CN113667613A - 一株重组毕赤酵母工程菌及其应用 - Google Patents
一株重组毕赤酵母工程菌及其应用 Download PDFInfo
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- CN113667613A CN113667613A CN202110706937.0A CN202110706937A CN113667613A CN 113667613 A CN113667613 A CN 113667613A CN 202110706937 A CN202110706937 A CN 202110706937A CN 113667613 A CN113667613 A CN 113667613A
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- pichia pastoris
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- hexose oxidase
- strain
- hox
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Abstract
本发明公开了一株重组毕赤酵母工程菌及其应用。所述重组毕赤酵母工程菌由毕赤酵母KM71基因组中插入多拷贝数的己糖氧化酶基因表达盒获得;所述己糖氧化酶基因核苷酸序列如SEQ ID NO.1所示。本发明提供了一株高拷贝数高活性的重组毕赤酵母工程菌,利用该重组毕赤酵母工程菌进行高密度发酵获得的己糖氧化酶,最适温度为50℃,最适反应pH为4.0,在pH为3.0~6.0时酶活较为稳定,催化的底物谱较广,能催化D‑果糖、D‑葡萄糖、麦芽糖、D‑半乳糖、乳糖和纤维二糖的氧化,在食品和畜牧养殖等方面具有很好的应用潜力。
Description
(一)技术领域
本发明涉及一株重组毕赤酵母工程菌及其应用。
(二)背景技术
己糖氧化酶(hexose oxidase,HOX)是一种底物谱广泛的糖类氧化还原酶,能催化D-葡萄糖、D-果糖、D-半乳糖、麦芽糖、乳糖等己糖和一些二糖的氧化,生成相应的内酯并释放出H2O2。己糖氧化酶具有除氧、酸化、提高蛋白交联等应用功能,极具新型绿色添加剂的潜力,可被应用于食品抗氧化、食品保鲜以及畜牧养殖等领域。
己糖氧化酶主要来源于红藻类物种,可经过提取得到天然来源的己糖氧化酶产品。但红藻类物种中的己糖氧化酶含量较低,直接提取,困难较大,成本偏高,且步骤繁琐。
毕赤酵母菌是美国FDA公认的一种Generally Recognized as Safe(GRAS)生物,在食品药品生产加工中被广泛应用。相较于酿酒酵母表达系统,毕赤酵母表达的蛋白糖基化程度较低,更有利于蛋白的活性保持。应用毕赤酵母作为宿主菌株,表达红藻类来源的己糖氧化酶,具有食品安全,易于发酵生产等优点。
(三)发明内容
本发明目的是提供一株产己糖氧化酶的重组毕赤酵母工程菌及其应用。
本发明采用的技术方案是:
一株重组毕赤酵母工程菌,由毕赤酵母KM71基因组中插入多拷贝数的己糖氧化酶基因表达盒获得;所述己糖氧化酶基因核苷酸序列如SEQ ID NO.1所示。本发明毕赤酵母工程菌用以表达己糖氧化酶。所述己糖氧化酶氨基酸序列来源于皱波角叉菜(Chondruscrispus),从GenBank数据库中搜索获得,其登录号为CDF77476,氨基酸序列如SEQ ID NO.2所示。所述酶编码基因是根据氨基酸序列经过密码子优化后获得,其优选的DNA序列如SEQID NO.1所示。
所述重组毕赤酵母是通过Zeocin抗性梯度平板筛选法、平板显色筛选法、基于实时荧光定量PCR分析的拷贝数检测等步骤,筛选而得到的高拷贝数高活性的转化子。
具体的,所述拷贝数为3~6,优选为6。
优选的,所述重组毕赤酵母工程菌按如下方法构建获得:
(1)利用表达质粒pPICZ-A,将序列如SEQ ID NO.1所示的hox基因克隆至甲醇诱导型启动下游,获得重组质粒pPICZ-HOX;
(2)重组质粒pPICZ-HOX转化至毕赤酵母菌株KM71感受态中,筛选hox基因拷贝数为3~6的重组菌株,即为所述重组毕赤酵母工程菌。
本发明还涉及所述重组毕赤酵母工程菌在微生物发酵制备己糖氧化酶中的应用。
具体的,所述应用为:将所述重组毕赤酵母工程菌经种子活化、种子培养获得种子液,种子液接种至BSMH培养基中培养至培养基中甘油耗尽,再以18.15mL/h/L速率进行甘油补料培养基的流加,甘油补料培养基流加结束后,继续饥饿培养20~30min;然后采用甲醇和山梨醇混合流加补料,将山梨醇补料培养基的流速设置为0.7~0.8g/L/h,同时设定甲醇补料培养基的补料速率为自动控制,保持培养基中甲醇浓度控制为5±0.1g/L,持续发酵85~96h后,获得重组菌株的高密度发酵液,发酵液经分离纯化获得所述己糖氧化酶。
所述分离纯化方法如下:将重组菌株的高密度发酵液离心,收集细胞沉淀重悬于pH7.4的磷酸钾缓冲液中,使用高压匀浆仪破碎菌液,离心去除沉淀,所得上清液进行镍柱纯化,获得纯化后的己糖氧化酶。
与现有的红藻类物种中直接提取己糖氧化酶的方法和已公开的CN1266275C、CN1190992A的方法相比,本发明提供生产己糖氧化酶毕赤酵母工程菌株的特点在于:①从GenBank数据库选择了一个新的己糖氧化酶氨基酸序列;②经过多重方法筛选和比较了大量的转化子后,得到产酶活较高且含有6个拷贝数的重组毕赤酵母KCX56菌珠,即基因型明确的毕赤酵母工程菌;③提供了甲醇-山梨醇混合共补料策略,并高密度发酵并获得己糖氧化酶发酵液,该方法易于放大生产;④此外,提供该重组己糖氧化酶的酶学特性,包括最适温度和最适pH,催化的底物谱较广,能催化D-果糖、D-葡萄糖、麦芽糖、D-半乳糖、乳糖和纤维二糖的氧化。
本发明的有益效果主要体现在:本发明提供了一株高拷贝数高活性的重组毕赤酵母工程菌,利用该重组毕赤酵母工程菌进行高密度发酵获得的己糖氧化酶,最适温度为50℃,最适反应pH为4.0,在pH为3.0~6.0时酶活较为稳定,催化的底物谱较广,能催化D-果糖、D-葡萄糖、麦芽糖、D-半乳糖、乳糖和纤维二糖的氧化,在食品和畜牧养殖等方面具有很好的应用潜力。
(四)附图说明
图1为pPICZ-HOX重组质粒图谱。
图2为平板显色法比较不同的重组毕赤酵母KCX菌株。
图3为Ni柱纯化的重组己糖氧化酶的SDS-PAGE图谱。
图4为重组己糖氧化酶的最适反应温度分析。
图5为重组己糖氧化酶的热稳定性分析。
图6为重组己糖氧化酶的最适pH分析。
图7为重组己糖氧化酶的pH稳定性分析。
图8为重组己糖氧化酶对不同糖类底物的催化活性比较。
(五)具体实施方式
为了加深对本发明的理解,下面将结合具体实施例对本发明做进一步详细描述,该实施例仅用于解释本发明,并不对本发明的保护范围构成限定。
实施例中如无特殊说明所用方法均为常规方法,所用试剂均可从商业途径获得。
BSM培养基:85%磷酸26.7mL/L,硫酸钙0.93g/L,硫酸钾18.2g/L,七水硫酸镁14.9g/L,氢氧化钾4.13g/L,甘油40g/L,PTM1 4.35mL/L,另加组氨酸2g/L。
诱导培养基1:100%甲醇、PTM1 12mL/L。
诱导培养基2:山梨醇500g/L。
PTM1微量盐溶液:硫酸铜6g/L,碘化钠0.08g/L,硫酸锰3g/L,钼酸钠0.2g/L,硼酸0.02g/L,氯化钴0.5g/L,硫酸亚铁65g/L,生物素0.2g/L,浓硫酸5ml/L,过滤除菌。
MMH平板:YNB 13.4g/L,甲醇5g/L,生物素4×10-4g/L,组氨酸4×10-2g/L,琼脂粉20g/L。
BMGY液体培养基:酵母粉10g/L,蛋白胨20g/L,甘油10g/L,YNB 13.4g/L,生物素4×10-4g/L,100mmol/L磷酸钾缓冲液(pH 6.0)。
BMMY液体培养基:酵母粉10g/L,蛋白胨20g/L,甲醇5g/L,YNB 13.4g/L,生物素4×10-4g/L,100mmol/L磷酸钾缓冲液(pH 6.0)。
BSMH培养基:85%H3PO4 26.7mL/L,CaSO4 0.93g/L,K2SO4 18.2g/L,MgSO4·7H2O14.9g/L,KOH 4.13g/L,2g/L组氨酸,甘油40g/L。
甘油流加培养基:50%(w/v)甘油,灭菌后加入12mL/L PTM1。
甲醇流加培养基:纯甲醇中加入12mL/L PTM1。
山梨醇流加培养基:500g/L山梨醇。
实施例1:毕赤酵母重组菌KCX的构建
从NCBI的GenBank数据库中,选择登入号CDF77476的己糖氧化酶基因,用于产己糖氧化酶的毕赤酵母工程菌。将其氨基酸序列提交至基因合成公司(华大基因(无锡)青兰生物科技有限公司),经过密码子优化后,进行人工合成基因,并直接克隆到表达载体pPIC9k的EcoRI/NotI之间,获得重组质粒pPIC9k-αHOX。并通过PCR扩增得到hox基因片段,应用一步克隆试剂盒(One Step Cloning Kit ClonExpressTM II,南京诺维赞生物科技有限公司)克隆至表达载体pPICZ-A,获得重组质粒pPICZ-HOX,如图1所示。将该质粒用限制性内切酶SacI线性化后,并胶回收纯化,转化至毕赤酵母菌株KM71感受态中,涂布含50μg/mL的Zeocin抗性的YPD平板,30℃倒置培养,直至出现转化子,将其命名为KCX菌株。
实施例2:抗生素梯度平板法筛选毕赤酵母重组菌KCX
用无菌牙签挑取重组KCX菌株的单菌落划线至抗生素浓度分别为100、500、1000μg/mL的Zeocin抗性平板上,30℃倒置培养。
通过该方法筛选得到54株抗1000μg/mL Zeocin的高抗性KCX菌株。
实施例3:平板显色法筛选毕赤酵母重组菌KCX
用无菌牙签挑取上述高抗性的重组KCX菌株分别点到具有编号的MMH平板。30℃倒置培养48h后倾倒显色液。
显色液:
溶液Ⅰ:邻联茴香胺储存液(0.1g邻联茴香胺溶于10mL甲醇);
溶液Ⅱ:18%葡萄糖溶液;
溶液Ⅲ:80U/mL辣根过氧化物酶。
先配制10mL 1%的琼脂糖溶液,微波炉加热溶化,待冷却至50-60℃后,加入2mL溶液Ⅱ,200μL溶液Ⅰ,400μL溶液Ⅲ即为显色液。显色液应现配现用。
通过该方法筛选得到46株显色菌株,如图2所示。
实施例4:实时荧光定量PCR检测重组菌KCX菌株的hox基因拷贝数
以毕赤酵母管家基因gap为内参,将其克隆至pUC18质粒上,得到重组质粒pUC18-GAP,作为标准质粒,用于双标准曲线法测定毕赤酵母重组菌株KCX中hox基因的拷贝数。
构建gap基因标准质粒pUC18-GAP。用酵母基因组DNA提取试剂盒,提取毕赤酵母KM71的基因组DNA;以此为模板,以Gap-pUC18-F和Gap-pUC18-R为引物,进行PCR扩增gap基因,并进行胶回收纯化;提取pUC18质粒,用BamHI和HindIII进行双酶切并进行胶回收纯化;以上两种产物用于一步克隆试剂盒进行连接反应,并转化到E.coli DH5α感受态中,涂布到100μg/mL氨苄青霉素抗性的LB平板上;挑取抗性平板上的转化子为模板,以pUC-M13-F和pUC-M13-R为引物,进行菌落PCR验证;挑取阳性克隆,接种至25mL含100μg/mL氨苄青霉素抗性的LB液体培养基中,37℃培养过夜,提取质粒并送样测序验证。
Gap-pUC18-F:
CGAATTCGAGCTCGGTACCCGGGGATCCTTTTTTGTAGAAATGTC
Gap-pUC18-R:CGACGGCCAGTGCCAAGCTTTTTAGATAAGGACAGGAGAGATG
pUC-M13-F:GTAAAACGACGGCCAGT
pUC-M13-R:CAGGAAACAGCTATGAC
提取各个重组菌KCX菌株的基因组DNA。针对Zeocin梯度抗性平板法和平板显色法筛选得到KCX菌株,将其分别接种于YPD液体培养基中,30℃过夜培养.取适量菌液,用酵母基因组DNA提取试剂盒提取各个菌株基因组DNA,将其作为实时荧光定量PCR的模板,从而检测各个菌株基因组上的hox基因的拷贝数。
以不同浓度的pUC18-GAP和pPICZ-HOX质粒为模板,以RT-gap-F/RT-gap-R和RT-HOX-F/RT-HOX-R为引物分别进行实时荧光定量PCR。以起始模板中质粒拷贝数的对数作为横坐标,测得的Ct值作为纵坐标,建立含gap和hox基因质粒的双标准曲线。
RT-gap-F:GGTCTTTTGAGTGGCGGTC
RT-gap-R:TTGTCGGTGTCAACGAGGAG
RT-HOX-F:GGCCAGGTGTGATTGGAAGA
RT-HOX-R:AACCAGCATGACCACCCAAA
以各个重组毕赤酵母KCX菌株基因组为模板,RT-HOX-F/RT-HOX-R为引物,进行实时荧光定量PCR,将Ct值分别代入两条标准曲线中,求出样品DNA中gap基因和hox基因的起始拷贝数。由于gap基因在毕赤酵母基因组中是单拷贝,因此样品中hox基因拷贝数=hox基因起始拷贝数/gap基因起始拷贝数。
经检测,重组菌株KCX56的hox基因拷贝数最高,为6拷贝,如表1所示。
表1:重组菌株KCX的拷贝数
实施例5:重组毕赤酵母菌在生产己糖氧化酶中的应用
一、己糖氧化酶的活性检测
在96孔板的每孔中分别加入0.28mM邻联茴香胺溶液120μL、10%葡萄糖溶液25μL、1U/mL辣根过氧化物酶溶液10μL,加入20μL待测样品。迅速混匀,置于酶标仪中。设定温度35℃,检测波长460nm,每隔1min测定一次吸光值,连续测定20min,计算每分钟吸光度增加值的平均值。
酶活定义为:温度35℃条件下,每分钟催化氧化1μmoL葡萄糖转化为葡萄糖酸并释放H2O2的量为一个酶活单位。
酶活=每分钟吸光度增加值的平均值×0.175/0.02×11.3
其中,0.175指用于分析的溶液体积,单位为mL。0.02指用于分析的样品制备溶液中的酶液体积,单位为mL。11.3指摩尔吸光系数ε,单位为mM-1·cm-1。
二、重组毕赤酵母菌摇瓶发酵产己糖氧化酶
将含有6个hox基因拷贝的重组菌株KCX56划线至YPD平板,30℃培养48h后,挑取单菌落接种于BMGY液体培养基中,200rpm,30℃过夜摇床培养。将菌液按5%(v/v)转接接种于新鲜配制的100mL BMGY液体培养基中,200rpm,30℃摇床培养至OD600=3.0。将菌液转移至无菌离心管中,3000×g,4℃离心10min,收集细胞,将细胞重悬于新鲜配制的100mL BMMY液体培养基中。200rpm,30℃摇床发酵培养,每24h补加0.5%甲醇。连续诱导96h后,获得KCX56的发酵液。
取100mL的KCX56发酵液,高压匀浆后,将破碎液上清直接用于HOX活性检测,结果表明KCX56发酵酶活为6.2×10-2U/mL。
三、重组毕赤酵母菌高密度发酵产己糖氧化酶
1.菌种活化:将筛选得到的重组毕赤酵母KCX56菌株划线至YPD平板,30℃倒置培养至长出单菌落。
2.种子液的制备:挑取YPD平板上的单菌落,接种于10mL YPD液体培养基中,200rpm,30℃过夜培养。将活化的菌液按5%(v/v)转接于100mL YPD液体培养基,200rpm,30℃培养至OD600=5.0,即得到接种到5L发酵罐的种子液。
3.5L发酵罐的高密度发酵:将步骤2中的种子液以10%的接种量转接到基础盐BSMH培养基中,在发酵罐中进行培养。发酵参数控制为:温度30℃,溶氧量DO值大于30%,用氨水调节发酵pH5.0。最初培养20h后,即培养基中甘油耗尽,开始以18.15mL/h/L速率进行甘油补料培养基的流加。甘油补料培养基流加结束后,继续饥饿培养30min;采用甲醇和山梨醇混合流加补料,将山梨醇补料培养基的流速设置为0.785g/L/h。同时设定甲醇补料培养基的补料速率为自动控制,即在甲醇自动检测仪的控制下,保持培养基中甲醇浓度控制约5g/L。持续发酵89h后,获得发酵液的细胞密度OD600为178,即得到KCX56菌株的高密度发酵液。
4.发酵液的HOX活性检测:将发酵液稀释至OD600为20,并采用高压匀浆仪破碎菌液。8000×g,4℃离心10min,去除沉淀,所得上清液即为粗酶液。将该粗酶液用于HOX活性检测,测得KCX56菌株的高密度发酵液的HOX酶活为0.25U/mL。
实施例6:重组毕赤酵母菌生产己糖氧化酶的功能分析
一、表达含C-端His-tag的己糖氧化酶的重组毕赤酵母菌构建
应用定点突变的方法,删除pPICZ-HOX上的hox基因的翻译终止密码子TAA,使其继续翻译,并在下游产生6×His标签后终止翻译,从而表达C-端融合His-tag的己糖氧化酶,其重组质粒为pPICZ-HOXHis。将该质粒用限制性内切酶SacI酶切线性化后,转化至毕赤酵母菌株KM71感受态中,并涂布含50μg/mL的Zeocin抗性的YPD平板,30℃倒置培养,生长的菌落被称为重组KCXH菌株。
筛选方法如实施例3~4所示。最终得到3拷贝菌株KCXH93,即获得表达C-端融合His-tag己糖氧化酶的重组菌,用于异源表达重组己糖氧化酶应用特性的表征。
二、重组毕赤酵母菌KCXH的发酵生产C-端融合His-tag的己糖氧化酶
重组毕赤酵母菌KCXH93摇瓶发酵和高密度发酵产己糖氧化酶的方法如实施例5所示。经96h的摇瓶发酵,KCXH93的酶活为3.7×10-2U/mL。采用甲醇/山梨醇混合补料诱导,经89h的高密度发酵,KCXH93的酶活为6.3×10-2U/mL。
三、C-端融合His-tag的己糖氧化酶的分离纯化:
将KCXH93菌株的高密度发酵液离心,4000×g,4℃离心10min,并将细胞沉淀重悬于50mmol/L磷酸钾缓冲液(pH7.4)中。使用高压匀浆仪破碎菌液,8000×g,4℃离心20min,去除沉淀,所得上清液即为纯化HOX的粗酶液,用于镍柱纯化。采用Takara公司的镍柱纯化凝胶(His60 Ni Superflow Resin and Gravity Columns)进行纯化,具体操作可参考使用说明书。
纯化所得的己糖氧化酶进行SDS-PAGE分析确定,并测定蛋白浓度为270mg/L。SDS-PAGE电泳检测图如图3所示。纯化的己糖氧化酶溶液经过SDS-PAGE分析表明具有3条带,大小分别为62kDa、40kDa和29kDa,分别是HOX的原酶和自我切割而形成的两个二聚体亚基。
四、重组己糖氧化酶的酶学性质分析
1、重组己糖氧化酶最适反应温度和热稳定性
最适反应温度的确定:将实施例5中所述的酶活检测的反应体系分别在20℃~80℃条件下保温5min,然后加入辣根过氧化酶和重组己糖氧化酶酶液进行反应,并测定各温度条件下己糖氧化酶的酶活力。结果如图4所示,最适温度为50℃。
热稳定性的分析:将纯化的己糖氧化酶酶液分别于20℃~80℃条件下保温2h,然后将其用于实施例5所述的HOX活力测定。结果如图5所示,在20~60℃时酶活较为稳定。
2、重组己糖氧化酶最适反应pH和pH稳定性
最适反应pH的确定:分别用50mM乙酸-乙酸钠缓冲液(pH 3.0~5.0)、50mM磷酸钾缓冲液(pH 6.0~8.0)、50mM Tris-HCl缓冲液(pH 9.0)配制邻联茴香胺溶液,然后加入重组HOX酶液进行反应,于35℃测定各pH条件下HOX的酶活力。结果如图6所示,重组己糖氧化酶最适pH为4.0。
pH稳定性的分析:将酶液分别于pH为3~9的条件下放置2h,加入重组己糖氧化酶酶液进行反应,测定各pH条件下己糖氧化酶的酶活力。结果如图7所示,在pH为3.0~6.0时酶活较为稳定。
3、重组己糖氧化酶对不同底物的氧化活性检测:在应温度50℃和pH4.0条件下,分别以葡萄糖、果糖、麦芽糖、乳糖、半乳糖、蔗糖和纤维二糖作为反应底物,进行如实施例5所述的HOX酶活测定。将最适底物的酶活设为100%,计算催化其他底物时的相对酶活。结果如图8所示,重组己糖氧化酶对果糖、葡萄糖、麦芽糖、半乳糖、乳糖和纤维二糖都有催化氧化的作用,其最适底物是葡萄糖。
序列表
<110> 浙江新银象生物工程有限公司
浙江工业大学
<120> 一株重组毕赤酵母工程菌及其应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1641
<212> DNA
<213> Chondrus crispus
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atggcaaccc taccccaaaa ggatcctggt tacattgtca ttgacgttaa tgccggaact 60
ccagataagc cagaccctag attaccttct atgaagcagg gtttcaaccg aagatggatc 120
ggtacaaaca ttgattttgt ttacgttgtt tacaccccac aaggagcctg tactgcattg 180
gatagggcaa tggaaaagtg ttctccaggt accgtgcgaa ttgtgtctgg tggtcattgt 240
tatgaagact ttgtattcga tgaatgtgtt aaggctataa ttaacgttac cggtctggta 300
gaatcaggat acgatgatga tagaggttat tttgtctcca gtggagacac taactgggga 360
tcctttaaga ccttgtttag agatcatgga agagttctgc caggtggatc atgttatagt 420
gttggcctgg gaggtcacat agttggtgga ggagatggta tcttagctcg tctgcatggt 480
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cacaccggag gaggcggtgg aaacttcggt attattacta aatactactt caaagatcta 660
cctatgtcac ctagaggtgt tatagccagt aatctgcact tcagttggga tggattcact 720
agagatgccc ttcaagacct actgacaaaa tactttaaac tggctcgatg tgactggaaa 780
aacactgttg gaaaatttca aatatttcat caagccgcag aggaatttgt tatgtactta 840
tacacctcat acagtaacga tgctgagagg gaagttgctc aggatcgtca ttatcactta 900
gaggctgaca tcgagcaaat atacaaaacc tgtgaaccaa caaaggcttt gggaggtcat 960
gccggttggg ccccttttcc agttagacct agaaagagac atacttcaaa aacatcttat 1020
atacatgatg aaactatgga ttatcctttt tacgccttga ctgaaactat aaatggttct 1080
ggccctaatc aacgtggtaa gtacaagtct gcctacatga taaaagattt tcctgatctg 1140
caaatagacg tcatttggaa gtacctgact gaggttcctg atggtcttac gagtgctgag 1200
atgaaagatg ctctgttgca ggtagatatg ttcggcggcg agatccacaa tgttgcatgg 1260
gatgctactg ccgtggccca aagaaaatat atcattaagt tgcagtatca aacatattgg 1320
caagaggaag acaaagacgc tgttaacttg aagtggatta gagactttta tgaagaaatg 1380
tacgaaccat acggtggtgt tcctgatccc aacacacagg tagaatcagg taagggtgtc 1440
ttcgaaggtt gttatttcaa ctatccagat gttgatttaa ataattggaa aaacggcaaa 1500
tatggagctc tagaactgta ttttttaggt aaccttaata gattaataaa ggctaaaaag 1560
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gagtacaagc aaactaaata a 1641
<210> 2
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Gly His Ile Val Gly Gly Gly Asp Gly Ile Leu Ala Arg Leu His Gly
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cgacggccag tgccaagctt tttagataag gacaggagag atg 43
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<212> DNA
<213> 未知(Unknown)
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gtaaaacgac ggccagt 17
<210> 6
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<212> DNA
<213> 未知(Unknown)
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<210> 7
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<212> DNA
<213> 未知(Unknown)
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ggtcttttga gtggcggtc 19
<210> 8
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<212> DNA
<213> 未知(Unknown)
<400> 8
ttgtcggtgt caacgaggag 20
<210> 9
<211> 20
<212> DNA
<213> 未知(Unknown)
<400> 9
ggccaggtgt gattggaaga 20
<210> 10
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<212> DNA
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aaccagcatg accacccaaa 20
Claims (6)
1.一株重组毕赤酵母工程菌,由毕赤酵母KM71基因组中插入多拷贝数的己糖氧化酶基因表达盒获得;所述己糖氧化酶基因核苷酸序列如SEQ ID NO.1所示。
2.如权利要求1所述的重组毕赤酵母工程菌,其特征在于所述拷贝数为3~6。
3.如权利要求1所述的重组毕赤酵母工程菌,其特征在于所述重组毕赤酵母工程菌按如下方法构建获得:
(1)利用表达质粒pPICZ-A,将序列如SEQ ID NO.1所示的hox基因克隆至甲醇诱导型启动下游,获得重组质粒pPICZ-HOX;
(2)重组质粒pPICZ-HOX转化至毕赤酵母菌株KM71感受态中,筛选hox基因拷贝数为3~6的重组菌株,即为所述重组毕赤酵母工程菌。
4.权利要求1所述重组毕赤酵母工程菌在微生物发酵制备己糖氧化酶中的应用。
5.如权利要求4所述的应用,其特征在于所述应用为:将所述重组毕赤酵母工程菌经种子活化、种子培养获得种子液,种子液接种至BSMH培养基中培养至培养基中甘油耗尽,再以18.15mL/h/L速率进行甘油补料培养基的流加,甘油补料培养基流加结束后,继续饥饿培养20~30min;然后采用甲醇和山梨醇混合流加补料,将山梨醇补料培养基的流速设置为0.7~0.8g/L/h,同时设定甲醇补料培养基的补料速率为自动控制,保持培养基中甲醇浓度控制为5±0.1g/L,持续发酵85~96h后,获得重组菌株的高密度发酵液,发酵液经分离纯化获得所述己糖氧化酶。
6.如权利要求5所述的应用,其特征在于所述分离纯化方法如下:将重组菌株的高密度发酵液离心,收集细胞沉淀重悬于pH7.4的磷酸钾缓冲液中,使用高压匀浆仪破碎菌液,离心去除沉淀,所得上清液进行镍柱纯化,获得纯化后的己糖氧化酶。
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