CN115161298A - 一种双加氧酶氨基酸序列、基因及其生物生香应用 - Google Patents
一种双加氧酶氨基酸序列、基因及其生物生香应用 Download PDFInfo
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Abstract
本发明公开了一种双加氧酶氨基酸序列、基因及其生物生香应用，其特征在于：该基因的氨基酸序列如序列1所示。本发明技术方案具有以下有益效果：(1)双加氧酶CADO处理后的烟草浸膏在吃味方面有明显改善。(2)经过双加氧酶CADO酶处理后的烟草浸膏，其中的特征类胡萝卜裂解香气产物含量有大幅提高。
Description
发明领域
本发明涉及一种双加氧酶氨基酸序列、基因及其生物生香应用，属于生物转化生香领域，特别是类胡萝卜氧化生香领域。
发明背景
双加氧酶与单加氧化酶是微生物及植物体内广泛存在，且非常重要的氧化酶，其广泛参与植物的多种代谢过程。它不仅对植物的生长发育具有重要意义,而且在许多药用植物功效及香气成分的生物合成中扮演重要的角色。双加氧酶是香豆素、黄酮类香气化合物核心的成环及羟基化衍生的重要酶，同时也是类胡萝卜素类香气前体的氧化裂解的生香的关键性酶。类胡萝卜素裂解双加氧酶可以作用于类胡萝卜素的双键位置，对类胡萝卜素进行氧化裂解，形成一系列的类胡萝卜素衍生物。高等植物中类胡萝卜素裂解双加氧酶根据系统发育和功能被分为5个家族，CCD1，CCD4，CCD7，CCD8以及NCED。目前已知植物CCD1酶的底物较多，在离体条件下，能够以八氢番茄红素、番茄红素、β-胡萝卜素、α-胡萝卜素、玉米黄质、紫黄质和新黄质等多种类胡萝卜素为反应底物，生成β-紫罗酮、3-羟基-β-紫罗酮、3-羟基-α-紫罗酮、香叶基丙酮、假紫罗酮、C27的环氧-脱辅基类胡萝卜醛和C27丙二烯脱辅基类胡萝卜醛等物质。而这些脱辅基类胡萝卜素参与了色素、风味、香气的形成。中国专利申请《一种与α- 紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因及其编码蛋白和应用》(CN202110653530.6)公开了一种来自于辣椒的类胡萝卜素裂解双加氧酶CaCCD1B，其表达后可提高辣椒中α-紫罗兰酮的含量进而改善辣椒的风味物质，提高辣椒的经济价值。中国专利申请《产生β-紫罗酮香气物质的枸杞类胡萝卜素裂解双加氧酶基因及应用》公开了一种来源于枸杞类胡萝卜素裂解双加氧酶基因，该基因可以通过大肠杆菌异源表达，提高紫罗兰酮的含量(CN201510826720.8)。专利申请《类胡萝卜素双加氧酶和在微生物和植物中以生物技术制备衍生自藏红花的化合物的方法》则公开了通过异源表达来自于玉米胚乳细胞类胡萝卜素双加氧酶裂解类胡萝卜素的双加氧酶活性以促进藏红花酸二醛和藏红花酸的分别产生(CN201580045027.5)。除了，采用依赖酮戊二酸的双加氧酶氧化裂解细胞色素C偶联的氧化酶同样可以氧化类胡萝卜。
烟叶中类胡萝卜含量的增加对于评吸结果是较为有利的。但烟叶的香味与类胡萝卜素的存在成反比，在调制陈化中类胡萝卜素不分解会使烟叶的香味不足。在高等级烟叶中通常类胡萝卜素及其降解产物含量较高。烟叶醇化过程中类胡萝卜素类物质因双键断裂的部位不同可产生很多致香物质，如大马酮、紫罗兰酮、二氢弥猴桃内酯等。提高烟叶质量，类胡萝卜素类降解产物的含量就明显增加，烟叶刺激性较小，香气质较好。专利《一种改善与提高烟草品质的烟草加工活性复合酶制剂及制备方法》公开了以氧化-还原酶类，包括葡萄糖氧化酶、叶绿素酶、脂氧合酶，作用于对烟草品质贡献较大的色质体(包括类胡萝卜素)以增加香气品质的方法(CN201010039187.8)。
本发明提供一种来分离于大花草莓(Fragaria grandiflora) 的双加氧酶，其具有能够显著提升类胡萝卜降解香气产物的丰度及香气质感的功能。
发明内容
本发明的目的是提供一种双加氧酶氨基酸序列、基因及其生物生香应用，以解决现有技术的上述技术问题。
本发明的目的是通过以下技术方案来实现的。
一种双加氧酶CADO基因，该基因的氨基酸序列如序列1所示。
所述的双加氧酶氨基酸序列来自于山椒双加氧酶基因翻译。
一种处理植物提取物以提高其类胡萝卜素降解香气成分的方法，其使用了双加氧酶CADO的重组酶，进行酶促催化处理。
一种提高烟草浸膏的品质的方法，使用了双加氧酶CADO对烟草浸膏进行酶促生香处理。
本发明技术方案具有以下有益效果：
(1)双加氧酶CADO处理后的烟草浸膏在吃味方面有明显改善。
(2)经过双加氧酶CADO酶处理后的烟草浸膏，其中的特征类胡萝卜裂解香气产物含量有大幅提高。
具体实施方式
下面结合具体实施例进一步阐述本发明的特点。
实施例1：氨基酸序列亲缘性比对
本发明申请提供的双加氧酶CADO的氨基酸序列输入BLAST (https://blast.ncbi.nlm.nih.gov/)进行序列比对，结果如表1 所示，其与辣椒的类胡萝卜素氧化裂解酶具有95.81％的相似性，与番茄的类胡萝卜素9,10(9',10')-裂解双加氧酶相似度有91.07％。
表1.双加氧酶CADO的亲缘相似性
实施例2：双加氧酶CADO的表达
将双加氧酶CADO按照大肠杆菌密码偏好性对其进行密码子优化，优化后的基因序列如序列2所示。将密码子优化后的CADO基因序列合成并亚克隆至大肠杆菌表达载体pET30a(+)(基因合成与亚克隆过程委托苏州金唯智公司完成，pET30a(+)为已公开的商品化大肠杆菌表达载体)。由本公司将带有CADO基因的重组质粒 pET30a-CADO转化大肠杆菌BL21(DE3)宿主，在含有氯霉素(34μg mL-1)的LB抗性固体培养基上过夜筛选。选择其中的阳性克隆单菌落挑选至摇瓶进行重组酶的表达，摇瓶培养采用LB培养基(蛋白胨10 g/L、酵母粉5g/L、氯化钠10g/L)，将原始酶重组菌株或变体酶重组菌株接种摇瓶后，在37℃培养至浊度OD600为0.6-1.0后，添加IPTG(异丙基硫代半乳糖苷)的诱导CADO表达(摇瓶内终浓度为 0.4mM)，同时降温至25℃培养8-14h。最后，离心收集菌体作为 CADO重组酶粗酶。所述的CADO粗酶可在4℃或-20℃保存备用，也可细胞破碎后用于纯酶的进一步制备，另外也能直接用于催化反应。
将所获得的不同阳性单克隆菌落所制备的重组酶粗酶，加入1 g/L的β-胡萝卜素进行催化反应，反应颜色从橙色逐渐蜕变为浅黄色，选择褪色速度最快的单克隆菌落菌株，作为Top1的重组酶菌株。
实施例3.利用双加氧酶CADO进行酶促生香反应
按照实施实例2所述的方法，步骤进行双加氧酶CADO的表达，获得Top1的重组酶菌株所表达的CADO重组酶粗酶(湿菌体)，将噬菌体按照2％(质量分数)加入烟草浸膏(波顿(上海)生物技术有限公司提供)中，在30℃下均匀搅拌反应24h。
分别选择辣椒类胡萝卜素氧化裂解酶(获取编号： NP_001311495.1)，烟草类胡萝卜素双加氧裂解酶1-1(获取编号： AIL30506.1)，番茄类胡萝卜素9,10(9',10')-裂解双加氧酶(获取编号：XP_015062345.1)按照实施实例2相同的方法克隆至pET30a (+)，然后在大肠杆菌BL21(DE3)中进行诱导表达。本实施实例中所有引述的酶的基因及氨基酸编码序列均可以从美国国家生物信息中心(https://www.ncbi.nlm.nih.gov/)通过相应的获取编号查询与获取。
将所表达的辣椒类胡萝卜素氧化裂解酶，烟草类胡萝卜素双加氧裂解酶1-1，番茄类胡萝卜素9,10(9',10')-裂解双加氧酶的粗酶，同样按照2％w/w比率加入烟草浸膏中在30℃下均匀搅拌反应24h，作为阴性对照。以加入2％w/w比率纯水，在30℃下均匀搅拌反应24h的烟草浸膏作为空白对照。
实施例4类胡萝卜素香气成分含量及感官评价 (a)香气成分相对含量变化
将实施实例3酶促生产反应所获取的烟草浸膏，以Agilent SPME 固相微萃取，吸附酶促反应后烟草浸膏的挥发性产物8h。将SPME 吸附的化合物进行GC-MS分析(Agilent7890AGC,Agilent,Santa Clara,CA)，同时配有5977A MS检测器(Agilent)。
HP-5MS(Agilent,30m×0.25mm；film thickness,0.25μm)柱子分离混合物。氦气作为GC流动相载气，并保持流速为1mL/min。炉温程序为：40℃保持2min，随后以每分钟上升5℃的速率上升至150℃。最后在260℃中保持10min。酶促生香效果选取，烟草中特征性的类胡萝卜素降解香气产物的相对锋面积变化作为表征(相对峰面积＝试验样产物峰面积/空白对照产品峰面积×100％)，结果如表2所示，仅有双加氧酶CADO粗酶处理后的烟草浸膏其中的特征类胡萝卜裂解香气产物含量有大幅提高。
表2类胡萝卜素特征裂解产物相对含量变化
(b)感官评价：将实施实例3酶促生产反应所获取的烟草浸膏用饮用水稀释100倍后，按0.1％w/w添加量喷到烟丝上，然后再恒温恒湿箱中控制烟丝含水率为11.5％，之后卷成烟支进行按烟草添加剂感官评吸方法(参照国标GB 5606.4)进行感官评价，结果如表3所示，双加氧酶CADO处理后的烟草浸膏在吃味方面有明显改善。
表3.不同双加氧酶处理后的烟丝感官评价
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<110> 上海龙殷生物科技有限公司
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Leu Ser Ala Val Ala Ala Ile Ala Ala Leu Thr Met Ser Ala Gly Pro
1045 1050 1055
Val Ala Val Val Gly Leu Pro Leu Ala Val Pro Thr Gly Pro His Ala
1060 1065 1070
Pro Pro Val Thr Gly Gly Gly Ile Leu Gly Gly Ala Leu Leu
1075 1080 1085
<210> 2
<211> 960
<212> DNA
<213> 草莓(Fragaria grandiflora)
<400> 2
atgggtgaaa aagaggaagg tgatgatggt gtggaacgtt ccgaaggtgc agttgttctg 60
gtcaacgaaa aaccgcgcaa gggcttctcc gctaaggccg tggatctgct ggaaaaaggc 120
atcgtgaaac tggtgcatga ctctagcaaa ccgctgcatt acctgcaggg taatttcgct 180
ccgaccgatg aaacccctcc gctgaacgat ctgctggtac acggtcacct gccggagtgc 240
ctgaacggcg aattcgtgcg cgttggtccg aacccgaaac cggcgccagt ggctggttac 300
cactggttcg acggtgacgg catgatccat ggcctgaaag acggcaaagc tacgtacgtt 360
agccgttatg ttcgcacttc tcgtctgaaa caagaagaat tcttcggtgg tgccaagttc 420
atgaaggtcg gtgatctgaa aggtctgttc ggtctgttta ccgtatatat gcagatgctg 480
cgcgctaagc tggaagtgct ggatatttcc tacggtaacg gtaccgcgaa cacggcactg 540
gcatatcatc atggtaaact gctggcgctg tccgaagcgg acaaaccgta cgccctgaaa 600
gttctgaaag atggtgatct gcagaccctg ggtgttctgg actacgataa gcgcctgacc 660
cactccttca ccgcccaccc gaaagttgac ccggttaccg gcgaaatgtt tactttcggc 720
tattctcacg acccaccata catcacctac cgtgttatca gctctgacgg tatcatgcag 780
gacccggttc cgatcaccat cccggaacct attatgatgc acgatttcgc aatcactgag 840
aactacgcta tcatgatgga cctgccgctg tgtttccgcc cgaaagaaat ggtggcgaac 900
aacaaactgg cgttcacctt cgatggcact aaaaaggctc gctttggcgt tctggcacgt 960
Claims (4)
1.一种双加氧酶CADO基因，其特征在于：该基因的氨基酸序列如序列1所示。
2.根据权利要求1所述的一种双加氧酶CADO基因，其特征在于：所述的双加氧酶基因的氨基酸序列来自于山椒双加氧酶基因翻译。
3.一种处理植物提取物以提高其类胡萝卜素降解香气成分的方法，其特征在于：其使用了双加氧酶CADO的重组酶，进行酶促催化处理。
4.一种提高烟草浸膏的品质的方法，其特征在于：使用双加氧酶CADO对烟草浸膏进行酶促生香处理。
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