CN114703158B - 一种蔗糖磷酸化酶突变体、编码基因及其应用 - Google Patents
一种蔗糖磷酸化酶突变体、编码基因及其应用 Download PDFInfo
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- CN114703158B CN114703158B CN202210230270.6A CN202210230270A CN114703158B CN 114703158 B CN114703158 B CN 114703158B CN 202210230270 A CN202210230270 A CN 202210230270A CN 114703158 B CN114703158 B CN 114703158B
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- sucrose phosphorylase
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N9/1048—Glycosyltransferases (2.4)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
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Abstract
本发明涉及一种提高转糖基活性和提升L‑抗坏血酸葡萄糖苷产物L‑抗坏血酸‑2‑α‑葡萄糖苷(2‑O‑α‑D‑glucopyranosyl‑L‑ascorbic acid,AA‑2G)纯度的蔗糖磷酸化酶突变体及其应用。本发明通过对SEQ ID.1所示氨基酸序列第134位脯氨酸、第341位亮氨酸和第343位亮氨酸中的一种或多种进行单点突变或多点组合突变,使得蔗糖磷酸化酶突变体比酶活提升1~2倍,合成AA‑2G浓度可达205 g/L,合成L‑抗坏血酸糖苷产物中AA‑2G占比达92~99%,这些突变体具有更好的工业应用特性。
Description
技术领域
本发明涉及一种蔗糖磷酸化酶突变体及其编码基因,以及其在酶法制备L-抗坏血酸葡萄糖苷中的应用。
背景技术
L-抗坏血酸-2-α-葡萄糖苷(2-O-α-D-glucopyranosyl-L-ascorbic acid, AA-2G),是稳定性显著提高的L-抗坏血酸(L-AA,又称维生素C)糖苷衍生物。该化合物的制备及技术最早于1990年由日本林原公司化学研究所与日本冈山大学药学系共同开发,是由L-AA的C2羟基被葡萄糖基取代后所得。由于L-AA结构中C2和C3是烯二醇结构,因此具有极强的还原性,在氧气、金属离子、光照以及碱性等条件下极其不稳定,使得其在工业化应用方面存在限制。而AA-2G本身不具有还原性,不易发生氧化反应,与L-AA相比有更好的稳定性,而且经过α-葡萄糖苷酶的水解作用生成L-AA和葡萄糖,发挥和L-AA一样的还原性和抗氧化性,与此同时,还延长了L-AA的作用时长,因此是L-AA良好的替代品。
L-抗坏血酸(L-AA)化学结构式
蔗糖磷酸化酶(SPase,EC 2.4.1.7)是糖苷水解酶GH13_18家族的成员,可以催化蔗糖的磷酸解反应生成1-磷酸-α-D-葡萄糖和D-果糖。除此之外,蔗糖磷酸化酶还能对多种小分子化合物进行糖基化反应,即将蔗糖分子中的葡萄糖基转移至受体分子上,合成相应的受体糖苷,若受体分子为水分子时,则实现了蔗糖的水解。蔗糖磷酸化酶具有催化活性高,底物混杂性,糖基供体蔗糖来源广泛且廉价易得等特点。其中一个重要应用是该酶可以以蔗糖为糖基供体高效催化L-AA糖基化一步法直接生成AA-2G。由于L-AA分子结构中含有4个羟基,SPase催化糖基化L-AA存在糖基化位点的区域选择特异性的问题,即合成多种L-抗坏血酸。针对L-AA分子上的不同羟基位点的糖基化分别生成AA-2G,AA-3G,AA-5G和AA-6G,甚至糖基化后的产物还可以作为糖基受体进行二次糖基化,形成AA-nGG (n代表2,3,5,6)。虽然在合适的催化条件下,SPase催化L-AA的糖基化产物以AA-2G为主,但是副产物的结构与AA-2G结构相似且稳定性不如AA-2G,难以下在游分离纯化过程中去除,并易转化成其他不稳定结构而引入新杂质等问题,这对下游纯化和产品质量控制存在极大的挑战。
发明内容
为解决上述问题,本发明提供一种蔗糖磷酸化酶突变体其在AA-2G生产过程中的应用,该突变体具有更高的L-AA的转化率和更高的AA-2G产物纯度,表现出更优的AA-2G工业化生产的应用价值。
本发明采用的技术方案是:
一种蔗糖磷酸化酶突变体,由氨基酸序列如SEQ ID NO.1所示的蔗糖磷酸化酶(BbrSPase)经单点突变或多点突变获得,所述单点突变或多点突变的突变位点为下列之一或其中两种以上:(1)第134位脯氨酸,(2)第341位亮氨酸,(3)第343位亮氨酸。
SEQ ID NO.1氨基酸序列如下:
MKNKVQLIAYADRLGDGTLSSMTDILRTRFDGVYDGVHILPFFTPFDGADAGFDPIDHTKVDPRLGSWDDVAELSKTHDIMVDAIVNHMSWESAQFQDVLKNGEHSEYYPMFLTMSSVFPNGATEEDLAGIYRPRPGLPFTHYKFAGKTRLVWVSFTPQQVDIDTDSAKGWEYLMSIFDQMAASHVRYIRLDAVGYGAKEAGTSCFMTPKTFKLISRLREEGVKRGLEILIEVHSYYKKQVEIASKVDRVYDFALPPLLLHSLFTGHVEPVVHWTEIRPNNAVTVLDTHDGIGVIDIGSDQLDRSLKGLVPDEDVDNLVNTIHANTHGESQAATGAAASNLDLYQVNSTYYSALGCNDQHYLAARAVQFFLPGVPQVYYVGALAGRNDMELLRKTNNGRDINRHYYSTAEIDENLERPVVKALNALAKFRNELPAFNGEFSYEADGDTSITFRWIAADGKTKAALIFEPGRGLGTDNTTPVASLAWTDAAGDHETDDLLSNPPIADID
其编码基因序列如下(SEQ ID NO.2):
atgaaaaaca aagtgcaact catcgcttac gccgatcgtc tcggcgatgg tactcttagc
tcgatgaccg acatcctgcg cacccgcttc gacggcgtgt atgacggcgt gcatatcctg
ccgttcttca ctccgttcga tggtgcggat gcaggctttg acccgatcga ccacaccaaa
gtcgacccgc gcctcggatc gtgggacgac gtcgccgagc tctccaaaac ccacgacatc
atggtcgatg ccatcgtcaa ccacatgagc tgggaatccg cccaattcca agacgtgctg
aagaacggcg agcactccga gtattacccg atgttcctga ccatgagttc cgttttcccg
aacggcgcca ccgaagagga tctcgccggc atctaccgcc cgcgcccggg cctgccgttc
acccactaca agttcgccgg caagacccgt ctggtatggg tcagcttcac cccacagcag
gtggacatcg acactgactc cgccaagggc tgggagtatc tgatgtccat ctttgatcag
atggcagcca gccatgtgcg ctacatccgt ctcgacgccg tgggctacgg cgccaaggag
gccggcacca gctgcttcat gacccccaag acctttaagc tcatctcacg cctacgcgag
gaaggcgtca agcgaggcct cgaaattctc attgaggtgc atagctacta caagaagcag
gttgaaatcg cctccaaggt ggaccgcgtc tacgacttcg ccctgcctcc gctgctcctg
cactcgctgt tcaccggtca cgtggaaccc gtggtccact ggaccgaaat ccgcccgaac
aacgccgtca ccgtgctcga tacgcacgac ggcatcggcg tgatcgacat cggctccgat
cagctcgacc gcagcctcaa gggcctcgtg cccgacgagg acgtcgataa tctggtcaac
accatccacg ccaacaccca cggcgaatcc caggccgcca ccggtgccgc cgccagcaac
ctcgacctct atcaggtcaa cagcacgtac tactccgcgc tcggctgcaa cgaccagcac
tatctggccg cccgcgcggt ccagttcttc ctgcccggag tgccgcaggt ctactacgtg
ggcgcgctcg ccggtcgcaa cgacatggaa ctgctgcgca agaccaacaa cggccgtgac
atcaatcgcc attactactc caccgccgaa atcgacgaaa acctcgagcg cccggtggtg
aaggccctga acgccctggc caagttccgc aacgaactgc ctgcattcaa tggcgagttc
agctacgaag ccgacggcga cacatccatc accttccgct ggatcgctgc cgacggcaag
accaaggccg ccctcatctt cgagcccggc cgcggactcg gcacggataa caccactccg
gtcgccagcc tcgcctggac cgatgccgcc ggtgaccacg agactgatga tctgctgagc
aacccgccga ttgccgatat cgactaa
进一步,所述点突变为下列之一或其中两种以上:(1)第134位脯氨酸突变为半胱氨酸,(2)第341位亮氨酸突变为异亮氨酸或缬氨酸,(3)第343位亮氨酸突变为异亮氨酸。
优选的,所述蔗糖磷酸化酶突变体为下列之一:
(1)氨基酸序列如SEQ ID NO.1所示蔗糖磷酸化酶第134位脯氨酸突变为半胱氨酸;
(2)氨基酸序列如SEQ ID NO.1所示蔗糖磷酸化酶第341位亮氨酸突变为异亮氨酸;
(3)氨基酸序列如SEQ ID NO.1所示蔗糖磷酸化酶第341位的亮氨酸突变为缬氨酸;
(4)氨基酸序列如SEQ ID NO.1所示蔗糖磷酸化酶第343位的亮氨酸突变为异亮氨酸;
(5)氨基酸序列如SEQ ID NO.1所示蔗糖磷酸化酶第341位的亮氨酸突变为异亮氨酸,第343位的亮氨酸突变为异亮氨酸;
(6)氨基酸序列如SEQ ID NO.1所示蔗糖磷酸化酶第341位的亮氨酸突变为异亮氨酸,第343位的亮氨酸突变为脯氨酸;
(7)氨基酸序列如SEQ ID NO.1所示蔗糖磷酸化酶第341位的亮氨酸突变为缬氨酸,第343位的亮氨酸突变为脯氨酸;
(8)氨基酸序列如SEQ ID NO.1所示蔗糖磷酸化酶第341位的亮氨酸突变为异亮氨酸,第343位的亮氨酸突变为天冬酰胺。
本发明还涉及所述蔗糖磷酸化酶突变体的编码基因。
优选的,所述编码基因核苷酸序列如SEQ ID No.3~10之一所示。
本发明还涉及含有所述编码基因的重组表达载体。这些重组载体可以用本领域常规方法将本发明的蔗糖磷酸化酶突变体核苷酸序列连接于各种载体上构建而成。所述载体可为本领域常规的各种载体,例如各种质粒、噬菌体或病毒载体等,优选pET-28a。
本发明还涉及含有所述编码基因的工程菌。作为一种重组表达载体的应用,可通过将本发明的重组表达载体转化至宿主微生物中获得基因工程菌。宿主微生物可为本领域常规的各种宿主微生物,主要满足重组表达载体可以稳定自我复制且所携带的本发明的蔗糖磷酸化酶突变体基因可以有效表达。本发明优选大肠杆菌,更优选为大肠杆菌E. coliBL21(DE3)。
本发明还涉及所述蔗糖磷酸化酶突变体在酶法制备L-抗坏血酸葡萄糖苷中的应用。
优选的,所述L-抗坏血酸葡萄糖苷为L-抗坏血酸-2-葡萄苷(AA-2G)。
具体的,所述应用为:以L-抗坏血酸和蔗糖为底物,加入所述蔗糖磷酸化酶突变体或其重组表达载体转化至宿主微生物中获得基因工程菌菌体细胞,在20~60℃条件下,于pH 4.0~6.5的催化体系中反应,得到含有L-抗坏血酸葡萄糖苷的催化液。
本发明的有益效果主要体现在:本发明提供的蔗糖磷酸化酶突变体,与未突变株相比具有更高的L-AA底物转化率以及更高的AA-2G产物纯度。结果表明:1)蔗糖磷酸化酶突变体对L-AA糖基化的比酶活是野生型的1~2倍,且催化合成AA-2G的浓度可达到205 g/L;2)底物L-AA转化率显著提高,达到38~51%,而野生型酶的转化率约为37%;3)合成的糖苷产物AA-2G纯度较野生型也显著提高,达到92~99%,而野生型酶合成的糖苷产物中AA-2G占比约90%。蔗糖磷酸化酶突变体可以更好地应用于生物催化领域,对AA-2G的工业化生产及应用有重要意义。
附图说明
图1为蔗糖磷酸化酶BbrSPase及其突变体诱导表达并分离纯化后的SDS-PAGE图谱。
图2为BbrSPase三维结构以及本发明涉及的3个突变位点相对位置。
图3为野生型BbrSPase和突变体L341I/L343F第48 h的催化液的液相色谱图比较。
具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
一种蔗糖磷酸化酶突变体,突变体为在BbrSPase氨基酸序列SEQ ID NO.1(核苷酸序列如序列表中SEQ ID NO.10所示)基础上含有如下几个位点的单点突变或多点组合突变:第134位脯氨酸(P134)、第341位亮氨酸(L341)和第343位亮氨酸(L343);这几个位点均位于蔗糖磷酸化酶BbrSPase催化活性中心的2个loop环结构上(如图2所示),这些位点的突变可以调整该loop结构的柔韧性,从而使得酶催化L-AA 糖基化活性提升,提高反应速率和底物转化率;此外该突变体对受体底物L-AA糖基化位点的区域选择性也显著提高,有效地降低副产物(AA-3G和AA-2GG)的含量。
本发明包括如下突变体:
突变体1,将如序列表中SEQ ID NO.1所示的氨基酸序列的第134位的脯氨酸替换为半胱氨酸;
突变体2,将如序列表中SEQ ID NO.1所示的氨基酸序列的第341位的亮氨酸替换为异亮氨酸;
突变体3 ,将如序列表中SEQ ID NO.1所示的氨基酸序列的第341位的亮氨酸替换为缬氨酸;
突变体4 ,将如序列表中SEQ ID NO.1所示的氨基酸序列的第343位的亮氨酸替换为异亮氨酸;
突变体5 ,将如序列表中SEQ ID NO.1所示的氨基酸序列的第341位的亮氨酸替换为异亮氨酸,第343位的亮氨酸替换成异亮氨酸;
突变体6 ,将如序列表中SEQ ID NO.1所示的氨基酸序列的第341位的亮氨酸替换成异亮氨酸,第343位的亮氨酸替换成脯氨酸;
突变体7 ,将如序列表中SEQ ID NO.1所示的氨基酸序列的第341位的亮氨酸替换为缬氨酸,第343位的亮氨酸替换成脯氨酸;
突变体8 ,将如序列表中SEQ ID NO.1所示的氨基酸序列的第341位的亮氨酸替换为异亮氨酸,第343位的亮氨酸替换成天冬酰胺。
任何上述突变体氨基酸序列中氨基酸经过缺失,插入或者替换一个或几个氨基酸且具有L-AA转糖基活性的,仍属于本发明的保护范围。
一种蔗糖磷酸化酶突变体的编码基因,其中突变体P134C氨基酸核苷酸序列如序列表中SEQ ID NO.3所示;突变体L341I核苷酸序列如序列表中SEQ ID NO.4所示;突变体L341V核苷酸序列如序列表中SEQ ID NO.5所示;突变体L343I核苷酸序列如序列表中SEQID NO.6所示;突变体L341I/L343I核苷酸序列如序列表中SEQ ID NO.7所示;突变体L341I/L343F核苷酸序列如序列表中SEQ ID NO.8所示;突变体L341V/L343F核苷酸序列如序列表中SEQ ID NO.9所示;突变体L341I/L343N核苷酸序列如序列表中SEQ ID NO.10所示。
一种重组蔗糖磷酸化酶突变体的制备方法,包括如下步骤:培养本发明的重组表达转化体,诱导获得重组蔗糖磷酸化酶突变体蛋白。其中,所述的培养重组表达转化体所用的培养基可以是本领域可使转化体生长并产生本发明的蔗糖磷酸化酶突变体蛋白的培养基,优选LB培养基:蛋白胨10 g/L,酵母粉5 g/L,氯化钠10 g/L,pH 6.8-7.2。培养方法和培养条件没有特殊限制,只要使转化体能够生长并产生蔗糖磷酸化酶突变体蛋白即可。优选方法如下:将本发明涉及的重组大肠杆菌接种至含50-100 mg/L卡那霉素的LB培养基中,当培养密度OD600nm=0.5~0.8时,在添加终浓度为0.25 ~0.5 mM异丙基-β-D-硫代吡喃半乳糖苷(IPTG)的诱导下,可高效表达本发明的重组蔗糖磷酸化酶突变体蛋白。
得到催化生产AA-2G的催化剂的制备方法如下:
种子活化:将含蔗糖磷酸化酶BbrSPase突变体编码基因的重组大肠杆菌涂布于含50~100 mg/L卡那霉素的LB固体培养基,在37℃静置培养12~20 h后获得单菌落;所述的LB固体培养基是在LB培养基中加入1.5~2.0%的琼脂。
种子培养:挑取LB固体培养基上的单菌落接种至含有50~100 mg/L卡那霉素的LB培养基中,37℃培养8~16 h后,获得种子液。
诱导表达: 将种子液以体积浓度1~2%的接种量接种至装有50 mL 含有终浓度为50~100 mg/L卡那霉素的LB培养基的250 mL摇瓶中,37℃摇床200 rpm培养至OD600nm=0.5-0.8,加入终浓度为0.25~0.5 mM的IPTG,于24-26℃摇床200 rpm诱导6-10 h。
本发明的应用:蔗糖磷酸化酶突变体或其基因工程菌可以以游离酶、固定化酶以及重组有力细胞的形式催化合成AA-2G。
应用方法如下:用去离子水溶解底物L-AA和蔗糖,底物初始浓度为0.1 ~2.0 mol/L用NaOH水溶液调节底物溶液的pH 4.0~6.5;加入上述突变体或基因工程菌,菌体的终浓度以OD600nm计为10~50;在20~60℃条件下反应,反应2~72 h后用高效液相色谱法检测反应液中AA-2G。
以下通过实验验证本发明的有益效果;
实验准备:
步骤一:突变体构建;
以含有突变点的寡核苷酸片段为引物(表1),采用Mut Express®II FastMutagenesis Kit V2试剂盒方法扩增含有BbrSPase基因的pET-28a重组质粒,该蔗糖磷酸化酶基因来源于短双歧杆菌(Bifidobacterium breve),由前期研究筛选获得。
表1: 突变体构建引物
备注:下划线标识为突变位点。
PCR反应体系: 2 ×Max Buffer 25 μL,dNTP Mix(各10 mM)1 μL, Phanta MaxSuper-Fidelity DNA Polymerase 1 μL, 上游引物2 μL,下游引物2 μL,模板质粒1 μL,加ddH2O至总体积为50 μL。
PCR程序:(1)95℃,30s;(2)95℃,15s;(3)70℃,15s;(4)72℃,45s;(5)72℃,6min。步骤(2)-(4)循环30次。
线性PCR产物经过DpnI消化后,消化产物在Exnase®II催化下使得线性DNA环化。
环化后的PCR产物转化至大肠杆菌BL21(DE3)中,得到相应的重组大肠杆菌,涂布于含卡那霉素的LB固体平板上,37℃培养过夜,随机挑选单菌落进行测序验证,结果表明含有蔗糖磷酸化酶BbrSPase突变体基因的重组表达载体成功转化至宿主E. coliBL21(DE3)中,最终获得突变株P134C,L341V,L341I,L343I,L341I/l343I,L341I/L343F,L341V/L343F和L341I/L343N。
步骤二:蔗糖磷酸化酶BbrSPase以及突变体的诱导表达;
将步骤一中得到的工程菌接种至含卡那霉素的LB培养基中,37℃,200 rpm过夜培养,再将培养好的菌液以2%接种量(v/v)接种至100 mL含卡那霉素的LB培养基中,37℃,200rpm培养至OD600nm~0.5左右,加入终浓度为0.25 mM的IPTG,24℃诱导表达6~8 h后,5000 rpm离心5 min后,收集菌体。
步骤三:蔗糖磷酸化酶BbrSPase以及突变体的分离纯化;
步骤二收集的菌体细胞悬浮于8 mL去离子水后,于冰上超声破碎(300 W功率, 工作1s,停3s,总工作时长10 min)。破碎液于12000 rpm离心10 min去除细胞碎片,搜集上清液用于酶的分离纯化。纯化柱为Ni-NTA, 装柱体积为4 mL, 先用10倍柱体积平衡缓冲液(20 mM磷酸钠,300 mM NaCl和20 mM咪唑,pH 7 .4)平衡Ni-NTA柱,后上样8 mL并封住柱子两端,于4℃冰箱缓慢震荡吸附1 h。竖直放置Ni-NTA,除去上样废液,用10倍柱体积洗涤缓冲液(20 mM磷酸钠,300 mM NaCl和40 mM咪唑,pH 7 .4)洗涤柱子。最后用洗脱缓冲液(20mM磷酸钠,300mM NaCl和300 mM咪唑,pH 7 .4)洗脱并收集目的蛋白。酶液用透析袋进行透析除盐,透析液为pH5.5-6.5的去离子水,除盐后的纯酶用PEG20000(平均分子量为20000的聚乙二醇)浓缩后于-20℃保存备用。纯化后的纯酶用SDS-PAGE进行分析。
图1为蔗糖磷酸化酶BbrSPase及其突变体诱导表达并分离纯化后的SDS-PAGE图。其中Lane 1,Marker;Lane 2,BbrSPase;Lane 3,P134C;Lane 4,L341I;Lane 5,L341V;Lane6,L343I;Lane 7,L341I/L343I;Lane 8,L341I/L343F;Lane 9,L341V/L343F;Lane 10,L341I/L343N。结果表明,经Ni-NTA亲和层析,得到电泳纯的重组蔗糖磷酸化酶及其突变体。
实验验证BbrSPase及其突变体的催化效率:
L-AA转化率由反应平衡后得到的AA-2G摩尔浓度除以初始L-AA的摩尔浓度所得;AA-2G的纯度是指AA-2G, AA-3G和AA-2GG三者在液相谱图上面积归一化后,AA-2G所占百分比,三种糖苷产物的HPLC图谱如图3所示。
底物以及催化产物的高效液相色谱检测方法:色谱柱:C18柱; 流动相:20 mMKH2PO4缓冲液(H3PO4调节pH 2.3);柱温:25℃;流速: 1 mL/min;检测波长:240 nm;
反应体系600 μL:
1.2 M L-AA终浓度,0.8 M蔗糖终浓度,调节底物pH5.0,加入终浓度为1.5 mg/mL的纯酶,于40℃,1000 rpm金属震荡仪中反应72 h。酶活定义:在上述条件下,每分钟生成1μmol AA-2G所需的酶量定义为一个酶活单位,计1 U。野生型BbrSPase及其突变体催化相应底物效率和AA-2G产物纯度,其结果如表2所示。
图3为野生型BbrSPase和L341I/L343F第48 h的催化液的液相色谱图,位于上方的液相色谱图对应于BbrSPase野生型,位于下方的液相色谱图对应于L341I/L343F,按照出峰时间顺序依次为:AA-3G,L-AA,AA-2G和AA-2GG。
表2 :BbrSPase及其突变体催化效率
实验结果分析:本发明所提供的蔗糖磷酸化酶BbrSPase突变体与野生型相比较,有更优良的催化活性,蔗糖磷酸化酶突变体催化合成AA-2G的浓度在153~205 g/L, 转化率达38~51%,产物纯度达92~99%,蔗糖磷酸化酶突变体可以更好地应用于生物催化领域,对AA-2G的工业化生产及应用有重要意义。
以上显示和描述了本发明的基本原理、主要特征和优点。本行业的技术人员应该了解,上述实施例不以任何形式限制本发明,凡采用等同替换或等效变换的方式所获得的技术方案,均落在本发明的保护范围内。
序列表
<110> 浙江工业大学
<120> 一种蔗糖磷酸化酶突变体、编码基因及其应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 508
<212> PRT
<213> Bifidobacterium breve
<400> 1
Met Lys Asn Lys Val Gln Leu Ile Ala Tyr Ala Asp Arg Leu Gly Asp
1 5 10 15
Gly Thr Leu Ser Ser Met Thr Asp Ile Leu Arg Thr Arg Phe Asp Gly
20 25 30
Val Tyr Asp Gly Val His Ile Leu Pro Phe Phe Thr Pro Phe Asp Gly
35 40 45
Ala Asp Ala Gly Phe Asp Pro Ile Asp His Thr Lys Val Asp Pro Arg
50 55 60
Leu Gly Ser Trp Asp Asp Val Ala Glu Leu Ser Lys Thr His Asp Ile
65 70 75 80
Met Val Asp Ala Ile Val Asn His Met Ser Trp Glu Ser Ala Gln Phe
85 90 95
Gln Asp Val Leu Lys Asn Gly Glu His Ser Glu Tyr Tyr Pro Met Phe
100 105 110
Leu Thr Met Ser Ser Val Phe Pro Asn Gly Ala Thr Glu Glu Asp Leu
115 120 125
Ala Gly Ile Tyr Arg Pro Arg Pro Gly Leu Pro Phe Thr His Tyr Lys
130 135 140
Phe Ala Gly Lys Thr Arg Leu Val Trp Val Ser Phe Thr Pro Gln Gln
145 150 155 160
Val Asp Ile Asp Thr Asp Ser Ala Lys Gly Trp Glu Tyr Leu Met Ser
165 170 175
Ile Phe Asp Gln Met Ala Ala Ser His Val Arg Tyr Ile Arg Leu Asp
180 185 190
Ala Val Gly Tyr Gly Ala Lys Glu Ala Gly Thr Ser Cys Phe Met Thr
195 200 205
Pro Lys Thr Phe Lys Leu Ile Ser Arg Leu Arg Glu Glu Gly Val Lys
210 215 220
Arg Gly Leu Glu Ile Leu Ile Glu Val His Ser Tyr Tyr Lys Lys Gln
225 230 235 240
Val Glu Ile Ala Ser Lys Val Asp Arg Val Tyr Asp Phe Ala Leu Pro
245 250 255
Pro Leu Leu Leu His Ser Leu Phe Thr Gly His Val Glu Pro Val Val
260 265 270
His Trp Thr Glu Ile Arg Pro Asn Asn Ala Val Thr Val Leu Asp Thr
275 280 285
His Asp Gly Ile Gly Val Ile Asp Ile Gly Ser Asp Gln Leu Asp Arg
290 295 300
Ser Leu Lys Gly Leu Val Pro Asp Glu Asp Val Asp Asn Leu Val Asn
305 310 315 320
Thr Ile His Ala Asn Thr His Gly Glu Ser Gln Ala Ala Thr Gly Ala
325 330 335
Ala Ala Ser Asn Leu Asp Leu Tyr Gln Val Asn Ser Thr Tyr Tyr Ser
340 345 350
Ala Leu Gly Cys Asn Asp Gln His Tyr Leu Ala Ala Arg Ala Val Gln
355 360 365
Phe Phe Leu Pro Gly Val Pro Gln Val Tyr Tyr Val Gly Ala Leu Ala
370 375 380
Gly Arg Asn Asp Met Glu Leu Leu Arg Lys Thr Asn Asn Gly Arg Asp
385 390 395 400
Ile Asn Arg His Tyr Tyr Ser Thr Ala Glu Ile Asp Glu Asn Leu Glu
405 410 415
Arg Pro Val Val Lys Ala Leu Asn Ala Leu Ala Lys Phe Arg Asn Glu
420 425 430
Leu Pro Ala Phe Asn Gly Glu Phe Ser Tyr Glu Ala Asp Gly Asp Thr
435 440 445
Ser Ile Thr Phe Arg Trp Ile Ala Ala Asp Gly Lys Thr Lys Ala Ala
450 455 460
Leu Ile Phe Glu Pro Gly Arg Gly Leu Gly Thr Asp Asn Thr Thr Pro
465 470 475 480
Val Ala Ser Leu Ala Trp Thr Asp Ala Ala Gly Asp His Glu Thr Asp
485 490 495
Asp Leu Leu Ser Asn Pro Pro Ile Ala Asp Ile Asp
500 505
<210> 2
<211> 1527
<212> DNA
<213> 未知(Unknown)
<400> 2
atgaaaaaca aagtgcaact catcgcttac gccgatcgtc tcggcgatgg tactcttagc 60
tcgatgaccg acatcctgcg cacccgcttc gacggcgtgt atgacggcgt gcatatcctg 120
ccgttcttca ctccgttcga tggtgcggat gcaggctttg acccgatcga ccacaccaaa 180
gtcgacccgc gcctcggatc gtgggacgac gtcgccgagc tctccaaaac ccacgacatc 240
atggtcgatg ccatcgtcaa ccacatgagc tgggaatccg cccaattcca agacgtgctg 300
aagaacggcg agcactccga gtattacccg atgttcctga ccatgagttc cgttttcccg 360
aacggcgcca ccgaagagga tctcgccggc atctaccgcc cgcgcccggg cctgccgttc 420
acccactaca agttcgccgg caagacccgt ctggtatggg tcagcttcac cccacagcag 480
gtggacatcg acactgactc cgccaagggc tgggagtatc tgatgtccat ctttgatcag 540
atggcagcca gccatgtgcg ctacatccgt ctcgacgccg tgggctacgg cgccaaggag 600
gccggcacca gctgcttcat gacccccaag acctttaagc tcatctcacg cctacgcgag 660
gaaggcgtca agcgaggcct cgaaattctc attgaggtgc atagctacta caagaagcag 720
gttgaaatcg cctccaaggt ggaccgcgtc tacgacttcg ccctgcctcc gctgctcctg 780
cactcgctgt tcaccggtca cgtggaaccc gtggtccact ggaccgaaat ccgcccgaac 840
aacgccgtca ccgtgctcga tacgcacgac ggcatcggcg tgatcgacat cggctccgat 900
cagctcgacc gcagcctcaa gggcctcgtg cccgacgagg acgtcgataa tctggtcaac 960
accatccacg ccaacaccca cggcgaatcc caggccgcca ccggtgccgc cgccagcaac 1020
ctcgacctct atcaggtcaa cagcacgtac tactccgcgc tcggctgcaa cgaccagcac 1080
tatctggccg cccgcgcggt ccagttcttc ctgcccggag tgccgcaggt ctactacgtg 1140
ggcgcgctcg ccggtcgcaa cgacatggaa ctgctgcgca agaccaacaa cggccgtgac 1200
atcaatcgcc attactactc caccgccgaa atcgacgaaa acctcgagcg cccggtggtg 1260
aaggccctga acgccctggc caagttccgc aacgaactgc ctgcattcaa tggcgagttc 1320
agctacgaag ccgacggcga cacatccatc accttccgct ggatcgctgc cgacggcaag 1380
accaaggccg ccctcatctt cgagcccggc cgcggactcg gcacggataa caccactccg 1440
gtcgccagcc tcgcctggac cgatgccgcc ggtgaccacg agactgatga tctgctgagc 1500
aacccgccga ttgccgatat cgactaa 1527
<210> 3
<211> 1527
<212> DNA
<213> 未知(Unknown)
<400> 3
atgaaaaaca aagtgcaact catcgcttac gccgatcgtc tcggcgatgg tactcttagc 60
tcgatgaccg acatcctgcg cacccgcttc gacggcgtgt atgacggcgt gcatatcctg 120
ccgttcttca ctccgttcga tggtgcggat gcaggctttg acccgatcga ccacaccaaa 180
gtcgacccgc gcctcggatc gtgggacgac gtcgccgagc tctccaaaac ccacgacatc 240
atggtcgatg ccatcgtcaa ccacatgagc tgggaatccg cccaattcca agacgtgctg 300
aagaacggcg agcactccga gtattacccg atgttcctga ccatgagttc cgttttcccg 360
aacggcgcca ccgaagagga tctcgccggc atctaccgct gccgcccggg cctgccgttc 420
acccactaca agttcgccgg caagacccgt ctggtatggg tcagcttcac cccacagcag 480
gtggacatcg acactgactc cgccaagggc tgggagtatc tgatgtccat ctttgatcag 540
atggcagcca gccatgtgcg ctacatccgt ctcgacgccg tgggctacgg cgccaaggag 600
gccggcacca gctgcttcat gacccccaag acctttaagc tcatctcacg cctacgcgag 660
gaaggcgtca agcgaggcct cgaaattctc attgaggtgc atagctacta caagaagcag 720
gttgaaatcg cctccaaggt ggaccgcgtc tacgacttcg ccctgcctcc gctgctcctg 780
cactcgctgt tcaccggtca cgtggaaccc gtggtccact ggaccgaaat ccgcccgaac 840
aacgccgtca ccgtgctcga tacgcacgac ggcatcggcg tgatcgacat cggctccgat 900
cagctcgacc gcagcctcaa gggcctcgtg cccgacgagg acgtcgataa tctggtcaac 960
accatccacg ccaacaccca cggcgaatcc caggccgcca ccggtgccgc cgccagcaac 1020
ctcgacctct atcaggtcaa cagcacgtac tactccgcgc tcggctgcaa cgaccagcac 1080
tatctggccg cccgcgcggt ccagttcttc ctgcccggag tgccgcaggt ctactacgtg 1140
ggcgcgctcg ccggtcgcaa cgacatggaa ctgctgcgca agaccaacaa cggccgtgac 1200
atcaatcgcc attactactc caccgccgaa atcgacgaaa acctcgagcg cccggtggtg 1260
aaggccctga acgccctggc caagttccgc aacgaactgc ctgcattcaa tggcgagttc 1320
agctacgaag ccgacggcga cacatccatc accttccgct ggatcgctgc cgacggcaag 1380
accaaggccg ccctcatctt cgagcccggc cgcggactcg gcacggataa caccactccg 1440
gtcgccagcc tcgcctggac cgatgccgcc ggtgaccacg agactgatga tctgctgagc 1500
aacccgccga ttgccgatat cgactaa 1527
<210> 4
<211> 1527
<212> DNA
<213> 未知(Unknown)
<400> 4
atgaaaaaca aagtgcaact catcgcttac gccgatcgtc tcggcgatgg tactcttagc 60
tcgatgaccg acatcctgcg cacccgcttc gacggcgtgt atgacggcgt gcatatcctg 120
ccgttcttca ctccgttcga tggtgcggat gcaggctttg acccgatcga ccacaccaaa 180
gtcgacccgc gcctcggatc gtgggacgac gtcgccgagc tctccaaaac ccacgacatc 240
atggtcgatg ccatcgtcaa ccacatgagc tgggaatccg cccaattcca agacgtgctg 300
aagaacggcg agcactccga gtattacccg atgttcctga ccatgagttc cgttttcccg 360
aacggcgcca ccgaagagga tctcgccggc atctaccgcc cgcgcccggg cctgccgttc 420
acccactaca agttcgccgg caagacccgt ctggtatggg tcagcttcac cccacagcag 480
gtggacatcg acactgactc cgccaagggc tgggagtatc tgatgtccat ctttgatcag 540
atggcagcca gccatgtgcg ctacatccgt ctcgacgccg tgggctacgg cgccaaggag 600
gccggcacca gctgcttcat gacccccaag acctttaagc tcatctcacg cctacgcgag 660
gaaggcgtca agcgaggcct cgaaattctc attgaggtgc atagctacta caagaagcag 720
gttgaaatcg cctccaaggt ggaccgcgtc tacgacttcg ccctgcctcc gctgctcctg 780
cactcgctgt tcaccggtca cgtggaaccc gtggtccact ggaccgaaat ccgcccgaac 840
aacgccgtca ccgtgctcga tacgcacgac ggcatcggcg tgatcgacat cggctccgat 900
cagctcgacc gcagcctcaa gggcctcgtg cccgacgagg acgtcgataa tctggtcaac 960
accatccacg ccaacaccca cggcgaatcc caggccgcca ccggtgccgc cgccagcaac 1020
atcgacctct atcaggtcaa cagcacgtac tactccgcgc tcggctgcaa cgaccagcac 1080
tatctggccg cccgcgcggt ccagttcttc ctgcccggag tgccgcaggt ctactacgtg 1140
ggcgcgctcg ccggtcgcaa cgacatggaa ctgctgcgca agaccaacaa cggccgtgac 1200
atcaatcgcc attactactc caccgccgaa atcgacgaaa acctcgagcg cccggtggtg 1260
aaggccctga acgccctggc caagttccgc aacgaactgc ctgcattcaa tggcgagttc 1320
agctacgaag ccgacggcga cacatccatc accttccgct ggatcgctgc cgacggcaag 1380
accaaggccg ccctcatctt cgagcccggc cgcggactcg gcacggataa caccactccg 1440
gtcgccagcc tcgcctggac cgatgccgcc ggtgaccacg agactgatga tctgctgagc 1500
aacccgccga ttgccgatat cgactaa 1527
<210> 5
<211> 1527
<212> DNA
<213> 未知(Unknown)
<400> 5
atgaaaaaca aagtgcaact catcgcttac gccgatcgtc tcggcgatgg tactcttagc 60
tcgatgaccg acatcctgcg cacccgcttc gacggcgtgt atgacggcgt gcatatcctg 120
ccgttcttca ctccgttcga tggtgcggat gcaggctttg acccgatcga ccacaccaaa 180
gtcgacccgc gcctcggatc gtgggacgac gtcgccgagc tctccaaaac ccacgacatc 240
atggtcgatg ccatcgtcaa ccacatgagc tgggaatccg cccaattcca agacgtgctg 300
aagaacggcg agcactccga gtattacccg atgttcctga ccatgagttc cgttttcccg 360
aacggcgcca ccgaagagga tctcgccggc atctaccgcc cgcgcccggg cctgccgttc 420
acccactaca agttcgccgg caagacccgt ctggtatggg tcagcttcac cccacagcag 480
gtggacatcg acactgactc cgccaagggc tgggagtatc tgatgtccat ctttgatcag 540
atggcagcca gccatgtgcg ctacatccgt ctcgacgccg tgggctacgg cgccaaggag 600
gccggcacca gctgcttcat gacccccaag acctttaagc tcatctcacg cctacgcgag 660
gaaggcgtca agcgaggcct cgaaattctc attgaggtgc atagctacta caagaagcag 720
gttgaaatcg cctccaaggt ggaccgcgtc tacgacttcg ccctgcctcc gctgctcctg 780
cactcgctgt tcaccggtca cgtggaaccc gtggtccact ggaccgaaat ccgcccgaac 840
aacgccgtca ccgtgctcga tacgcacgac ggcatcggcg tgatcgacat cggctccgat 900
cagctcgacc gcagcctcaa gggcctcgtg cccgacgagg acgtcgataa tctggtcaac 960
accatccacg ccaacaccca cggcgaatcc caggccgcca ccggtgccgc cgccagcaac 1020
gtcgacctct atcaggtcaa cagcacgtac tactccgcgc tcggctgcaa cgaccagcac 1080
tatctggccg cccgcgcggt ccagttcttc ctgcccggag tgccgcaggt ctactacgtg 1140
ggcgcgctcg ccggtcgcaa cgacatggaa ctgctgcgca agaccaacaa cggccgtgac 1200
atcaatcgcc attactactc caccgccgaa atcgacgaaa acctcgagcg cccggtggtg 1260
aaggccctga acgccctggc caagttccgc aacgaactgc ctgcattcaa tggcgagttc 1320
agctacgaag ccgacggcga cacatccatc accttccgct ggatcgctgc cgacggcaag 1380
accaaggccg ccctcatctt cgagcccggc cgcggactcg gcacggataa caccactccg 1440
gtcgccagcc tcgcctggac cgatgccgcc ggtgaccacg agactgatga tctgctgagc 1500
aacccgccga ttgccgatat cgactaa 1527
<210> 6
<211> 1527
<212> DNA
<213> 未知(Unknown)
<400> 6
atgaaaaaca aagtgcaact catcgcttac gccgatcgtc tcggcgatgg tactcttagc 60
tcgatgaccg acatcctgcg cacccgcttc gacggcgtgt atgacggcgt gcatatcctg 120
ccgttcttca ctccgttcga tggtgcggat gcaggctttg acccgatcga ccacaccaaa 180
gtcgacccgc gcctcggatc gtgggacgac gtcgccgagc tctccaaaac ccacgacatc 240
atggtcgatg ccatcgtcaa ccacatgagc tgggaatccg cccaattcca agacgtgctg 300
aagaacggcg agcactccga gtattacccg atgttcctga ccatgagttc cgttttcccg 360
aacggcgcca ccgaagagga tctcgccggc atctaccgcc cgcgcccggg cctgccgttc 420
acccactaca agttcgccgg caagacccgt ctggtatggg tcagcttcac cccacagcag 480
gtggacatcg acactgactc cgccaagggc tgggagtatc tgatgtccat ctttgatcag 540
atggcagcca gccatgtgcg ctacatccgt ctcgacgccg tgggctacgg cgccaaggag 600
gccggcacca gctgcttcat gacccccaag acctttaagc tcatctcacg cctacgcgag 660
gaaggcgtca agcgaggcct cgaaattctc attgaggtgc atagctacta caagaagcag 720
gttgaaatcg cctccaaggt ggaccgcgtc tacgacttcg ccctgcctcc gctgctcctg 780
cactcgctgt tcaccggtca cgtggaaccc gtggtccact ggaccgaaat ccgcccgaac 840
aacgccgtca ccgtgctcga tacgcacgac ggcatcggcg tgatcgacat cggctccgat 900
cagctcgacc gcagcctcaa gggcctcgtg cccgacgagg acgtcgataa tctggtcaac 960
accatccacg ccaacaccca cggcgaatcc caggccgcca ccggtgccgc cgccagcaac 1020
ctcgacatct atcaggtcaa cagcacgtac tactccgcgc tcggctgcaa cgaccagcac 1080
tatctggccg cccgcgcggt ccagttcttc ctgcccggag tgccgcaggt ctactacgtg 1140
ggcgcgctcg ccggtcgcaa cgacatggaa ctgctgcgca agaccaacaa cggccgtgac 1200
atcaatcgcc attactactc caccgccgaa atcgacgaaa acctcgagcg cccggtggtg 1260
aaggccctga acgccctggc caagttccgc aacgaactgc ctgcattcaa tggcgagttc 1320
agctacgaag ccgacggcga cacatccatc accttccgct ggatcgctgc cgacggcaag 1380
accaaggccg ccctcatctt cgagcccggc cgcggactcg gcacggataa caccactccg 1440
gtcgccagcc tcgcctggac cgatgccgcc ggtgaccacg agactgatga tctgctgagc 1500
aacccgccga ttgccgatat cgactaa 1527
<210> 7
<211> 1527
<212> DNA
<213> 未知(Unknown)
<400> 7
atgaaaaaca aagtgcaact catcgcttac gccgatcgtc tcggcgatgg tactcttagc 60
tcgatgaccg acatcctgcg cacccgcttc gacggcgtgt atgacggcgt gcatatcctg 120
ccgttcttca ctccgttcga tggtgcggat gcaggctttg acccgatcga ccacaccaaa 180
gtcgacccgc gcctcggatc gtgggacgac gtcgccgagc tctccaaaac ccacgacatc 240
atggtcgatg ccatcgtcaa ccacatgagc tgggaatccg cccaattcca agacgtgctg 300
aagaacggcg agcactccga gtattacccg atgttcctga ccatgagttc cgttttcccg 360
aacggcgcca ccgaagagga tctcgccggc atctaccgcc cgcgcccggg cctgccgttc 420
acccactaca agttcgccgg caagacccgt ctggtatggg tcagcttcac cccacagcag 480
gtggacatcg acactgactc cgccaagggc tgggagtatc tgatgtccat ctttgatcag 540
atggcagcca gccatgtgcg ctacatccgt ctcgacgccg tgggctacgg cgccaaggag 600
gccggcacca gctgcttcat gacccccaag acctttaagc tcatctcacg cctacgcgag 660
gaaggcgtca agcgaggcct cgaaattctc attgaggtgc atagctacta caagaagcag 720
gttgaaatcg cctccaaggt ggaccgcgtc tacgacttcg ccctgcctcc gctgctcctg 780
cactcgctgt tcaccggtca cgtggaaccc gtggtccact ggaccgaaat ccgcccgaac 840
aacgccgtca ccgtgctcga tacgcacgac ggcatcggcg tgatcgacat cggctccgat 900
cagctcgacc gcagcctcaa gggcctcgtg cccgacgagg acgtcgataa tctggtcaac 960
accatccacg ccaacaccca cggcgaatcc caggccgcca ccggtgccgc cgccagcaac 1020
atcgacatct atcaggtcaa cagcacgtac tactccgcgc tcggctgcaa cgaccagcac 1080
tatctggccg cccgcgcggt ccagttcttc ctgcccggag tgccgcaggt ctactacgtg 1140
ggcgcgctcg ccggtcgcaa cgacatggaa ctgctgcgca agaccaacaa cggccgtgac 1200
atcaatcgcc attactactc caccgccgaa atcgacgaaa acctcgagcg cccggtggtg 1260
aaggccctga acgccctggc caagttccgc aacgaactgc ctgcattcaa tggcgagttc 1320
agctacgaag ccgacggcga cacatccatc accttccgct ggatcgctgc cgacggcaag 1380
accaaggccg ccctcatctt cgagcccggc cgcggactcg gcacggataa caccactccg 1440
gtcgccagcc tcgcctggac cgatgccgcc ggtgaccacg agactgatga tctgctgagc 1500
aacccgccga ttgccgatat cgactaa 1527
<210> 8
<211> 1527
<212> DNA
<213> 未知(Unknown)
<400> 8
atgaaaaaca aagtgcaact catcgcttac gccgatcgtc tcggcgatgg tactcttagc 60
tcgatgaccg acatcctgcg cacccgcttc gacggcgtgt atgacggcgt gcatatcctg 120
ccgttcttca ctccgttcga tggtgcggat gcaggctttg acccgatcga ccacaccaaa 180
gtcgacccgc gcctcggatc gtgggacgac gtcgccgagc tctccaaaac ccacgacatc 240
atggtcgatg ccatcgtcaa ccacatgagc tgggaatccg cccaattcca agacgtgctg 300
aagaacggcg agcactccga gtattacccg atgttcctga ccatgagttc cgttttcccg 360
aacggcgcca ccgaagagga tctcgccggc atctaccgcc cgcgcccggg cctgccgttc 420
acccactaca agttcgccgg caagacccgt ctggtatggg tcagcttcac cccacagcag 480
gtggacatcg acactgactc cgccaagggc tgggagtatc tgatgtccat ctttgatcag 540
atggcagcca gccatgtgcg ctacatccgt ctcgacgccg tgggctacgg cgccaaggag 600
gccggcacca gctgcttcat gacccccaag acctttaagc tcatctcacg cctacgcgag 660
gaaggcgtca agcgaggcct cgaaattctc attgaggtgc atagctacta caagaagcag 720
gttgaaatcg cctccaaggt ggaccgcgtc tacgacttcg ccctgcctcc gctgctcctg 780
cactcgctgt tcaccggtca cgtggaaccc gtggtccact ggaccgaaat ccgcccgaac 840
aacgccgtca ccgtgctcga tacgcacgac ggcatcggcg tgatcgacat cggctccgat 900
cagctcgacc gcagcctcaa gggcctcgtg cccgacgagg acgtcgataa tctggtcaac 960
accatccacg ccaacaccca cggcgaatcc caggccgcca ccggtgccgc cgccagcaac 1020
atcgacttct atcaggtcaa cagcacgtac tactccgcgc tcggctgcaa cgaccagcac 1080
tatctggccg cccgcgcggt ccagttcttc ctgcccggag tgccgcaggt ctactacgtg 1140
ggcgcgctcg ccggtcgcaa cgacatggaa ctgctgcgca agaccaacaa cggccgtgac 1200
atcaatcgcc attactactc caccgccgaa atcgacgaaa acctcgagcg cccggtggtg 1260
aaggccctga acgccctggc caagttccgc aacgaactgc ctgcattcaa tggcgagttc 1320
agctacgaag ccgacggcga cacatccatc accttccgct ggatcgctgc cgacggcaag 1380
accaaggccg ccctcatctt cgagcccggc cgcggactcg gcacggataa caccactccg 1440
gtcgccagcc tcgcctggac cgatgccgcc ggtgaccacg agactgatga tctgctgagc 1500
aacccgccga ttgccgatat cgactaa 1527
<210> 9
<211> 1527
<212> DNA
<213> 未知(Unknown)
<400> 9
atgaaaaaca aagtgcaact catcgcttac gccgatcgtc tcggcgatgg tactcttagc 60
tcgatgaccg acatcctgcg cacccgcttc gacggcgtgt atgacggcgt gcatatcctg 120
ccgttcttca ctccgttcga tggtgcggat gcaggctttg acccgatcga ccacaccaaa 180
gtcgacccgc gcctcggatc gtgggacgac gtcgccgagc tctccaaaac ccacgacatc 240
atggtcgatg ccatcgtcaa ccacatgagc tgggaatccg cccaattcca agacgtgctg 300
aagaacggcg agcactccga gtattacccg atgttcctga ccatgagttc cgttttcccg 360
aacggcgcca ccgaagagga tctcgccggc atctaccgcc cgcgcccggg cctgccgttc 420
acccactaca agttcgccgg caagacccgt ctggtatggg tcagcttcac cccacagcag 480
gtggacatcg acactgactc cgccaagggc tgggagtatc tgatgtccat ctttgatcag 540
atggcagcca gccatgtgcg ctacatccgt ctcgacgccg tgggctacgg cgccaaggag 600
gccggcacca gctgcttcat gacccccaag acctttaagc tcatctcacg cctacgcgag 660
gaaggcgtca agcgaggcct cgaaattctc attgaggtgc atagctacta caagaagcag 720
gttgaaatcg cctccaaggt ggaccgcgtc tacgacttcg ccctgcctcc gctgctcctg 780
cactcgctgt tcaccggtca cgtggaaccc gtggtccact ggaccgaaat ccgcccgaac 840
aacgccgtca ccgtgctcga tacgcacgac ggcatcggcg tgatcgacat cggctccgat 900
cagctcgacc gcagcctcaa gggcctcgtg cccgacgagg acgtcgataa tctggtcaac 960
accatccacg ccaacaccca cggcgaatcc caggccgcca ccggtgccgc cgccagcaac 1020
gtggacttct atcaggtcaa cagcacgtac tactccgcgc tcggctgcaa cgaccagcac 1080
tatctggccg cccgcgcggt ccagttcttc ctgcccggag tgccgcaggt ctactacgtg 1140
ggcgcgctcg ccggtcgcaa cgacatggaa ctgctgcgca agaccaacaa cggccgtgac 1200
atcaatcgcc attactactc caccgccgaa atcgacgaaa acctcgagcg cccggtggtg 1260
aaggccctga acgccctggc caagttccgc aacgaactgc ctgcattcaa tggcgagttc 1320
agctacgaag ccgacggcga cacatccatc accttccgct ggatcgctgc cgacggcaag 1380
accaaggccg ccctcatctt cgagcccggc cgcggactcg gcacggataa caccactccg 1440
gtcgccagcc tcgcctggac cgatgccgcc ggtgaccacg agactgatga tctgctgagc 1500
aacccgccga ttgccgatat cgactaa 1527
<210> 10
<211> 1527
<212> DNA
<213> 未知(Unknown)
<400> 10
atgaaaaaca aagtgcaact catcgcttac gccgatcgtc tcggcgatgg tactcttagc 60
tcgatgaccg acatcctgcg cacccgcttc gacggcgtgt atgacggcgt gcatatcctg 120
ccgttcttca ctccgttcga tggtgcggat gcaggctttg acccgatcga ccacaccaaa 180
gtcgacccgc gcctcggatc gtgggacgac gtcgccgagc tctccaaaac ccacgacatc 240
atggtcgatg ccatcgtcaa ccacatgagc tgggaatccg cccaattcca agacgtgctg 300
aagaacggcg agcactccga gtattacccg atgttcctga ccatgagttc cgttttcccg 360
aacggcgcca ccgaagagga tctcgccggc atctaccgcc cgcgcccggg cctgccgttc 420
acccactaca agttcgccgg caagacccgt ctggtatggg tcagcttcac cccacagcag 480
gtggacatcg acactgactc cgccaagggc tgggagtatc tgatgtccat ctttgatcag 540
atggcagcca gccatgtgcg ctacatccgt ctcgacgccg tgggctacgg cgccaaggag 600
gccggcacca gctgcttcat gacccccaag acctttaagc tcatctcacg cctacgcgag 660
gaaggcgtca agcgaggcct cgaaattctc attgaggtgc atagctacta caagaagcag 720
gttgaaatcg cctccaaggt ggaccgcgtc tacgacttcg ccctgcctcc gctgctcctg 780
cactcgctgt tcaccggtca cgtggaaccc gtggtccact ggaccgaaat ccgcccgaac 840
aacgccgtca ccgtgctcga tacgcacgac ggcatcggcg tgatcgacat cggctccgat 900
cagctcgacc gcagcctcaa gggcctcgtg cccgacgagg acgtcgataa tctggtcaac 960
accatccacg ccaacaccca cggcgaatcc caggccgcca ccggtgccgc cgccagcaac 1020
atcgacaact atcaggtcaa cagcacgtac tactccgcgc tcggctgcaa cgaccagcac 1080
tatctggccg cccgcgcggt ccagttcttc ctgcccggag tgccgcaggt ctactacgtg 1140
ggcgcgctcg ccggtcgcaa cgacatggaa ctgctgcgca agaccaacaa cggccgtgac 1200
atcaatcgcc attactactc caccgccgaa atcgacgaaa acctcgagcg cccggtggtg 1260
aaggccctga acgccctggc caagttccgc aacgaactgc ctgcattcaa tggcgagttc 1320
agctacgaag ccgacggcga cacatccatc accttccgct ggatcgctgc cgacggcaag 1380
accaaggccg ccctcatctt cgagcccggc cgcggactcg gcacggataa caccactccg 1440
gtcgccagcc tcgcctggac cgatgccgcc ggtgaccacg agactgatga tctgctgagc 1500
aacccgccga ttgccgatat cgactaa 1527
Claims (8)
1.一种蔗糖磷酸化酶突变体,其特征在于所述蔗糖磷酸化酶突变体为下列之一:
(1)氨基酸序列如SEQ ID NO.1所示蔗糖磷酸化酶第134位脯氨酸突变为半胱氨酸;
(2)氨基酸序列如SEQ ID NO.1所示蔗糖磷酸化酶第341位亮氨酸突变为异亮氨酸;
(3)氨基酸序列如SEQ ID NO.1所示蔗糖磷酸化酶第341位的亮氨酸突变为缬氨酸;
(4)氨基酸序列如SEQ ID NO.1所示蔗糖磷酸化酶第343位的亮氨酸突变为异亮氨酸;
(5)氨基酸序列如SEQ ID NO.1所示蔗糖磷酸化酶第341位的亮氨酸突变为异亮氨酸,第343位的亮氨酸突变为异亮氨酸;
(6)氨基酸序列如SEQ ID NO.1所示蔗糖磷酸化酶第341位的亮氨酸突变为异亮氨酸,第343位的亮氨酸突变为脯氨酸;
(7)氨基酸序列如SEQ ID NO.1所示蔗糖磷酸化酶第341位的亮氨酸突变为缬氨酸,第343位的亮氨酸突变为脯氨酸;
(8)氨基酸序列如SEQ ID NO.1所示蔗糖磷酸化酶第341位的亮氨酸突变为异亮氨酸,第343位的亮氨酸突变为天冬酰胺。
2.权利要求1所述蔗糖磷酸化酶突变体的编码基因。
3.如权利要求2所述的编码基因,其特征在于所述编码基因核苷酸序列如SEQ ID No.3~10之一所示。
4.含有权利要求2所述编码基因的重组表达载体。
5.含有权利要求2所述编码基因的工程菌。
6.权利要求1所述蔗糖磷酸化酶突变体在酶法制备L-抗坏血酸葡萄糖苷中的应用。
7.如权利要求6所述的应用,其特征在于所述L-抗坏血酸葡萄糖苷为L-抗坏血酸-2-葡萄苷。
8.如权利要求6或7所述的应用,其特征在于所述应用为:以L-抗坏血酸和蔗糖为底物,加入所述蔗糖磷酸化酶突变体或其重组表达载体转化至宿主微生物中获得基因工程菌菌体细胞,在20~60℃条件下,于pH 4.0~6.5的催化体系中反应,得到含有L-抗坏血酸葡萄糖苷的催化液。
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