CN1699577A - 一种β－甘露聚糖酶基因和高效制备 - Google Patents
一种β－甘露聚糖酶基因和高效制备 Download PDFInfo
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- CN1699577A CN1699577A CN 200510081649 CN200510081649A CN1699577A CN 1699577 A CN1699577 A CN 1699577A CN 200510081649 CN200510081649 CN 200510081649 CN 200510081649 A CN200510081649 A CN 200510081649A CN 1699577 A CN1699577 A CN 1699577A
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
一种β－甘露聚糖酶基因和高效制备，属于微生物基因工程技术领域。本发明提供了来源于地衣芽孢杆菌(Bacillus licheniformis)ATCC14580中的β－甘露聚糖酶基因man，提供了man的核苷酸序列和相应蛋白质的氨基酸序列，提供了含有基因man的克隆载体pSK－man和芽孢杆菌表达载体pBL－man，以及在大肠杆菌或芽孢杆菌中的高效表达。本发明可用于β－甘露聚糖酶工业化生产的基因工程菌的构建和现有β－甘露聚糖酶基因的分子改造，提高微生物发酵法生产β－甘露聚糖酶的水平和质量。
Description
技术领域
一种β-甘露聚糖酶基因和高效制备，属于微生物基因工程技术领域。
背景技术
β-1，4-D-甘露聚糖酶又简称为β-甘露聚糖酶(1，4-β-D-mannanmannanohydrolases，EC 3.2.1.78)，是一类能够水解含有β-1，4-D-甘露糖苷键的甘露寡糖、甘露多糖的内切水解酶，它属于半纤维素酶类。
β-甘露聚糖在自然界的广泛分布为β-甘露聚糖酶的开发和应用带来空间。随着近来对自然界半纤维素资源的开发和甘露寡糖药用价值的发现，β-甘露聚糖酶的研究和开发进入一个新阶段，它将在食品、医药、造纸、石油开采，生物漂白及饲料加工等领域得到广泛应用。
β-甘露聚糖酶主要来源于微生物。在微生物生产的β-甘露聚糖酶中，依据最适作用pH值范围可分为酸性β-甘露聚糖酶、中性β-甘露聚糖酶和碱性β-甘露聚糖酶。依据最适作用温度又可分为低温β-甘露聚糖酶、中温β-甘露聚糖酶和高温β-甘露聚糖酶。目前，通过菌种筛选和传统菌种改良获得的β-甘露聚糖酶产酶水平一般在100u/mL(杨文博等，1995，微生物学通报)，发酵水平尚不能满足工业化生产需要。
发明内容
本发明的目的是提供一种β-甘露聚糖酶基因和高效制备，寻找一种β-甘露聚糖酶编码基因，因此首先从培养液中纯化了地衣芽孢杆菌中的β-甘露聚糖酶，接着对其酶学性质进行分析，再利用基因扩增等手段克隆了该酶编码基因，最后在大肠杆菌或芽孢杆菌中高效表达和制备重组β-甘露聚糖酶。本发明的技术方案：根据已报道的芽孢杆菌β-甘露聚糖酶基因保守序列设计引物BL0-manS1(ggcttgaggcgatgctgagcaaaat)和引物BL0-manS2(aaaggagagatatgggacggcgattc)，以地衣芽孢杆菌ATCC14580染色体DNA为模板，扩增条件是95℃5min；94℃30s，56℃30s，72℃1min 35次循环；72℃10min；PCR扩增出550bp大小的片段，测序后，经比对分析此扩增片段为β-甘露聚糖酶编码基因。以此为基础设计引物BL0-manS3 taagggagcggacaccctctatctt，BL0-manS4aacgggcacgccttcattttcaa。用Sau3AI部分酶切地衣芽孢杆菌ATCC14580染色体DNA，回收3-4kb的DNA片段，自身环化后用作PCR模板，用引物BL0-manS3和BL0-manS4扩增，扩增条件是95℃5min；94℃30s，56℃30s，68℃5min35次循环；68℃10min；扩增出3.2kb大小的条带，测序。通过软件对550bp和3.2kb的两段核苷酸序列进行拼接，获得1011bp全长man基因的核苷酸序列。
接下来设计克隆表达方案，如图1，首先，根据拼接得到的全长man基因的核苷酸序列设计下列引物：引物BLP1：acggaattccacaccgtttctccggtgaacc；引物BLP2：gggttattccacgacaggcgtcaaagaat，为方便操作两条引物分别设计EcoR I位点和Sma I位点的一部分ggg。然后，以地衣芽胞杆菌ATCC14580染色体DNA为模板进行PCR扩增，扩增条件是95℃5min；94℃30s，56℃30s，72℃1min 35次循环；72℃10min。扩增出编码β-甘露聚糖酶成熟肽的一段DNA，大小1011bp，所得PCR产物经EcoR I酶切后，插入经EcoR I和Sma I双酶切载体pBlueScriptII SK(-)中，得到重组质粒pSK-man。重组质粒pSK-man送到上海鼎安生物科技有限公司进行序列测定。最后，重组质粒pSK-man经EcoR I和Sma I双酶切，割胶分离得到的基因man，插入到载体pBL-WZX的EcoR I和Sma I位点，得到重组质粒pBL-man。将重组质粒pBL-man转入大肠杆菌或芽孢杆菌分别获得重组菌EC-man或BL-man。重组质粒pBL-man转入大肠杆菌JM109，通过氨苄青霉素和卡那霉素双抗性筛选，得到重组菌EC-man，取其中一株做发酵实验。采用LB(Amp+Kan)培养基，装液量35ml(250ml三角瓶)，37℃，200r/min培养。12h后每隔6h取样分析酶活。
来源于地衣芽孢杆菌(Bacillus licheniformis)ATCC14580中的耐热β-甘露聚糖酶基因man的核苷酸序列如下：
cacaccgttt ctccggtgaa cccgaatgcc cagccgacga cgaaagcggt gatgaactgg 60
ctcgcccacc tgcccaatcg gacggaaagc cgggtgatgt ccggggcgtt cggaggatac 120
agcctcgaca cattttcaac ggctgaagcc gaccggatca aacaggcaac aggacagctg 180
ccggccatat acggctgcga ttatgcaaga ggatggctgg agccggaaaa gatcgccgat 240
acgattgact acagctgcaa ccgtgatttg atcgcatact ggaaaagcgg aggcattccg 300
caaatcagca tgcacctcgc aaaccccgcg tttacttcgg gtcattataa aactcagatt 360
tcaaacagcc agtatgagag aattttagat tcttccactc ctgaaggaaa gcggcttgag 420
gcgatgctga gcaaaatcgc ggacggcctc caggagcttg aaaatgaagg cgtgcccgtt 480
ctattcagac cccttcacga aatgaacggc gaatggttct ggtgggggct gacgcaatat 540
aatcaaaaag acagcgaaag aatctccttg tacaaacagc tctatgtgaa aatctatgac 600
tatatgacaa aaacaagagg cctggatcat ctcttgtggg tgtatgcgcc ggacgccaac 660
agagacttta aaacggactt ttatccgggc gcatcatatg tggacattgt cgggcttgac 720
gcttattttg atgacccgta cgccattgat ggctacgaag agctcacatc gctgaacaag 780
ccgtttgcct ttacagaagt cggaccgcag acgacaaacg gcgggctgga ttacgcgcgg 840
tttatccatg ccatcaaaga aaaatacccg aaaacgacgt acttcctggc gtggaacgat 900
gagtggagcc cggctgtgaa taagggagcg gacaccctct atcttcatcc atggacgctg 960
aataaaggag agatatggga cggcgattct ttgacgcctg tcgtggaata a 1011
其中taa为终止密码子。
根据以上核苷酸序列，地衣芽孢杆菌ATCC14580的β-甘露聚糖酶的氨基酸序列如下：
His Thr Val Ser Pro Val Asn Pro Asn Ala Gln Pro Thr Thr Lys
1 5 10 15
Ala Val Met Asn Trp Leu Ala His Leu Pro Asn Arg Thr Glu Ser
20 25 30
Arg Val Met Ser Gly Ala Phe Gly Gly Tyr Ser Leu Asp Thr Phe
35 40 45
Ser Thr Ala Glu Ala Asp ArgIle Lys Gln Ala Thr Gly Gln Leu
50 55 60
Pro Ala Ile Tyr Gly Cys Asp Tyr Ala Arg Gly Trp Leu Glu Pro
65 70 75
Glu Lys Ile Ala Asp Thr Ile Asp Tyr Ser Cys Asn Arg Asp Leu
80 85 90
Ile Ala Tyr Trp Lys Ser Gly Gly Ile Pro Gln Ile Ser Met His
95 100 105
Leu Ala Asn Pro Ala Phe Thr Ser Gly His Tyr Lys Thr Gln Ile
110 115 120
Ser Asn Ser Gln Tyr Glu Arg Ile Leu Asp Ser Ser Thr Pro Glu
125 130 135
Gly Lys Arg Leu Glu Ala Met Leu Ser Lys Ile Ala Asp Gly Leu
140 145 150
Gln Glu Leu Glu Asn Glu Gly Val Pro Val Leu Phe Arg Pro Leu
155 160 165
His Glu MET Asn Gly Glu Trp Phe Trp Trp Gly Leu Thr Gln Tyr
170 175 180
Ash Gln Lys Asp Ser Glu Arg Ile Ser Leu Tyr Lys Gln Leu Tyr
185 190 195
Val Lys Ile Tyr Asp Tyr Met Thr Lys Thr Arg Gly Leu Asp His
200 205 210
Leu Leu Trp Val Tyr Ala Pro Asp Ala Asn Arg Asp Phe Lys Thr
215 220 225
Asp Phe Tyr Pro Gly Ala Ser Tyr Val Asp Ile Val Gly Leu Asp
230 235 240
Ala Tyr Phe Asp Asp Pro Tyr Ala Ile Asp Gly Tyr Glu Glu Leu
245 250 255
Thr Ser Leu Asn Lys Pro Phe Ala Phe Thr Glu Val Gly Pro Gln
260 265 270
Thr Thr Asn Gly Gly Leu Asp Tyr Ala Arg Phe Ile His Ala Ile
275 280 285
Lys Glu Lys Tyr Pro Lys Thr Thr Tyr Phe Leu Ala Trp Asn Asp
290 295 300
Glu Trp Ser Pro Ala Val Asn Lys Gly Ala Asp Thr Leu Tyr Leu
305 310 315
His Pro Trp Thr Leu Asn Lys Gly Glu Ile Trp Asp Gly Asp Ser
320 325 330
Leu Thr Pro Val Val Glu
335 336
该β-甘露聚糖酶由336个氨基酸残基组成，分子量为38005，酶比活达到2826u/mg，80℃的半衰期为1h，在pH4.0-9.0范围内都有活性，pH5.0-6.0为酶的最适作用范围，酶在pH4.0-9.0范围内比较稳定。
本发明的有益效果：本发明提供了一种来源于地衣芽孢杆菌ATCC14580中热稳定性好的β-甘露聚糖酶基因man核苷酸序列及其相应的氨基酸序列。提供了含有地衣芽孢杆菌ATCC14580β-甘露聚糖酶基因的克隆载体pSK-man，在大肠杆菌或芽孢杆菌中高效表达的表达载体pBL-man及其基因重组菌EC-man或BL-man。利用本发明成果，可以用于β-甘露聚糖酶工业化生产的基因工程菌的构建及其规模化制备。
本发明的β-甘露聚糖酶具有明显的高比酶活，比酶活达到2826u/mg，分别是B.lichenformis NK-27和B.subtilis BM9602β-甘露聚糖酶40和4倍。
本发明的β-甘露聚糖酶具有耐高温性能，在Mg2+存在下，50℃以下1h以内稳定，80℃的半衰期为1h，而B.lichenformis NK-27β-甘露聚糖酶经30-80℃30min后，30-40℃区间酶的稳定性最佳，超过70℃酶活几乎全部失活。
本发明的β-甘露聚糖酶具有广pH有效性和稳定性，pH对酶的影响实验表明，此酶在pH4.0-9.0范围内都有活性，pH5.0-6.0为酶的最适作用范围，酶在pH4.0-9.0范围内比较稳定。
本发明的β-甘露聚糖酶作用于底物槐豆胶产生大量的寡聚糖如甘露二糖、甘露三糖等，利用此特性可用该酶制备低聚甘露聚糖。
附图说明
图1重组表达质粒pBL-man构建方案
图2重组菌EC-man表达β-甘露聚糖酶的SDS-PAGE分析。1.蛋白质分子量标准；2.重组菌EC-man；3.大肠杆菌JM109(pBL-WZX)
图3重组β-甘露聚糖酶的最适作用pH
图4重组β-甘露聚糖酶的最适作用温度
图5重组β-甘露聚糖酶的热稳定性
图6重组β-甘露聚糖酶的酸碱稳定性
具体实施方式
实施例1地衣芽孢杆菌ATCC14580产β-甘露聚糖酶的条件和纯化
在无机盐溶液基础上，添加15-25g/L槐豆胶作为碳源，5-20g/L蛋白胨为氮源，发酵温度为37-40℃，接种量在任选的情况下可选取1％，装液量为70mL(250mL三角瓶)，pH在任选的情况下可选取7.0，在此工艺条件下摇瓶发酵12h的平均酶活为41.92u/mL，收集上清培养液即粗酶液，经纯化后比酶活达到2862.4u/mg。
β-甘露聚糖酶活性分析用槐豆胶作底物。反应混合液体中含有1.5ml用0.1mol/L的醋酸钠缓冲液(pH5.8)配制的槐豆胶溶液(5g/L)和0.02ml适当稀释(约20U/ml)的酶液。该混合液于65℃保温15分钟，用3，5-二硝基水杨酸(DNS)法测定反应产生的还原糖。酶单位定义为：每分钟释放1μmol还原糖(相当于甘露糖)的酶量为一个酶活力单位。蛋白质浓度测定按Bradford法进行，以BSA为标准参照。
实施例2：β-甘露聚糖酶基因的克隆和重组质粒的构建
根据已报道的芽孢杆菌β-甘露聚糖酶基因保守序列设计引物BL0-manS1ggcttgaggcgatgctgagcaaaat和BL0-manS2aaaggagagatatgggacggcgattc，以地衣芽孢杆菌ATCC14580染色体DNA为模板，PCR扩增出550bp大小的片段，测序后，经比对分析此扩增片段为β-甘露聚糖酶编码基因。以此为基础设计引物BL0-manS3taagggagcggacaccctctatctt，BL0-manS4aacgggcacgccttcattttcaa。用Sau3AI部分酶切地衣芽孢杆菌ATCC14580染色体DNA，回收3-4kb的DNA片段，自身环化后用作PCR模板，用引物BL0-manS3和BL0-manS4扩增出3.2kb大小的条带，测序并与550bp的DNA序列进行拼接获得全长man基因的核苷酸序列。
以地衣芽胞杆菌ATCC14580染色体DNA为模板，用引物BLP1acggaattccacaccgtttctccggtgaacc，BLP2gggttattccacgacaggcgtcaaagaat进行PCR扩增得到产物man，大约1011bp，同预期大小相符。此片段经EcoR I酶切纯化后插入克隆载体的EcoR I和Sma I之间，以氨苄青霉素为筛选标记得到重组菌JM109(pSK-man)。重组质粒pSK-man用EcoR I和Sma I双酶切验证，确定目的片段man已插入pBlueScript IISK(-)中。为了表达编码β-甘露聚糖酶的成熟肽基因片断，构建产β-甘露聚糖酶重组菌，首先用EcoR I和Sma I消化一定量的重组质粒pSK-man，回收目的片段，以相同的酶切位点插入到表达载体pBL-WZX中，获得重组质粒pBL-man。
实施例3：β-甘露聚糖酶基因在大肠杆菌中表达
重组质粒pBL-man转化大肠杆菌JM109，通过氨苄青霉素和卡那霉素双抗性筛选，得到重组大肠杆菌EC-man。
重组大肠杆菌EC-man在液体LB(Amp+Kan)培养基中培养，装液量35ml(250ml三角瓶)，37℃，200r/min培养。培养48h后，离心取上清液进行酶活分析，重组大肠杆菌EC-man产β-甘露聚糖酶最高水平达到0.9mg/ml，大约是地衣芽孢杆菌ATCC14580在最佳培养条件下酶活的59倍。以大肠杆菌JM109(pBL-WZX)为对照，对重组菌EC-man的12h培养物进行全细胞SDS-PAGE分析，结果如图2所示，样品在38kD处有一条明显的表达条带，而对照样品却明显没有此条带。SDS-PAGE分析时，采用5％浓缩胶、10％分离胶的不连续垂直板电泳，25mA电泳4h。考马斯亮蓝R-250染色。
实施例4重组β-甘露聚糖酶的酶学性质
酶的最适作用pH。用下列不同pH值的缓冲液配制底物(5g/L槐豆胶)，测定酶活力。pH值的缓冲液：0.1mol/L HAc-NaAc(pH3.6、4.0、5.0、5.5、5.8)；0.1mol/LNa2HPO4-NaH2PO4(pH6.5、7.0、8.0)；0.1mol/L甘氨酸-氢氧化钠缓冲液(pH9.0、10.0)。计算相对酶活确定最适pH(图3)。此酶在pH4.0-9.0范围内都有活性，pH5.0-6.0为酶的最适作用范围。
酶的最适反应温度的测定。分别在25、30、37、45、50、55、60、65、70、80℃测定纯化酶的酶活。计算相对酶活确定最适温度(图4)。该酶在30-100℃之间都有活性，最适温度是70-75℃。
酶的热稳定性测定。将适当稀释(约20u/mL)的纯化酶分别在30、40、50、60、70、80、90℃保温60min，立即取出，冰浴30min，然后测定酶活，并计算相对酶活(图5)。80℃的半衰期恰好为1h。
酶酸碱稳定性的测定。将纯化酶分别用pH 2.0、2.5、3.0、3.5、4.0、4.5、5.0、5.5、6.0、6.5、7.0、7.5、8.0、8.5、9.0、9.5缓冲液适当稀释，室温放置1h，然后测定酶活，并计算相对酶活(图6)。该酶在pH4.0-9.0范围内比较稳定。
Claims (3)
1、一种来源于地衣芽孢杆菌(Bacillus licheniformis)ATCC 14580中的耐热β-甘露聚糖酶基因man，其核苷酸序列为：
cacaccgttt ctccggtgaa cccgaatgcc cagccgacga cgaaagcggt gatgaactgg 60
ctcgcccacc tgcccaatcg gacggaaagc cgggtgatgt ccggggcgtt cggaggatac 120
agcctcgaca cattttcaac ggctgaagcc gaccggatca aacaggcaac aggacagctg 180
ccggccatat acggctgcga ttatgcaaga ggatggctgg agccggaaaa gatcgccgat 240
acgattgact acagctgcaa ccgtgatttg atcgcatact ggaaaagcgg aggcattccg 300
caaatcagca tgcacctcgc aaaccccgcg tttacttcgg gtcattataa aactcagatt 360
tcaaacagcc agtatgagag aattttagat tcttccactc ctgaaggaaa gcggcttgag 420
gcgatgctga gcaaaatcgc ggacggcctc caggagcttg aaaatgaagg cgtgcccgtt 480
ctattcagac cccttcacga aatgaacggc gaatggttct ggtgggggct gacgcaatat 540
aatcaaaaag acagcgaaag aatctccttg tacaaacagc tctatgtgaa aatctatgac 600
tatatgacaa aaacaagagg cctggatcat ctcttgtggg tgtatgcgcc ggacgccaac 660
agagacttta aaacggactt ttatccgggc gcatcatatg tggacattgt cgggcttgac 720
gcttattttg atgacccgta cgccattgat ggctacgaag agctcacatc gctgaacaag 780
ccgtttgcct ttacagaagt cggaccgcag acgacaaacg gcgggctgga ttacgcgcgg 840
tttatccatg ccatcaaaga aaaatacccg aaaacgacgt acttcctggc gtggaacgat 900
gagtggagcc cggctgtgaa taagggagcg gacaccctct atcttcatcc atggacgctg 960
aataaaggag agatatggga cggcgattct ttgacgcctg tcgtggaa
1011
其中，
为终止密码子。
2、根据权利要求1所述基因man，其氨基酸组成为：
His Thr Val Ser Pro Val Asn Pro Asn Ala Gln Pro Thr Thr Lys
1 5 10 15
Ala Val Met Asn Trp Leu Ala His Leu Pro Asn Arg Thr Glu Ser
20 25 30
Arg Val Met Ser Gly Ala Phe Gly Gly Tyr Ser Leu Asp Thr Phe
35 40 45
Ser Thr Ala Glu Ala Asp Arg Ile Lys Gln Ala Thr Gly Gln Leu
50 55 60
Pro Ala Ile Tyr Gly Cys Asp Tyr Ala Arg Gly Trp Leu Glu Pro
65 70 75
Glu Lys Ile Ala Asp Thr Ile Asp Tyr Ser Cys Asn Arg Asp Leu
80 85 90
Ile Ala Tyr Trp Lys Ser Gly Gly Ile Pro Gln Ile Ser Met His
95 100 105
Leu Ala Asn Pro Ala Phe Thr Ser Gly His Tyr Lys Thr Gln Ile
110 115 120
Ser Asn Ser Gln Tyr Glu Arg Ile Leu Asp Ser Ser Thr Pro Glu
125 130 135
Gly Lys Arg Leu Glu Ala Met Leu Ser Lys Ile Ala Asp Gly Leu
140 145 150
Gln Glu Leu Glu Asn Glu Gly Val Pro Val Leu Phe Arg Pro Leu
155 160 165
His Glu MET Asn Gly Glu Trp Phe Trp Trp Gly Leu Thr Gln Tyr
170 175 180
Asn Gln Lys Asp Ser Glu Arg Ile Ser Leu Tyr Lys Gln Leu Tyr
185 190 195
Val Lys Ile Tyr Asp Tyr Met Thr Lys Thr Arg Gly Leu Asp His
200 205 210
Leu Leu Trp Val Tyr Ala Pro Asp Ala Asn Arg Asp Phe Lys Thr
215 220 225
Asp Phe Tyr Pro Gly Ala Ser Tyr Val Asp Ile Val Gly Leu Asp
230 235 240
Ala Tyr Phe Asp Asp Pro Tyr Ala Ile Asp Gly Tyr Glu Glu Leu
245 250 255
Thr Ser Leu Asn Lys Pro Phe Ala Phe Thr Glu Val Gly Pro Gln
260 265 270
Thr Thr Asn Gly Gly Leu Asp Tyr Ala Arg Phe Ile His Ala Ile
275 280 285
Lys Glu Lys Tyr Pro Lys Thr Thr Tyr Phe Leu Ala Trp Asn Asp
290 295 300
Glu Trp Ser Pro Ala Val Asn Lys Gly Ala Asp Thr Leu Tyr Leu
305 310 315
His Pro Trp Thr Leu Asn Lys Gly Glu Ile Trp Asp Gly Asp Ser
320 325 330
Leu Thr Pro Val Val Glu
335 336
3、如权利要求1所述β-甘露聚糖酶基因man的克隆方法，其特征是
A)引物设计
BLO-manS1 ggcttgaggcgatgctgagcaaaat
BLO-manS2 aaaggagagatatgggacggcgattc
BLO-manS3 taagggagcggacaccctctatctt
BLO-manS4 aacgggcacgccttcattttcaa
BLP1 acggaattccacaccgtttctccggtgaacc
BLP2 gggttattccacgacaggcgtcaaagaat
B)通过引物BLO-manS1 ggcttgaggcgatgctgagcaaaat和引物BLO-manS2aaaggagagatatgggacggcgattc，以地衣芽孢杆菌ATCC14580染色体DNA为模板，扩增条件是95℃ 5min；94℃ 30s，56℃ 30s，72℃ 1min 35次循环；72℃ 10min；PCR扩增出550bp大小的片段；
C)用Sau3AI部分酶切地衣芽孢杆菌ATCC14580染色体DNA，回收3-4kb的DNA片段，自身环化后用作PCR模板，用引物BLO-manS3taagggagcggacaccctctatctt和引物BLO-manS4 aacgggcacgccttcattttcaa扩增，扩增条件是95℃ 5min；94℃ 30s，56℃ 30s，68℃ 5min 35次循环；68℃ 10min；PCR扩增出3.2kb大小的条带；
D)测序并通过软件与步骤B)获得的550bp DNA序列进行拼接，获得全长man基因的核苷酸序列；
E)引物BLP1 acggaattccacaccgtttctccggtgaacc和引物BLP2gggttattccacgacaggcgtcaaagaat，两条引物分别设计EcoR I位点和Sma I位点的一部分ggg，以地衣芽胞杆菌ATCC14580染色体DNA为模板进行PCR扩增，扩增条件是95℃ 5min；94℃ 30s，56℃ 30s，72℃ 1min 35次循环；72℃ 10min；扩增出编码β-甘露聚糖酶成熟肽的一段DNA，大小1011bp；
F)步骤E)所得PCR产物经EcoR I酶切后，插入经EcoR I和Sma I双酶切载体pBlueScript II SK(-)中，得到重组质粒pSK-man；
G)重组质粒pSK-man经EcoR I和Sma I双酶切，胶分离得到基因man，插入到载体pBL-WZX的EcoR I和Sma I位点，得到重组质粒pBL-man；
H)重组表达质粒pBL-man转化大肠杆菌或芽孢杆菌分别获得基因重组菌EC-man或BL-man；
I)重组菌做发酵实验，采用LB培养基，装液量35ml，250ml三角瓶，37℃，200r/min培养，进行重组β-甘露聚糖酶的酶活水平测定，实现β-甘露聚糖酶的高效制备。
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| CN100370030C (zh) * | 2006-01-25 | 2008-02-20 | 暨南大学 | 一种β-甘露聚糖酶基因及其编码产物的氨基酸序列和制备方法 |
| CN100420744C (zh) * | 2006-04-10 | 2008-09-24 | 中国农业大学 | β-甘露聚糖酶及其表达方法与专用工程菌 |
| WO2012079222A1 (en) * | 2010-12-14 | 2012-06-21 | Fujian Fuda Biotech Co. Ltd. | Mannanase, coding gene and production thereof |
| CN102732493A (zh) * | 2011-04-07 | 2012-10-17 | 中国农业大学 | 耐热β-甘露聚糖酶、其编码基因、重组菌和应用 |
| CN103184206A (zh) * | 2011-08-16 | 2013-07-03 | 青岛蔚蓝生物集团有限公司 | 甘露聚糖酶及其突变体 |
| CN103740682A (zh) * | 2014-01-17 | 2014-04-23 | 北京科为博生物科技有限公司 | 一种耐高温酸性β-甘露聚糖酶Man-L30及其基因和应用 |
| CN110004166A (zh) * | 2018-01-05 | 2019-07-12 | 中国科学院天津工业生物技术研究所 | 高效表达分泌β-甘露聚糖酶的重组枯草芽孢杆菌菌株及其获得方法 |
| CN111040966A (zh) * | 2019-12-23 | 2020-04-21 | 河北科技大学 | 一种地衣芽孢杆菌KD-1、其生产的β-甘露聚糖酶及其应用 |
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| CN100420744C (zh) * | 2006-04-10 | 2008-09-24 | 中国农业大学 | β-甘露聚糖酶及其表达方法与专用工程菌 |
| WO2012079222A1 (en) * | 2010-12-14 | 2012-06-21 | Fujian Fuda Biotech Co. Ltd. | Mannanase, coding gene and production thereof |
| CN102732493A (zh) * | 2011-04-07 | 2012-10-17 | 中国农业大学 | 耐热β-甘露聚糖酶、其编码基因、重组菌和应用 |
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| CN103184206B (zh) * | 2011-08-16 | 2015-04-15 | 青岛蔚蓝生物集团有限公司 | 甘露聚糖酶及其突变体 |
| CN103740682A (zh) * | 2014-01-17 | 2014-04-23 | 北京科为博生物科技有限公司 | 一种耐高温酸性β-甘露聚糖酶Man-L30及其基因和应用 |
| CN103740682B (zh) * | 2014-01-17 | 2016-01-06 | 北京科为博生物科技有限公司 | 一种耐高温酸性β-甘露聚糖酶Man-L30及其基因和应用 |
| CN110004166A (zh) * | 2018-01-05 | 2019-07-12 | 中国科学院天津工业生物技术研究所 | 高效表达分泌β-甘露聚糖酶的重组枯草芽孢杆菌菌株及其获得方法 |
| CN111040966A (zh) * | 2019-12-23 | 2020-04-21 | 河北科技大学 | 一种地衣芽孢杆菌KD-1、其生产的β-甘露聚糖酶及其应用 |
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