CN113862240A - 一种密码子优化的糖苷酶swmu-f2-2及其制备方法与应用 - Google Patents
一种密码子优化的糖苷酶swmu-f2-2及其制备方法与应用 Download PDFInfo
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- CN113862240A CN113862240A CN202111137767.5A CN202111137767A CN113862240A CN 113862240 A CN113862240 A CN 113862240A CN 202111137767 A CN202111137767 A CN 202111137767A CN 113862240 A CN113862240 A CN 113862240A
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Abstract
本发明公开了一种密码子优化的糖苷酶SWMU‑F2‑2及其制备方法与应用，其通过将密码子优化的糖苷酶基因SWMU‑F2‑2进行全基因合成，得到糖苷酶基因SWMU‑F2‑2的全长片段；重组质粒的制备：将糖苷酶基因SWMU‑F2‑2的全长片段与载体pET‑28a(+)连接，获得重组质粒pET‑28a(+)/SWMU‑F2‑2；糖苷酶SWMU‑F2‑2的制备：将重组质粒pET‑28a(+)/SWMU‑F2‑2转化至E.coli BL21感受态细胞中，得到重组E.coli BL21细胞，然后加入异丙基硫代半乳糖苷IPTG进行诱导表达，得到菌液，离心，取上清液进一步纯化，得到糖苷酶SWMU‑F2‑2的酶溶液，糖苷酶SWMU‑F2‑2的DNA序列如SEQ ID NO.1所示，氨基酸序列如SEQ ID NO.2所示。
Description
技术领域
本发明属于生物技术领域，具体涉及一种密码子优化的糖苷酶SWMU-F2-2及其制备方法与应用。
背景技术
糖苷酶即糖苷水解酶(Glycoside hydrolases,GH,EC3.2.1)，是一类水解糖苷键(glycosidic bonds)的酶，在生物体糖和糖缀合物的水解与合成过程中扮演着重要角色。糖苷酶在催化糖苷反应时，如果水分子的氧原子进攻受体葡萄糖上的异头碳，即发生水解反应，但如果是葡萄糖羟基上的氧原子进攻受体葡萄糖上的异头碳，即发生转糖基反应。
糖苷酶几乎存在于所有的生物体中，是一类以内切或外切方式水解各种含糖化合物(包括单糖苷、寡糖、多糖、皂甙和糖蛋白等)中的糖苷键,生成单糖、寡糖或糖复合物的酶。糖苷酶在寡糖合成、烷基糖苷和芳香基糖苷的合成、氨基酸和多肽的糖基化以及抗生素的糖基化方面发挥了重要作用。
人参为我国传统名贵中草药，被誉为“百草之王”，有效成分包括人参多糖、人参挥发油、氨基酸和人参皂苷，主要有效成分是人参皂苷。人参皂苷属于四环三萜皂苷，结构上由苷元与糖连接而成，分为原二醇型人参皂苷、原三醇型人参皂苷和齐墩果酸型人参皂苷，其中，Rb1、Rb2、Rc、Rd、Re、Rg1、Rf等为含量丰富的天然人参皂苷，Rh1、Rh2、F1、F2、S-Rg3、Rb3、compound K、PPT等为稀有人参皂苷，稀有人参皂苷具有更高的药理活性，且更易被胃肠道吸收。因此，如何提高稀有人参皂苷的含量成为现代研究的热点。目前，人参皂苷的体外转化方法主要包括物理方法、化学方法和微生物转化法，物理方法主要为加热法，此方法反应条件要求高、副产物多且耗能巨大；化学方法主要是酸碱法，此类方法反应剧烈难以控制，且效率低污染环境；微生物转化法主要包括酶解法和微生物发酵法，其主要原理在于微生物所分泌的糖苷酶对人参皂苷进行转化，酶解法反应条件温和、耗能低、产物单一、提取率高、绿色环保，然而酶法成本高，不稳定易失活不易保存，因此微生物液体发酵是目前工业生产皂苷的主要手段。
根据人参皂苷的C3位和C20位葡萄糖基数量和分布，人参皂苷Rd在C3有两个葡萄糖基，在C20位有一个葡萄糖基，若水解掉C3位上其中一个葡萄糖基，则可得到稀有人参皂苷F2。
发明内容
针对上述的不足，本发明的第一目的是提供一种密码子优化的糖苷酶SWMU-F2-2；
本发明的第二目的是提供一种密码子优化的糖苷酶SWMU-F2-2的制备方法；
本发明的第三目的是提供一种密码子优化的糖苷酶SWMU-F2-2在水解人参皂苷Rd中的应用。
为实现上述目的，本发明采取以下技术方案：
一种密码子优化的糖苷酶，所述糖苷酶SWMU-F2-2的DNA序列如SEQ ID NO.1所示，其来源于粘质沙雷氏菌SGAir 0764染色体基因密码子优化。
进一步，所述糖苷酶SWMU-F2-2的氨基酸序列如SEQ ID NO.2所示。
进一步，所述粘质沙雷氏菌SGAir 0764染色体基因密码子优化的方法为：基于基因密码的简并性，使用GeneOptimizer算法，在单次运算中考虑到了DNA序列中特定功能Motif、重复序列、GC含量、mRNA二级结构、密码子偏好性等相关优化参数，在不改变其所编码的蛋白序列的前提下，对原有基因序列进行优化，得到密码子优化的糖苷酶基因SWMU-F2-2。
进一步，具体步骤如下：
1)将密码子优化的糖苷酶基因SWMU-F2-2进行全基因合成，得到糖苷酶基因SWMU-F2-2的全长片段；
2)重组质粒的制备：将糖苷酶基因SWMU-F2-2的全长片段与载体pET-28a(+)连接，获得重组质粒pET-28a(+)/SWMU-F2-2；
3)糖苷酶SWMU-F2-2的制备：将重组质粒pET-28a(+)/SWMU-F2-2转化至E.coliBL21感受态细胞中，得到重组E.coli BL21细胞，然后加入异丙基硫代半乳糖苷IPTG进行诱导表达，得到菌液后，超声破碎菌体，离心，取上清液进一步纯化，得到糖苷酶SWMU-F2-2的酶溶液。
进一步，所述的一种密码子优化的糖苷酶SWMU-F2-2在水解人参皂苷Rd中的应用。
采用以上方案，本发明具有如下优点：
本发明首次将来源于粘质沙雷氏菌SGAir 0764染色体基因进行密码子优化，得到糖苷酶SWMU-F2-2，糖苷酶SWMU-F2-2与粘质沙雷氏菌SGAir 0764染色体基因序列的一致性为82.40％，糖苷酶SWMU-F2-2将人参皂苷Rd水解为稀有人参皂苷F2，其没有其他副产物产生，有效的提高了产物的收率，且生产成本低、反应条件温和、耗能低、产物单一、提取率高、绿色环保、稳定性好、容易保存。
本发明的其他优点、目标和特征在某种程度上将在随后的说明书中进行阐述，并且在某种程度上，基于对下文的考察研究对本领域技术人员而言将是显而易见的，或者可以从本发明的实践中得到教导。本发明的目标和其他优点可以通过下面的说明书和权利要求书来实现和获得。
附图说明
图1为SWMU-CK-1的SDS-PAGE凝胶电泳图；
图2为温度对GH活性的影响；
图3为温度对SWMU-F2-2活性的影响；
图4为pH对GH活性的影响；
图5为pH对SWMU-F2-2活性的影响；
图6为SWMU-F2-2转化结果TLC色谱图。
具体实施方式
下面结合附图和实施例对本发明的进行详细的描述，但实施例并不对本发明作任何形式的限定，除非特别说明，本发明所涉及的试剂、方法和设备为本技术领域常规试剂、方法和设备。
实施例1：糖苷酶基因SWMU-F2-2的获取。
1、实验方法
根据对NCBI数据库中的粘质沙雷氏菌SGAir 0764染色体基因序列(GenebanNo.CP027300.1:916652-918034)采用密码子优化技术进行优化，即基于基因密码的简并性，使用GeneOptimizer算法，在单次运算中考虑到了DNA序列中特定功能Motif、重复序列、GC含量、mRNA二级结构、密码子偏好性等相关优化参数，在不改变其所编码的蛋白序列的前提下，对原有基因序列进行优化，得到密码子优化的糖苷酶基因SWMU-F2-2，将密码子优化的糖苷酶基因SWMU-F2-2由生工生物工程(上海)股份有限公司进行全基因合成，得到糖苷酶基因SWMU-F2-2全长片段，其DNA序列如SEQ ID NO.1所示。
2、实验结果
密码子优化后的糖苷酶基因SWMU-F2-2与粘质沙雷氏菌SGAir 0764染色体基因(Geneban No.CP027300.1:916652-918034)的序列一致性为82.40％。
实施例2：重组质粒的构建。
1、载体构建：由生工生物工程(上海)股份有限公司将糖苷酶基因SWMU-F2-2的序列全长连接在载体pET-28a(+)上，酶切位点选用NdeI/XhoI；
2、转化E.coliDH5α感受态细胞：将感受态细胞E.coliDH5α放置冰上融化，将步骤1所得的连接体系加入融化的E.coliDH5α感受态中，冰上30min，然后42℃热处理90s，然后冰上静置2min，加入LB培养基600μL，摇床温度37℃，摇床摇速150rpm，时间45min，得到含目的基因的菌液，吸取200μL菌液，涂布于卡那霉素(Kan+)抗性LB平板培养基上，37℃过夜培养。
3、阳性克隆鉴定：挑选步骤2中培养好的抗性LB平板培养基上的白色单菌落，接种于LB/Kan+的液体培养基中，200rpm、37℃培养8小时，8000rpm离心5min获取菌体，使用OMEGA Plasmid Mini Kit I质粒提取试剂盒，按照说明书中的操作步骤提取质粒，使用双酶切法，确定基因片段成功插入pET-28a(+)载体中，将鉴定正确的重组质粒送上海生工公司测序，将测序结果比对正确的重组质粒pET-28a(+)/SWMU-F2-2作为表达载体。
实施例3：糖苷酶基因SWMU-F2-2的表达。
质粒转化E.coliBL21细胞：-80℃中取出E.coliBL21感受态细胞冰上放置，加入2μL表达载体pET-28a(+)/SWMU-F2-2，冰上放置30min，42℃热处理90s，冰上放置2min，复苏，加入600μL LB培养基，于37℃，150rpm摇床培养45min，得到含表达载体的菌液，吸取200μL菌液，涂布于Kan+LB平板培养基上，37℃培养过夜，得到菌种；
蛋白表达：将菌种接种入无菌LB液体培养基中，卡纳霉素终浓度为50μg/mL，37℃，180rpm摇床培养，待菌液OD600≈0.8时，加入终浓度为0.2mM的异丙基硫代半乳糖苷(IPTG)，16℃，220rpm摇床培养诱导12h，8000rpm，5min收集菌体，加入10倍体积的超纯水重悬菌体，260W超声破碎30min至菌液澄清，12000rpm离心20min，取上清，得到目的蛋白；
蛋白纯化：采用Ni-NTA Resin对含His标签的目的蛋白进行分离纯化，为避免蛋白变性，操作空间温度应保持在4℃左右或在低温环境中进行：
a.装柱：将配套空的层析柱组装好，重悬介质后向层析柱加入4ml填料，静置；
b.平衡：用5倍柱体积的平衡缓冲液平衡层析柱；
c.上样：将所得含目的蛋白的上清液使用0.45μm过滤器过滤后，加入层析柱中，盖好盖子，充分摇匀10使His标签与Ni+充分结合；
d.洗涤：上样完毕后，放掉杂质蛋白，再加入5倍体积平衡缓冲液冲洗掉未结合的杂蛋白，收集流出液；
e.洗脱：先使用20mM咪唑洗脱与Ni+结合较弱的杂蛋白，再使用40mM咪唑洗脱目的蛋白，收集流出液；
f.透析：在4℃使用分子筛大小为20000的纤维素透析袋对洗脱液进行透析，每6h更换一次纯水，透析2至3天后，得到最终纯化后的酶溶液，即糖苷酶SWMU-F2-2，其蛋白质序列如SEQ ID NO.2所示。
实施例4：糖苷酶SWMU-F2-2的分子量检测。
1、实验方法：
将实施例3中所得纯化后的糖苷酶SWMU-F2-2溶液样品进行SDS-PAGE，鉴定其分子量大小及纯度，使用BCA试剂盒测定纯化蛋白的浓度。
2、实验结果
如图1所示，糖苷酶SWMU-F2-2的蛋白质分子量约为52.94kDa，纯化后蛋白浓度为2.75mg/ml，且由图1可看出目的蛋白条带单一无杂带，其纯度较好。
实施例5：糖苷酶SWMU-F2-2水解人参皂苷Rd。
1、实验材料
实施例3中的条件下获得的糖苷酶SWMU-F2-2、人参皂苷Rd、醋酸-醋酸钠缓冲液、水饱和正丁醇溶液、色谱纯甲醇。
2、实验方法
室温条件下，将1.0ml浓度为2.0mg/ml人参皂苷Rd溶液(0.20mol/L pH＝5.0醋酸-醋酸钠缓冲液)与1.0ml糖苷酶SWMU-F2-2酶液混合，置于30℃恒温培养箱中反应24h。反应结束后，轻微振荡摇匀后，取两份摇匀后的溶液，每份溶液为1.0ml，其中一份溶液加入2.0ml水饱和正丁醇溶液涡旋30s终止酶促反应，8000rpm离心15min取上层有机相，吹干后加1.0ml色谱纯甲醇复溶，使用TLC色谱分析法对其进行测定分析，另一份溶液用DNS法测定转化率。
3、实验结果
通过TLC色谱分析法对产物进行测定，由图6证实本发明糖苷酶SWMU-F2-2基因产物为一种新型糖苷酶，能有效水解人参皂苷Rd C3位上的葡萄糖侧链，使其转化为稀有人参皂苷F2；图6中1为人参皂苷CK、F2、Rd混点；2为反应产物；3为人参皂苷CK标准品；4为人参皂苷F2标准品；5为人参皂苷Rd标准品；
DNS法通过测定产物中所含葡萄糖的数量，来推算每个人参皂苷分子被水解的葡萄糖基的数量，结合产物，从而测定人参皂苷的转化率，因此人参皂苷Rd的24h反应产物为F2，其转化率为23.25％。
实施例6：糖苷酶SWMU-F2-2的活性测定。
1、实验材料
实施例3中的条件下获得的糖苷酶SWMU-F2-2、人参皂苷Rd、醋酸-醋酸钠缓冲液、氢氧化钠溶液、DNS试剂。
2、实验方法
取糖苷酶SWMU-F2-2酶溶液1.0ml，加入4ml 10mmol/L的人参皂苷Rd溶液(用0.2mol/L pH5.0的醋酸醋酸钠缓冲液配制)，30℃水浴反应80min后，加入1.0ml 2mol/L氢氧化钠溶液终止反应，加入2ml DNS试剂，80℃恒温水浴10min后用流动水冷却，于540nm处测定吸光度，以测得的吸光度计算酶活力。
3、实验结果
酶活力定义为在30℃，pH5.0的条件下，1mg酶每1min水解Rd生成1mg葡萄糖醛酸的酶活力为一个酶活力单位。
实施例7：糖苷酶SWMU-F2-2与现有的糖苷水解酶GH的酶学性质比较。
糖苷酶SWMU-F2-2与糖苷水解酶GH催化的底物均为人参皂苷Rd，产物均为稀有人参皂苷F2。
1、实验材料
实施例3中的条件下获得的糖苷酶SWMU-F2-2、现有的糖苷水解酶GH(编码基因GenBank NO.BAN05876)、人参皂苷Rd、醋酸-醋酸钠缓冲液、氢氧化钠溶液、DNS试剂。
2、实验方法
2.1温度对酶活性的影响
分别取四份SWMU-F2-2、糖苷水解酶GH的酶溶液1.0ml，分别加入4ml 10mmol/L的人参皂苷Rd溶液(用0.2mol/L pH5.0的醋酸醋酸钠缓冲液配制)，将分别含有SWMU-F2-2、糖苷水解酶GH的八份人参皂苷Rd溶液分别在温度为20℃、30℃、40℃、50℃的条件下水浴反应80min后，分别加入1.0ml 2mol/L氢氧化钠溶液终止反应，加入2ml DNS试剂，80℃恒温水浴10min后用流动水冷却，于540nm处测定吸光度，以测得的吸光度计算酶活力；
2.2pH对酶活性的影响
分别取五份SWMU-F2-2、糖苷水解酶GH的酶溶液1.0ml，分别加入4ml 10mmol/L的Rd溶液(用pH分别为3、4、5、6、7、8的0.2mol/L醋酸醋酸钠缓冲液配制)，30℃水浴反应80min后，加入1.0ml 2mol/L氢氧化钠溶液终止反应，加入2ml DNS试剂，80℃恒温水浴10min后用流动水冷却，于540nm处测定吸光度，以测得的吸光度计算酶活力；
3、实验结果
3.1温度对酶活性的影响
由图2和图3比较得知，本发明表达的糖苷酶SWMU-F2-2在低温情况下活性保持情况低于GH，但在温度稍高的条件下，活性保持情况明显高于糖苷酶GH；
3.2pH对酶活性的影响
由图4和图5比较得知，pH的变化对本发明SWMU-F2-2表达酶的活性相较于糖苷水解酶GH基因表达的糖苷酶活性影响较小，本发明表达的糖苷酶SWMU-F2-2可以在较大的pH范围内保持相对较高的活性；
最后用说明的是：以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行同等替换。凡在本发明的精神和原则之内，所作的任何修改、同等替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 西南医科大学
<120> 一种密码子优化的糖苷酶SWMU-F2-2及其制备方法与应用
<160> 2
<170> SIPOSequenceListing 1.0
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gaaggcgcgg cggcgcgtga cggtaaatct cgtaacatct tcgactactg gtacgaaatc 120
gcaccggaac gtttccacga tcgtgttggc ccgaccgaag cgagcacctt ctacgatcac 180
tttcgtaccg atatcggcct gctgaaaacc ctgggccaca acaccttccg taccagcatt 240
tcttggtctc gtctgatccc ggatggtgat ggtgaagtga acccgcaggc ggttgcgttc 300
tacaacgcca tgatcgatga actgctggcg cagggtatca ccccgttcat caacctgtac 360
catttcgaca tgccgctgtg catgcagcag cgtggtggtt gggaaagccg tgcggttgtt 420
gaagcgtacg cgcgttacgc ggatacctgc ttcggtctgt tcggtgaccg cgttacccac 480
tggttcacct tcaacgaacc gatcgttccg gtagaagcgg gttacctgaa cgatctgcac 540
tacccgtgcg tggttgactt caaacgtgcg gtgaccgttg cgtaccactc cgttctggcg 600
cacgcgatgg cggttcagcg tttccgtgcg cgtcgtctgc cgggcagcat cggcattatc 660
ctgaacctga gcccgaccta cccgcgttct gatgcgccgg aagatcgcca ggcggcgcac 720
gacgctgatc tgctgctgaa ccgtagcttc ctcgatccgg ttgcgaaagg ccgctacccg 780
gcggcgctgc tgcacctgct ggaacgtcac ggcctgatgc cgtactgtga accgcaggat 840
gcgcagctga tcgaaggtgg cgttgttgat atcctgggtg tgaactacta ccagccgcgt 900
cgcgtgcagg ccaaagctgg ccgtcgcgcg gaaggcccga tcgcgagccc ggaagatctc 960
ttctcctatt acgcgatgcc gggccgtaaa atcaacccgc accgtggttg ggaaatctac 1020
gaaaaaggtc tgtacgatat cctgatggat ctgaaagaaa actacggcaa cctgccgtgc 1080
tatatcagcg aaaacggcat gggtgttgaa ggtgaagaag cgttcatcgg cgcggatggt 1140
cgtgttgaag atgattaccg tatcgatttc atccgtgaac acctgaaatg gctgcaccgt 1200
gcgctggcgg aaggcagcca gtgcaaaggc taccacctgt ggaccttcat cgattgctgg 1260
tcttggctga acgcgtacaa aaaccgttac ggcctggttc gtctggatcg tgctgatcag 1320
cgtcgtacca tcaaaaaatc tggctactgg ttcgcggaag ctgcgcgtcg taacggcttc 1380
gactaa 1386
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<211> 461
<212> PRT
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Met Glu Tyr Gln Phe Ala Asp Gly Phe Trp Trp Gly Ser Ala Thr Ser
1 5 10 15
Ala Pro Gln Ser Glu Gly Ala Ala Ala Arg Asp Gly Lys Ser Arg Asn
20 25 30
Ile Phe Asp Tyr Trp Tyr Glu Ile Ala Pro Glu Arg Phe His Asp Arg
35 40 45
Val Gly Pro Thr Glu Ala Ser Thr Phe Tyr Asp His Phe Arg Thr Asp
50 55 60
Ile Gly Leu Leu Lys Thr Leu Gly His Asn Thr Phe Arg Thr Ser Ile
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Ser Trp Ser Arg Leu Ile Pro Asp Gly Asp Gly Glu Val Asn Pro Gln
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Ala Val Ala Phe Tyr Asn Ala Met Ile Asp Glu Leu Leu Ala Gln Gly
100 105 110
Ile Thr Pro Phe Ile Asn Leu Tyr His Phe Asp Met Pro Leu Cys Met
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Gln Gln Arg Gly Gly Trp Glu Ser Arg Ala Val Val Glu Ala Tyr Ala
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Arg Tyr Ala Asp Thr Cys Phe Gly Leu Phe Gly Asp Arg Val Thr His
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Trp Phe Thr Phe Asn Glu Pro Ile Val Pro Val Glu Ala Gly Tyr Leu
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Asn Asp Leu His Tyr Pro Cys Val Val Asp Phe Lys Arg Ala Val Thr
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Val Ala Tyr His Ser Val Leu Ala His Ala Met Ala Val Gln Arg Phe
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Arg Ala Arg Arg Leu Pro Gly Ser Ile Gly Ile Ile Leu Asn Leu Ser
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Pro Thr Tyr Pro Arg Ser Asp Ala Pro Glu Asp Arg Gln Ala Ala His
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Asp Ala Asp Leu Leu Leu Asn Arg Ser Phe Leu Asp Pro Val Ala Lys
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Gly Arg Tyr Pro Ala Ala Leu Leu His Leu Leu Glu Arg His Gly Leu
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Met Pro Tyr Cys Glu Pro Gln Asp Ala Gln Leu Ile Glu Gly Gly Val
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Val Asp Ile Leu Gly Val Asn Tyr Tyr Gln Pro Arg Arg Val Gln Ala
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Lys Ala Gly Arg Arg Ala Glu Gly Pro Ile Ala Ser Pro Glu Asp Leu
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Phe Ser Tyr Tyr Ala Met Pro Gly Arg Lys Ile Asn Pro His Arg Gly
325 330 335
Trp Glu Ile Tyr Glu Lys Gly Leu Tyr Asp Ile Leu Met Asp Leu Lys
340 345 350
Glu Asn Tyr Gly Asn Leu Pro Cys Tyr Ile Ser Glu Asn Gly Met Gly
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Val Glu Gly Glu Glu Ala Phe Ile Gly Ala Asp Gly Arg Val Glu Asp
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Asp Tyr Arg Ile Asp Phe Ile Arg Glu His Leu Lys Trp Leu His Arg
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Ala Leu Ala Glu Gly Ser Gln Cys Lys Gly Tyr His Leu Trp Thr Phe
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Ile Asp Cys Trp Ser Trp Leu Asn Ala Tyr Lys Asn Arg Tyr Gly Leu
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Val Arg Leu Asp Arg Ala Asp Gln Arg Arg Thr Ile Lys Lys Ser Gly
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Tyr Trp Phe Ala Glu Ala Ala Arg Arg Asn Gly Phe Asp
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Claims (5)
1.一种密码子优化的糖苷酶，其特征在于，所述糖苷酶SWMU-F2-2的DNA序列如SEQ IDNO.1所示，其来源于粘质沙雷氏菌SGAir 0764染色体基因密码子优化。
2.根据权利要求1所述的一种密码子优化的糖苷酶，其特征在于，所述糖苷酶SWMU-F2-2的蛋白质序列如SEQ ID NO.2所示。
3.根据权利要求2所述的一种密码子优化的糖苷酶，其特征在于，所述粘质沙雷氏菌SGAir 0764染色体基因密码子优化的方法为：基于基因密码的简并性，使用GeneOptimizer算法，在单次运算中考虑到了DNA序列中特定功能Motif、重复序列、GC含量、mRNA二级结构、密码子偏好性等相关优化参数，在不改变其所编码的蛋白序列的前提下，对原有基因序列进行优化，得到密码子优化的糖苷酶基因SWMU-F2-2。
4.根据权利要求3所述的一种密码子优化的糖苷酶的制备方法，其特征在于，具体步骤如下：
1)将密码子优化的糖苷酶基因SWMU-F2-2进行全基因合成，得到糖苷酶基因SWMU-F2-2的全长片段；
2)重组质粒的制备：将糖苷酶基因SWMU-F2-2的全长片段与载体pET-28a(+)连接，获得重组质粒pET-28a(+)/SWMU-F2-2；
3)糖苷酶SWMU-F2-2的制备：将重组质粒pET-28a(+)/SWMU-F2-2转化至E.coli BL21感受态细胞中，得到重组E.coli BL21细胞，然后加入异丙基硫代半乳糖苷IPTG进行诱导表达，得到菌液后，超声破碎菌体，离心，取上清液进一步纯化，得到糖苷酶SWMU-F2-2的酶溶液。
5.根据权利要求1或2所述的一种密码子优化的糖苷酶SWMU-F2-2在水解人参皂苷Rd中的应用。
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