CN114395540A - 一种冠突曲霉葡萄糖氧化酶在改善面粉加工品质中的应用 - Google Patents
一种冠突曲霉葡萄糖氧化酶在改善面粉加工品质中的应用 Download PDFInfo
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- CN114395540A CN114395540A CN202210087611.9A CN202210087611A CN114395540A CN 114395540 A CN114395540 A CN 114395540A CN 202210087611 A CN202210087611 A CN 202210087611A CN 114395540 A CN114395540 A CN 114395540A
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- glucose oxidase
- aspergillus
- guani
- flour
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Abstract
本发明公开一种冠突曲霉葡萄糖氧化酶在改善面粉加工品质中的应用,属于基因工程和谷物科学领域。本发明采用真核表达的方法,获得了具有生物活性的重组冠突曲霉葡萄糖氧化酶,具有表达量高,纯化简便,易于放大,适合工业化应用等特点。本发明提供了一种单独添加冠突曲霉葡萄糖氧化酶改善面粉加工品质的方法,直接添加冠突曲霉葡萄糖氧化酶能显著降低面筋蛋白巯基含量,强化面筋网络结构,增加面包的比容,降低面包的硬度和咀嚼性,是一种潜在的新型面粉改良剂。
Description
技术领域
本发明属于基因工程和谷物科学领域,具体涉及一种毕赤酵母重组冠突曲霉葡萄糖氧化酶及其制备方法,以及毕赤酵母重组冠突曲霉葡萄糖氧化酶在改善面粉加工品质中的应用。
背景技术
随着生活水平的提高,人们对面包等面制品的需求逐渐上升,对优质面粉的需求也逐渐增加。我国国产小麦虽然品种较多,但是生产出来的面粉普遍存在“高筋不强,低筋不弱”的特点,难以达到优质面粉的标准。酶制剂作为绿色天然的面粉改良剂,具有广泛的应用前景。
目前葡萄糖氧化酶正逐步应用于面包烘焙行业,然而优质的葡萄糖氧化酶确鲜有报道。葡萄糖氧化酶的来源较为广泛,目前用于面粉加工行业的葡萄糖氧化酶主要来自黑曲霉和青霉,但黑曲霉和青霉生产产量低,纯化工艺复杂。如何提供一种葡萄糖氧化酶,并提供一种优良的生产菌株是本领域技术人员亟需解决的问题。
冠突曲霉又名冠突散囊菌,俗称“金花”,是茯砖茶中的一种优势益生菌。冠突曲霉葡萄糖氧化酶具有典型葡萄糖氧化酶的性质,是一种黄素依赖型蛋白,属于葡萄糖/甲醇/胆碱氧化还原酶家族。目前,国内外对于冠突曲霉葡萄糖氧化酶的报道较少,而将该酶作为面粉改良酶制剂,用于面制品品质改良中尚未见报道。
发明内容
为了克服现有技术的缺点与不足,本发明的目的在于提供一种冠突曲霉葡萄糖氧化酶在改善面粉加工品质中的应用。
本发明的目的通过下述技术方案实现:
本发明提供一种冠突曲霉葡萄糖氧化酶在改善面粉加工品质中的应用,特别是单独添加以冠突曲霉葡萄糖氧化酶为主的酶制剂在改善面粉加工品质中的应用。
具体的,冠突曲霉葡萄糖氧化酶在改善面筋及面包品质中的应用。
单独添加冠突曲霉葡萄糖氧化酶即能达到改善面包烘焙品质的目的。
所述的冠突曲霉葡萄糖氧化酶在面粉中的添加水平为2ppm~8ppm(面粉基),进一步为4ppm~8ppm(面粉基),可以提高面包的比容,降低面包的硬度和咀嚼性。
所述的冠突曲霉葡萄糖氧化酶的氨基酸序列如SEQ ID NO.1所示。本发明还提供了编码冠突曲霉葡萄糖氧化酶的DNA分子,其碱基序列如SEQ ID NO.2所示。
优选的,将冠突曲霉葡萄糖氧化酶、面粉、糖、盐、植物油、酵母粉和水搅拌均匀,形成的面团经分割称重、成形以及醒发后,焙烤得到面包成品;
进一步的,所述各组分添加量为:面粉90~110重量份,酵母粉0.6~1.8重量份,盐1~3重量份,水50~70重量份,糖5~10重量份,植物油2~5重量份。
所述的冠突曲霉葡萄糖氧化酶可以通过对转化有真核重组表达载体的毕赤酵母进行发酵获得。
所述的真核重组表达载体为包含冠突曲霉葡萄糖氧化酶基因的真核重组表达载体。
优选的,所述的真核重组表达载体的出发载体包括但不限于pPICZαA;
优选的,所述的毕赤酵母包括但不限于毕赤酵母X33。
所述的冠突曲霉葡萄糖氧化酶的毕赤酵母重组表达方法,包括如下步骤:
(1)利用全基因合成的方法获得了密码子优化的冠突曲霉葡萄糖氧化酶基因(序列如SEQ ID NO.2所示),将其与真核表达载体连接,将获得的真核重组表达载体转化到毕赤酵母感受态细胞中,获得重组表达菌;
所述的真核表达载体包括但不限于pPICZαA,所述的毕赤酵母包括但不限于毕赤酵母X33。
(2)将步骤(1)构建的重组表达菌接种至BMGY液体培养集中,过夜振荡培养;常温离心,收集菌体沉淀;将菌体沉淀转接至BMMY液体培养基中,转接后的菌液OD600为0.5~1.0;继续培养,期间每隔24h向培养基中添加甲醇至0.5%~2.0%v/v,固液分离后,收集发酵上清液;
(3)收集步骤(2)的发酵上清液,利用Ni-NTA柱层析,获得纯化的重组冠突曲霉葡萄糖氧化酶蛋白。
所述的BMGY液体培养基包含胰蛋白胨20g/L、酵母提取物10g/L、甘油10mL/L、无氨基酵母氮源13.4g/L、磷酸盐0.1mol/L。
所述的BMMY液体培养基包含胰蛋白胨20g/L、酵母提取物10g/L、无氨基酵母氮源13.4g/L、磷酸盐0.1mol/L、甲醇10mL/L。
步骤(2)中,
所述的过夜振荡培养为25~35℃,150~250rpm条件下过夜振荡培养;进一步为30℃,250rpm条件下过夜振荡培养;
所述的常温离心的条件为2500~3000g条件下常温离心2~5min;进一步为3000g条件下常温离心2min。
所述的继续培养的条件为25~35℃,150~250rpm条件下继续培养24~144h;进一步为30℃,250rpm条件下继续培养24~144h;
优选的,添加甲醇至1.0%v/v。
本发明相对于现有技术具有如下的优点及效果:
(1)本发明采用真核表达的方法,获得了具有生物活性的重组冠突曲霉葡萄糖氧化酶,具有表达量高,纯化简便,易于放大,适合工业化应用等特点。
(2)本发明提供了一种单独添加冠突曲霉葡萄糖氧化酶改善面粉加工品质的方法,直接添加冠突曲霉葡萄糖氧化酶能显著降低面筋蛋白巯基含量,强化面筋网络结构,增加面包的比容,降低面包的硬度和咀嚼性,是一种潜在的新型面粉改良剂。
附图说明
图1是实施例1重组表达质粒的双酶切结果;其中,泳道M:分子量Marker;泳道1:重组表达质粒双酶切产物。
图2是实施例2重组冠突曲霉葡萄糖氧化酶Ni-NTA亲和层析后SDS-PAGE分析;其中,泳道M:分子量Marker;泳道1:经还原剂β-巯基乙醇处理;泳道2:无还原剂处理。
图3是实施例3重组冠突曲霉葡萄糖氧化酶酶学性质法分析;其中,A:最适反应温度;B:最适反应pH;C:温度稳定性;D:pH稳定性。
图4是实施例5中重组冠突曲霉葡萄糖氧化酶对面包比容的影响;其中,实验重复3次,每一列不同上标字母表示差异性显著(p<0.05)。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
下列实施例中未注明具体实验条件的试验方法,通常按照常规实验条件或按照制造厂所建议的实验条件。除有特别说明,本发明中用到的各种试剂、原料均为可以从市场上购买的商品或者可以通过公认的方法制得的产品。
实施例中所用的BMGY液体培养基包含胰蛋白胨20g/L、酵母提取物10g/L、甘油10mL/L、无氨基酵母氮源13.4g/L、磷酸盐0.1mol/L。
所用的BMMY液体培养基包含胰蛋白胨20g/L、酵母提取物10g/L、无氨基酵母氮源13.4g/L、磷酸盐0.1mol/L、甲醇10mL/L。
实施例1葡萄糖氧化酶基因密码子优化及克隆
利用全基因合成技术,合成了毕赤酵母密码子优化的冠突曲霉(Aspergilluscristatus)葡萄糖氧化酶的全基因序列。其中,冠突曲霉葡萄糖氧化酶的氨基酸序列如SEQID NO.1所示,编码冠突曲霉葡萄糖氧化酶的碱基序列如SEQ ID NO.2所示。之后利用EcoRI和NotI对冠突曲霉葡萄糖氧化酶序列和载体质粒pPICZαA进行双酶切,并用T4 DNA酶进行连接反应,连接产物转化至大肠杆菌DH5α感受态细胞中。挑取单菌落,然后提取质粒电泳检测,并在-20℃保存质粒。再利用EcoRI与NotI酶切检测目的片段(图1),之后送质粒由公司进行测序,测序正确的质粒即为密码子优化的冠突曲霉葡萄糖氧化酶重组表达质粒pPICZαA-cgod。
实施例2葡萄糖氧化酶的诱导表达及纯化
1.诱导表达
将实施例1获得的上述重组表达载体pPICZαA-cgod用SacI进行线性化,利用电转化的方法将其转化至宿主细胞毕赤酵母X33中。对转化的抗性筛选平板上的单菌落进行菌落PCR验证,确保外源基因的整合。接种筛选出来的毕赤酵母重组子于10mL BMGY液体培养基中,30℃,250rpm,振荡培养过夜;常温3000g离心2min后,收集菌体,将其转接至BMMY培养基中,转接后的菌液OD600为0.5~1.0;30℃,250rpm条件下继续培养24~144h,期间每隔24h取样并向培养基中添加甲醇至1.0%v/v,培养完成后,固液分离获得发酵上清液。SDS-PAGE分析表明,在24~144h的发酵时间内,该重组菌都能够诱导表达重组冠突曲霉葡萄糖氧化酶。
2.亲和纯化
收集发酵上清液,然后使用AKTA蛋白纯化系统对获得的发酵上清液进行Ni-NTA柱层析。用含有100mM咪唑的20mM磷酸盐缓冲液洗脱,用超滤管(Millipore)浓缩收集目标蛋白溶液,通过SDS-PAGE分析判断蛋白质纯度(图2)。结果表明通过Ni-NTA一步纯化,可以获得电泳纯的冠突曲霉葡萄糖氧化酶目的蛋白。
实施例3葡萄糖氧化酶酶学性质测定
1.酶活力的测定
取1.25mL 0.2mol/L磷酸盐缓冲液(PH6.0),1.25mL 0.21mmol/L的还原型邻联茴香胺,0.3mL 18mg/mL的葡萄糖,100μL 60U/mL辣根过氧化物酶溶液和100μL适当稀释的酶液充分混合,在40℃条件下测定其在500nm处吸光度的变化。酶活定义为:在上述条件下,每分钟催化1μmol D-葡萄糖生成D-葡萄糖酸和H2O2所需的酶量定义为一个单位U。
2.酶学性质测定
按照上述酶活测定方法,测定实施例2所制备的重组葡萄糖氧化酶在不同的温度(20~70℃)下的酶活和不同pH(3.0~8.0)下的酶活,确定其最适反应温度和最适反应pH。将酶液分别置于不同温度(4℃~80℃)中保温1h后,测定其残余酶活力,确定其温度稳定性。将酶液置于不同pH(3.0~10.0)的缓冲液中,室温下处理12h,测定残余酶活性,以研究葡萄糖氧化酶的pH稳定性。所用缓冲液pH3.0~6.0范围内采用100mM柠檬酸盐缓冲液(Citrate buffer),pH6.0~8.0范围内采用100mM磷酸盐缓冲液(Phosphate buffer),pH8.0~9.0范围内采用100mM的Tris-HCl缓冲液(Tris-HCl buffer),pH9.0~10.0采用100mM甘氨酸-氢氧化钠缓冲液(Glycine-NaOH buffer)。
结果表明,冠突曲霉葡萄糖氧化酶的最适温度为40℃(图3A),最适反应pH为6.0(图3B);在温度小于60℃时处理1h,重组冠突曲霉葡萄糖氧化酶能保持80%以上的酶活力(图3C);在pH4.0~9.0范围内处理12h,葡萄糖氧化酶能保持80%以上的酶活力(图3D)。
实施例4应用重组冠突曲霉葡萄糖氧化酶降低面筋蛋白巯基含量
1.面筋蛋白的制备
将100g全面粉,58g纯净水,重组冠突曲霉葡萄糖氧化酶加入到和面钵中,搅拌后形成均匀的面团。将和好的面团置于30℃醒发30min后,用水冲洗面团中的淀粉,剩下的部分则认为是面筋蛋白。收集面筋蛋白进行冷冻干燥后,研磨成粉末备用,以不加酶组为对照组。
2.面筋蛋白巯基含量的测定
25mg面筋蛋白加入到0.5mL Tris-甘氨酸蛋白质变性缓冲液(86mM三羟甲基氨基甲烷Tris,90mM甘氨酸,4mM乙二胺四乙酸二钠,8M尿素,pH 8.0)中,漩涡振荡10min后,15,000g离心5min。随后,取100μL上清液加入到150μL Tris-甘氨酸蛋白质变性缓冲液和50μL新鲜配置的4mg/mL的5,5'-二硫双(2-硝基苯甲酸)溶液中。吸取100μL混合液加入到96孔板中,30℃孵育30min后,测定412nm处的吸光值。以相同体积的缓冲液作为空白对照。以还原性谷胱甘肽为标准样品,配置一系列标准浓度,按上述方法测定吸光值,绘制标准曲线。
结果表明(表1),与对照组相比,添加重组冠突曲霉葡萄糖氧化酶后,面筋蛋白中自由巯基含量从148.79μM降低到了116.36μM,降低了21.80%。面筋网络结构的增强往往与面筋蛋白分子间二硫键数量的增多有关,巯基含量的减少可以间接反映面筋蛋白中二硫键的增加。因此,巯基含量测定结果表明,添加重组冠突曲霉葡萄糖氧化酶促进了面筋蛋白中二硫键的交联,从而强化了面筋网络结构,提高了面粉加工品质。
表1重组冠突曲霉葡萄糖氧化酶对面筋蛋白巯基含量的影响
重组冠突曲霉葡萄糖氧化酶添加量(面粉基) | 巯基含量(μM) |
对照组 | 148.79±3.78a |
2ppm | 134.24±1.05b |
4ppm | 116.36±0.91c |
8ppm | 117.58±2.92c |
注:实验重复3次,每一列不同上标字母表示差异性显著(p<0.05)。
实施例5应用重组冠突曲霉葡萄糖氧化酶改善面包的比容
1.面包制备
面包烘焙的配方为:面粉100重量份,酵母粉1重量份,盐1.6重量份,水58重量份,糖6重量份,植物油3重量份,重组冠突曲霉葡萄糖氧化酶。以不加酶组为对照组。
面包制备步骤为:将称量好的水、小麦粉(面粉)、白砂糖、食用盐、酵母粉和葡萄糖氧化酶加入到和面钵中,搅拌18min,使其形成均匀的面团。将和好的面团置于天平上,分成50g/个,揉成圆形,至于38℃发酵60min,发酵好的面团放入180℃烤箱中焙烤10min,得到面包成品。将制得的面包室温下冷却2h,测定面包的质量和体积。其中面包的体积采用菜籽替代法,比容为面包的体积/面包的质量。
结果表明(图4),与对照组相比,加入冠突曲霉葡萄糖氧化酶后,面包比容增加,表明单独添加冠突曲霉葡萄糖氧化酶可以提高面包的品质。
实施例6应用重组冠突曲霉葡萄糖氧化酶改善面包的质构
1.面包制备
面包烘焙的配方为:面粉100重量份,酵母粉1重量份,盐1.6重量份,水58重量份,糖6重量份,植物油3重量份,重组冠突曲霉葡萄糖氧化酶。以不加酶组为对照组。
面包制备步骤为:将称量好的水、小麦粉(面粉)、白砂糖、食用盐、酵母粉和葡萄糖氧化酶加入到和面钵中,搅拌18min,使其形成均匀的面团。将和好的面团置于天平上,分成50g/个,揉成圆形,至于38℃发酵60min,发酵好的面团放入180℃烤箱中焙烤10min,得到面包成品。将制得的面包室温下冷却2h,测量面包的质构。
面包质构测定条件为:面包切成2cm/块,采用P/25探头,测前和测试速度设置为1mm/s,测后速度设置为5mm/s,压缩比50%,下压间隔10s。记录面包的硬度和咀嚼性。
结果表明(表2),与对照组相比,加入冠突曲霉葡萄糖氧化酶后,面包的硬度和咀嚼性显著性下降,表明添加冠突曲霉葡萄糖氧化酶可以提高面包的质构特性。
表2重组冠突曲霉葡萄糖氧化酶对面包质构特性的影响
注:实验重复3次,每一列不同上标字母表示差异性显著(p<0.05)。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110> 华南理工大学
<120> 一种冠突曲霉葡萄糖氧化酶在改善面粉加工品质中的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 592
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 冠突曲霉葡萄糖氧化酶的氨基酸序列
<400> 1
Leu Pro Lys Phe Pro Arg Asp His Leu Gly Val Glu Pro Gln Leu Leu
1 5 10 15
Thr Asp Pro Thr Val Leu Ala Asn Thr Thr Val Asp Tyr Ile Ile Ala
20 25 30
Gly Gly Gly Leu Thr Gly Leu Thr Val Ala Ala Arg Leu Thr Glu Asp
35 40 45
Pro Asn Ile Lys Val Leu Val Ile Glu Ser Gly Tyr Phe Glu Ser Asn
50 55 60
Arg Gly Pro Ile Ile Glu Asp Leu Asn Arg Tyr Gly Glu Ile Phe Gly
65 70 75 80
Thr Glu Val Asp His Ala Phe Glu Thr Val Gln Leu Ala Val Asn Asn
85 90 95
Arg Thr Glu Ile Ile Arg Ser Gly Asn Gly Leu Gly Gly Ser Thr Leu
100 105 110
Ile Asn Gly Gly Thr Trp Thr Arg Pro His Lys Val Gln Val Asp Ser
115 120 125
Trp Glu Thr Val Phe Gly Asn Gln Gly Trp Asn Trp Asp Asp Leu Leu
130 135 140
Pro Tyr Met Leu Lys Ile Glu Lys Ala Arg Pro Pro Asn Gln Arg Gln
145 150 155 160
Ile Glu Ala Gly His Tyr Phe Asn Pro Gln Cys His Gly Phe Asn Gly
165 170 175
Ser Val His Ala Gly Pro Arg Asp Thr Gly Glu Pro Tyr Ser Pro Ile
180 185 190
Met Arg Ala Leu Met Asp Thr Val Ser Ala Glu Gly Val Pro Val Arg
195 200 205
Lys Asp Leu Cys Cys Gly Asp Pro His Gly Val Ser Met Phe Leu Asn
210 215 220
Thr Leu Tyr Pro Ser Gln Ile Arg Ala Asp Ala Ala Arg Glu Tyr Leu
225 230 235 240
Val Pro Asn Tyr His Arg Pro Asn Phe Gln Val Leu Thr Gly Gln Arg
245 250 255
Val Gly Lys Val Leu Leu Asp Lys Thr Val Pro Gly Ser Pro Lys Ala
260 265 270
Ile Gly Val Glu Phe Gly Thr His Arg Thr Arg Lys Tyr Glu Ala Tyr
275 280 285
Ala Arg Arg Glu Val Leu Leu Ala Ala Gly Ser Thr Ile Ser Pro Thr
290 295 300
Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys Ser Val Leu Asp Ser Val
305 310 315 320
Gly Ile Glu Gln Val Val Glu Leu Pro Val Gly Val Asn Leu Gln Asp
325 330 335
Gln Thr Thr Leu His Val Glu Ser Arg Ile Thr Pro Ala Gly Ala Gly
340 345 350
Gln Gly Gln Ala Ala Tyr Phe Ala Thr Phe Asn Glu Thr Phe Gly Asp
355 360 365
Phe Ala Pro Gln Ala His Glu Leu Leu Asn Thr Lys Leu Asp Gln Trp
370 375 380
Ala Glu Glu Val Val Ala Arg Gly Gly Phe His Asn Ala Thr Ala Leu
385 390 395 400
Arg Ile Gln Tyr Glu Asn Tyr Arg Asn Trp Leu Val Asn Asn Asn Val
405 410 415
Ala Phe Ser Glu Leu Phe Leu Asp Thr Ala Gly Lys Ile Ser Phe Asp
420 425 430
Val Trp Asp Leu Ile Pro Phe Thr Arg Gly Tyr Val His Ile Ala Asp
435 440 445
Lys Asp Pro Tyr Leu Arg Arg Leu Tyr Asn Asn Pro Gln Tyr Phe Leu
450 455 460
Asn Glu Leu Asp Val Leu Gly Glu Ala Ala Ala Ser Lys Leu Ala Arg
465 470 475 480
Glu Leu Ser Ser Lys Gly Ala Met Ala Gln Tyr Tyr Ala Gly Glu Thr
485 490 495
Val Pro Gly Phe Asp Gln Leu Pro Ala Asp Ala Ser Leu Arg Asp Trp
500 505 510
Ala Lys Tyr Val Lys Asp Arg Phe Arg Pro Asn Tyr His Ala Val Ser
515 520 525
Thr Cys Ala Met Met Ser Lys Glu Leu Gly Gly Val Val Asp Ser Ala
530 535 540
Ala Arg Val Tyr Asp Val Glu Arg Leu Arg Val Val Asp Gly Ser Ile
545 550 555 560
Pro Pro Thr Gln Val Ser Ser His Val Met Thr Val Phe Tyr Gly Met
565 570 575
Ala Glu Lys Ile Ala Glu Ala Ile Leu Gln Asp Tyr His Ala Arg Lys
580 585 590
<210> 2
<211> 1776
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 编码冠突曲霉葡萄糖氧化酶的DNA分子
<400> 2
ttgccaaagt ttccaagaga tcatttgggt gttgaaccac aattgttgac tgatccaact 60
gttttggcta acactactgt tgattacatt attgctggag gtggtttgac tggtttgact 120
gttgctgcta gattgactga agatcctaac attaaggttt tggttattga atccggttac 180
tttgaatcta acagaggtcc tattattgaa gatttgaaca gatacggaga aatttttggt 240
actgaagttg atcatgcttt tgaaactgtt caattggctg ttaacaacag aactgaaatt 300
attagatcag gtaacggatt gggaggttcc actttgatta acggaggtac ttggactaga 360
ccacataagg ttcaagttga ttcctgggaa actgtttttg gtaaccaagg atggaactgg 420
gatgatttgt tgccttacat gttgaagatt gaaaaggcta gacctccaaa ccaaagacaa 480
attgaagctg gacattactt taacccacaa tgtcatggat ttaacggatc agttcatgct 540
ggacctagag atactggaga accttactcc cctattatga gagctttgat ggatactgtt 600
tccgctgaag gagttccagt tagaaaggat ttgtgttgtg gtgatcctca tggagtttct 660
atgtttttga acactttgta cccatcccaa attagagctg atgctgctag agaatacttg 720
gttcctaact accatagacc taactttcaa gttttgactg gtcaaagagt tggtaaggtt 780
ttgttggata agactgttcc aggttcacct aaggctattg gtgttgaatt tggaactcat 840
agaactagaa agtacgaagc ctacgctaga agagaagttt tgttggctgc tggatctact 900
atttcaccta ctattttgga atactccggt attggtatga agtccgtttt ggattccgtt 960
ggtattgaac aagttgttga attgccagtt ggagttaact tgcaagatca aactactttg 1020
catgttgaat ccagaattac tccagctgga gctggtcaag gacaagctgc ttactttgct 1080
acttttaacg aaacttttgg tgattttgct cctcaagctc atgaattgtt gaacactaag 1140
ttggatcaat gggctgaaga agttgttgct agaggaggat ttcataacgc tactgctttg 1200
cgtatccaat acgaaaacta cagaaactgg ttggttaaca acaacgttgc tttttccgaa 1260
ttgtttttgg atactgctgg taagatttcc tttgatgttt gggatttgat tccatttact 1320
agaggttacg ttcatattgc tgataaggac ccttacttga gaagattgta caacaaccca 1380
caatactttt tgaacgaatt ggatgttttg ggtgaagctg ctgcttctaa gttggctaga 1440
gaattgtctt ctaagggagc tatggctcaa tactacgctg gtgaaactgt tccaggattt 1500
gatcaattgc cagctgatgc ttccttgaga gattgggcta agtacgttaa ggatagattt 1560
agaccaaact accatgctgt ttccacttgt gctatgatgt ctaaggaatt gggtggagtt 1620
gttgattcag ctgctagagt ttacgatgtt gaaagattga gagttgttga tggttctatt 1680
cctccaactc aagtttcttc acatgttatg actgtttttt acggtatggc tgaaaagatt 1740
gctgaagcta ttttgcaaga ttaccatgct agaaag 1776
Claims (10)
1.冠突曲霉葡萄糖氧化酶在改善面粉加工品质中的应用。
2.根据权利要求1所述的应用,其特征在于:
单独添加以冠突曲霉葡萄糖氧化酶为主的酶制剂在改善面粉加工品质中的应用。
3.根据权利要求1或2所述的应用,其特征在于:
冠突曲霉葡萄糖氧化酶在改善面筋及面包品质中的应用。
4.根据权利要求1或2所述的应用,其特征在于:
所述的冠突曲霉葡萄糖氧化酶在面粉中的添加水平为2ppm~8ppm,面粉基;进一步为4ppm~8ppm,面粉基。
5.根据权利要求1或2所述的应用,其特征在于:
所述的冠突曲霉葡萄糖氧化酶的氨基酸序列如SEQ ID NO.1所示。
6.根据权利要求5所述的应用,其特征在于:
编码冠突曲霉葡萄糖氧化酶的DNA分子,其碱基序列如SEQ ID NO.2所示。
7.根据权利要求1或2所述的应用,其特征在于:
将冠突曲霉葡萄糖氧化酶、面粉、糖、盐、植物油、酵母粉和水搅拌均匀,形成的面团经分割称重、成形以及醒发后,焙烤得到面包成品。
8.根据权利要求1或2所述的应用,其特征在于:
所述的冠突曲霉葡萄糖氧化酶通过对转化有真核重组表达载体的毕赤酵母进行发酵获得;
所述的真核重组表达载体为包含冠突曲霉葡萄糖氧化酶基因的真核重组表达载体。
9.根据权利要求8所述的应用,其特征在于:
所述的冠突曲霉葡萄糖氧化酶的毕赤酵母重组表达方法,包括如下步骤:
(1)利用全基因合成的方法获得了密码子优化的冠突曲霉葡萄糖氧化酶基因,将其与真核表达载体连接,将获得的真核重组表达载体转化到毕赤酵母感受态细胞中,获得重组表达菌;
(2)将步骤(1)构建的重组表达菌接种至BMGY液体培养集中,过夜振荡培养;常温离心,收集菌体沉淀;将菌体沉淀转接至BMMY液体培养基中,转接后的菌液OD600为0.5~1.0;继续培养,期间每隔24h向培养基中添加甲醇至0.5%~2.0%v/v,固液分离后,收集发酵上清液;
(3)收集步骤(2)的发酵上清液,利用Ni-NTA柱层析,获得纯化的重组冠突曲霉葡萄糖氧化酶蛋白。
10.根据权利要求9所述的应用,其特征在于:
步骤(1)中,所述的真核表达载体为pPICZαA,所述的毕赤酵母为毕赤酵母X33;
步骤(2)中,所述的过夜振荡培养为25~35℃,150~250rpm条件下过夜振荡培养;
步骤(2)中,所述的常温离心的条件为2500~3000g条件下常温离心2~5min;
步骤(2)中,所述的继续培养的条件为25~35℃,150~250rpm条件下继续培养24~144h。
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NA: "GenBank: ODM18347.1", 《GENBANK》 * |
聂金梅等: "黑曲霉葡萄糖氧化酶基因改造及其在毕赤酵母中的表达", 《江苏农业科学》 * |
邓家珞等: "葡萄糖氧化酶和过氧化氢酶对面团与面包品质的影响", 《现代食品科技》 * |
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WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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