msb2基因失活的黑曲霉菌SJ1,所述的黑曲霉菌SJ1已保藏于中国典型培养物保藏中心,保藏时间为2019年02月28日,保藏编号为CCTCC NO:M2019115;
所述Msb2基因的核苷酸序列如SEQ ID NO.1所示,所述Msb2基因失活后的核苷酸序列如SEQ ID NO.2所示。
2.权利要求1所述产木聚糖酶的黑曲霉基因工程菌的构建方法,其特征在于,包括如下步骤:
(1)提取黑曲霉SJ1基因组;
(2)构建基因敲除片段△Msb2,所述基因敲除片段的核苷酸序列如SEQ ID NO.3所示;
(3)制备黑曲霉原生质体;
(4)将基因敲除片段导入原生质体中进行同源重组,得到Msb2基因失活黑曲霉基因工程菌。
3.权利要求1所述产木聚糖酶的黑曲霉菌基因工程菌在制备木聚糖酶中的应用。
4.根据权利要求3所述的应用,其特征在于,包括如下步骤:以产木聚糖的黑曲霉基因工程菌为发酵菌剂,多孔纤维材料为固定化载体,发酵制备木聚糖酶,所述的固定化载体为聚氨酯。
5.根据权利要求4所述的应用,其特征在于,所述的固定化载体通过如下方法进行预处理:
将固定化载体用0.5~2M的NaOH浸泡30min~2h,用水冲洗至pH为中性,在0.5~2M HCl中浸泡30min~2h,用水冲洗至pH为中性,烘干,得到经过预处理的固定化载体。
6.根据权利要求4所述的应用,其特征在于,所述的发酵,其发酵条件如下:
发酵培养基: 木聚糖 10~70g/L、NaNO3 2~6 g/L、KH2PO4 0.3~2 g/L、MgSO4·7H2O 0.1~0.8 g/L、吐温-80 0.1~0.8 g/L,以麸皮浸出液配制;
所述的麸皮浸出液按照如下方法制备:称取15~60g/L的麸皮,加入800mL水,煮沸30min~2h后,过滤得滤液,用水再定容至1L;
发酵条件如下:28~32°C、150~300 r/min培养3~6d。
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CN201910284360.1A CN110004070B (zh) | 2019-04-10 | 2019-04-10 | 一株产木聚糖酶黑曲霉基因工程菌及其构建方法与应用 |
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