罗汉果苷ⅢE的酶法定点糖基化修饰
|
摘要
罗汉果苷是由罗汉果Siraitia grosvenorii生物合成的四环三萜类皂苷。基于甜度高、热量低和口感好等优点,罗汉果苷已成为开发全新非营养型天然甜味剂的重要来源。本研究以甜度为蔗糖300倍且口味较好的罗汉果苷ⅢE为模型化合物,利用糖基转移酶对11位羟基进行糖基化修饰,研究其与罗汉果苷类化合物甜度和口味的构效关系。通过糖基转移酶库的筛选,获得了能够区域选择性糖基化罗汉果苷ⅢE 11位羟基的糖基转移酶HXSW-GT-2,然后经诱导表达条件优化实现了该酶在大肠埃希菌中的高效可溶性表达。在此基础上,通过对HXSW-GT-2反应条件如反应pH、温度、二磷酸尿苷葡萄糖二钠盐用量、反应时间等系统优化,罗汉果苷ⅢE糖基化的转化率被提高至85%以上,扩大反应体系后纯化得纯度大于95%的糖基化产物MG-ⅢE-Glu。口感测试结果表明,与罗汉果苷ⅢE和5%蔗糖溶液相比,MG-ⅢE-Glu的甜度基本消失,苦涩味明显。本研究初步阐释了11位羟基与罗汉果苷甜度和口感的构效关系,为该类天然甜味剂的构效改造和开发提供理论指导。
Mogrosides,the main sweet components isolated from Siraitia grosvenorii,are a family of cucurbitane-type tetracyclic triterpenoid saponins.Given that the high sweetness,low calorie and excellent taste,mogrosides have become the important resource for the development of natural non-nutritive sweeteners.As reported,11α-hydroxyl group in the structural skeleton of mogrosides was closely related to sweetness and taste,but it had not been confirmed experimentally.In this work,we used mogroside ⅢE as a model compound,which was 300 times sweeter than 5% sucrose and tasted better,and modified its 11α-hydroxyl group through glycosyltransferase to elucidate the relations between structure and sweetness of mogroside compounds.The glycosyltransferase HXSW-GT-2 was obtained to regio-selectively glycosylate the 11α-hydroxyl group of mogroside ⅢE through the screening of glycosyltransferase library.And then,the soluble expression of HXSW-GT-02 in Escherichia coli was efficiently achieved by optimizing the induction conditions.Subsequently,the yield of glycosylated mogroside ⅢE(MG-ⅢE-Glu) was increased to > 85% through optimizing reaction pH,temperature,UDP-G dosage and biocatalyst loading.The product MG-ⅢE-Glu was bio-prepared at a 0.5 L scale and the final purity was 97.8%.A "mouth feel" test showed that MG-ⅢE-Glu had no sweetness and displayed obvious bitterness through the comparison with mogroside ⅢE and 5% sucrose.In conclusion,the function of the 11α-hydroxyl group of mogrosides in sweetness and taste was preliminarily elucidated which would be beneficial for the structural modification and development of mogroside sweeteners.
Mogrosides,the main sweet components isolated from Siraitia grosvenorii,are a family of cucurbitane-type tetracyclic triterpenoid saponins.Given that the high sweetness,low calorie and excellent taste,mogrosides have become the important resource for the development of natural non-nutritive sweeteners.As reported,11α-hydroxyl group in the structural skeleton of mogrosides was closely related to sweetness and taste,but it had not been confirmed experimentally.In this work,we used mogroside ⅢE as a model compound,which was 300 times sweeter than 5% sucrose and tasted better,and modified its 11α-hydroxyl group through glycosyltransferase to elucidate the relations between structure and sweetness of mogroside compounds.The glycosyltransferase HXSW-GT-2 was obtained to regio-selectively glycosylate the 11α-hydroxyl group of mogroside ⅢE through the screening of glycosyltransferase library.And then,the soluble expression of HXSW-GT-02 in Escherichia coli was efficiently achieved by optimizing the induction conditions.Subsequently,the yield of glycosylated mogroside ⅢE(MG-ⅢE-Glu) was increased to > 85% through optimizing reaction pH,temperature,UDP-G dosage and biocatalyst loading.The product MG-ⅢE-Glu was bio-prepared at a 0.5 L scale and the final purity was 97.8%.A "mouth feel" test showed that MG-ⅢE-Glu had no sweetness and displayed obvious bitterness through the comparison with mogroside ⅢE and 5% sucrose.In conclusion,the function of the 11α-hydroxyl group of mogrosides in sweetness and taste was preliminarily elucidated which would be beneficial for the structural modification and development of mogroside sweeteners.
引文
[1] Li C,Lin LM,Sui F,et al.Chemistry and pharmacology of Siraitia grosvenorii:a review[J].China J Nat Med,2014,12(2):89-102.
[2] Chen L,Chen YJ,Wang SZ,et al.Enzymatic synthesis of mogroside IIIE[J].J China Pharm Univ(中国药科大学学报),2018,49(3):354-359.
[3] Wang L,Yang Z,Lu F,et al.Cucurbitane glycosides derived from mogroside IIE:structure-taste relationships,antioxidant activity,and acute toxicity[J].Molecules,2014,19(8):12676-12689.
[4] Di R,Huang MT,Ho CT,et al.Anti-inflammatory activities of mogrosides from Momordica grosvenori in murine macrophages and a murine ear edema model[J].J Agr Food Chem,2011,59(13):7474-7481.
[5] Takeo E,Yoshida H,Tada N,et al.Sweet elements of Siraitia grosvenori inhibit oxidative modification of low-density lipoprotein[J].J Atheroscler Thromb,2002,9(2):114-120.
[6] Suzuki YA,Murata Y,Inui H,et al.Triterpene glycosides of Siraitia grosvenori inhibit rat intestinal maltase and suppress the rise in blood glucose level after a single oral administration of maltose in rats[J].J Agr Food Chem,2005,53(8):2941-2946.
[7] Lin GP,Jiang T,Hu XB,et al.Effect of Siraitia grosvenorii polysaccharide on glucose and lipid of diabetic rabbits induced by feeding high fat/high sucrose chow[J].Exp Diabetes Res,2007,2007:67435.
[8] Matsumoto S,Jin M,Dewa Y,et al.Suppressive effect of Siraitia grosvenorii extract on dicyclanil-promoted hepatocellular prolife-rative lesions in male mice[J].J Toxicol Sci,2009,34(1):109-118.
[9] Pawar RS,Krynitsky AJ,Rader JI,et al.Sweeteners from plants-with emphasis on Stevia rebaudiana(Bertoni) and Siraitia grosvenorii(Swingle)[J].Anal Bioanal Chem,2013,405(13):4397-4407.
[10] Kaiser R,Matsumoto K,Nie RL,et al.Glycosides from Chinese medicinal plant,Hemsleyapanacis-scandens and structure-taste relationship of cucurbitane-glycosides[J].Chem Pharm Bull,1988,36(1):234-243.
[11] Zhou M,Thorson,Jon S,et al.Asymmetric enzymatic glycosylation of mitoxantrone[J].Org Lett,2011,13(10):2786-2788.
[12] Gantt RW,Peltier-Pain P,Singh S,et al.Broadening the scope of glycosyltrans ferase-catalyzed sugar nucleotide synthesis[J].Proc Natl Acad Sci U S A,2013,110(19):7648-7653.
[13] Rosano GL,Ceccarelli EA,Leandro,et al.Recombinant protein expression in Escherichia coli:advances and challenges[J].Front Microbiol,2014,5:172.
[14] Wu XR,Gou XD,Chen YJ,et al.Enzymatic preparation of t-butyl-6-cyano-(3R,5R)-dihydroxyhexanoate by a whole-cell biocatalyst co-expressing carbonyl reductase and glucose dehydrogenase[J].Process Biochem,2015,50(1):104-110.
[15] Vera C,Guerrero C,Wilson L,et al.Optimization of reaction conditions and the donor substrate in the synthesis of hexyl-β-d-galactoside[J].Process Biochem,2017,58:128-136.
[16] Choi SH,Ryu M,Yoon YJ,et al.Glycosylation of various flavonoids by recombinant oleandomycin glycosyltransferase from Streptomyces antibioticus in batch and repeated batch modes[J].Biotechnol Lett,2012,34(3):499-505.
[2] Chen L,Chen YJ,Wang SZ,et al.Enzymatic synthesis of mogroside IIIE[J].J China Pharm Univ(中国药科大学学报),2018,49(3):354-359.
[3] Wang L,Yang Z,Lu F,et al.Cucurbitane glycosides derived from mogroside IIE:structure-taste relationships,antioxidant activity,and acute toxicity[J].Molecules,2014,19(8):12676-12689.
[4] Di R,Huang MT,Ho CT,et al.Anti-inflammatory activities of mogrosides from Momordica grosvenori in murine macrophages and a murine ear edema model[J].J Agr Food Chem,2011,59(13):7474-7481.
[5] Takeo E,Yoshida H,Tada N,et al.Sweet elements of Siraitia grosvenori inhibit oxidative modification of low-density lipoprotein[J].J Atheroscler Thromb,2002,9(2):114-120.
[6] Suzuki YA,Murata Y,Inui H,et al.Triterpene glycosides of Siraitia grosvenori inhibit rat intestinal maltase and suppress the rise in blood glucose level after a single oral administration of maltose in rats[J].J Agr Food Chem,2005,53(8):2941-2946.
[7] Lin GP,Jiang T,Hu XB,et al.Effect of Siraitia grosvenorii polysaccharide on glucose and lipid of diabetic rabbits induced by feeding high fat/high sucrose chow[J].Exp Diabetes Res,2007,2007:67435.
[8] Matsumoto S,Jin M,Dewa Y,et al.Suppressive effect of Siraitia grosvenorii extract on dicyclanil-promoted hepatocellular prolife-rative lesions in male mice[J].J Toxicol Sci,2009,34(1):109-118.
[9] Pawar RS,Krynitsky AJ,Rader JI,et al.Sweeteners from plants-with emphasis on Stevia rebaudiana(Bertoni) and Siraitia grosvenorii(Swingle)[J].Anal Bioanal Chem,2013,405(13):4397-4407.
[10] Kaiser R,Matsumoto K,Nie RL,et al.Glycosides from Chinese medicinal plant,Hemsleyapanacis-scandens and structure-taste relationship of cucurbitane-glycosides[J].Chem Pharm Bull,1988,36(1):234-243.
[11] Zhou M,Thorson,Jon S,et al.Asymmetric enzymatic glycosylation of mitoxantrone[J].Org Lett,2011,13(10):2786-2788.
[12] Gantt RW,Peltier-Pain P,Singh S,et al.Broadening the scope of glycosyltrans ferase-catalyzed sugar nucleotide synthesis[J].Proc Natl Acad Sci U S A,2013,110(19):7648-7653.
[13] Rosano GL,Ceccarelli EA,Leandro,et al.Recombinant protein expression in Escherichia coli:advances and challenges[J].Front Microbiol,2014,5:172.
[14] Wu XR,Gou XD,Chen YJ,et al.Enzymatic preparation of t-butyl-6-cyano-(3R,5R)-dihydroxyhexanoate by a whole-cell biocatalyst co-expressing carbonyl reductase and glucose dehydrogenase[J].Process Biochem,2015,50(1):104-110.
[15] Vera C,Guerrero C,Wilson L,et al.Optimization of reaction conditions and the donor substrate in the synthesis of hexyl-β-d-galactoside[J].Process Biochem,2017,58:128-136.
[16] Choi SH,Ryu M,Yoon YJ,et al.Glycosylation of various flavonoids by recombinant oleandomycin glycosyltransferase from Streptomyces antibioticus in batch and repeated batch modes[J].Biotechnol Lett,2012,34(3):499-505.