Series enzyme composition capable of isomerizing flavone oxygen glycoside compounds and flavone C-glycoside compounds into different types of flavone C-glycoside compounds

Abstract

The invention relates to a series of enzyme compositions capable of isomerizing flavone oxygen glycoside compounds and flavone C-glycoside compounds into different types of flavone C-glycoside compounds and application of the series of enzyme compositions, the series of enzyme compositions are from Enterococcus avium W974-1 and Enterococcus gallinarum P581a of human, and are W974-1 c-Glic1B, 2A / 3A and 2B / 3B; w974-1 c-Glic1B, 2A / 3A, W974-1 c-Glic1B; the chemical formula is W974-1 c-Glic1B, 2B / 3B; the chemical formula is P581a c-Glic1B, 2A / 3A, and W974-1 c-Glic2B / 3B. The series of enzyme compositions can catalyze the conversion of flavone oxygen glycoside compounds to flavone carbon glycoside compounds; and mutual transformation between flavone C-glycoside compounds. The method for producing the flavone C-glycoside compound by using the enzyme provided by the invention has the characteristics of simple steps, low cost, environmental protection and the like, and can be used for the purposes of manufacturing, research and development of medicines, foods and chemical raw materials and the like.

Description

Technical Field

[0001] The invention relates to a series of enzyme compositions capable of isomerizing flavonoid oxygen glycoside compounds and flavonoid carbon glycoside compounds into different types of flavonoid carbon glycoside compounds and applications thereof. Background Art

[0002] Flavonoid carbon glycoside compounds are a class of compounds that exist naturally in nature. Their main feature is that the sugar group is directly connected to the flavonoid nucleus by a CC bond (Wu Xin'an et al. Journal of PLA Pharmacy, 2005, 21(2): 135-138). The sugar group is often connected to the C-6 or C-8 position of the flavonoid A ring to form a very stable glycoside structure. This type of compound is widely distributed in flowering plants. The most common aglycones are luteolin and apigenin, whose C-6 or C-8 position is connected to a glucose group to form vitexin, orientin, isovitexin, isoorientin, etc. (Gong Jinyan et al. Natural Product Research and Development, 2010, 22(3): 525-530). Flavonoid carbon glycoside compounds are mainly obtained by separation and purification from plants, or chemical synthesis. The separation and purification of flavonoid glycoside compound vitexin from plants requires multiple steps such as extraction, separation and chromatography (Xie Yunchang, Jiang Xiaohua. CN201610283820.5); if chemical synthesis is used to obtain isorientin, at least 10 steps are required and the yield is only 15% (KUMAZAWA T, et al. Carbohydrate Research, 2000, 329(3): 507-513.). According to literature reports (K. Nakamura, et al. Biol Pharm Bull 2019, 42, 417-423; Z. Tao, et al. J Gen Appl Microbiol 2022, 68, 125-133; S. Wang, et al. Arch Microbiol 2022, 204, 310), in addition to the proteins DgpA, DgpB and DgpC expressed by the genes dgpA, dgpB and dgpC contained in the Clostridium strain PUE derived from human feces, which can deglycosylate puerarin to produce soybean aglycone and also have a deglycosylation effect on oxygen glycoside compounds (Nakamura K, et al. Biol Pharm Bull, 2019, 42 (3): 417-423; Nakamura K, et al. Appl Environ Microbiol, 2020, 86(14): e00607-20), enzymes with similar effects were also found in many other strains. Summary of the invention

[0003] The present invention provides a series of enzyme compositions capable of isomerizing flavonoid oxygen glycoside compounds and flavonoid carbon glycoside compounds into different types of flavonoid carbon glycoside compounds, which are respectively from human Enterococcus avium 2020-W974-1 and Enterococcus gallinarum 2016-P581a, namely W974-1 c-Glic1B, 2A / 3A, 2B / 3B (enzyme composition 1); W974-1 c-Glic1B, 2A / 3A (enzyme composition 2); W974-1 c-Glic1B, 2B / 3B (enzyme composition 3); P581a c-Glic1B, 2A / 3A, W974-1 c-Glic2B / 3B (enzyme composition 4) (" / " represents that the two proteins before and after it together form an enzyme complex, for example, "2A / 3A" means that 2A and 3A together form an enzyme complex named 2A / 3A to act). The series of enzyme compositions can be used for the preparation and structural identification of flavonoid carbon glycoside compounds.

[0004] The two strains of bacteria involved in the present invention are from patent CN113956994A, and their specific biological preservation information is as follows:

[0005] Enterococcus avium 2020-W974-1(Deposit number: CGMCC No.22234)

[0006] Enterococcus gallinarum 2016-P581a(Deposit number: CGMCC No.22235)

[0007] This series of enzyme compositions can convert flavonoid oxygen glycoside compounds such as luteolin and apigenin-7-O-glucoside into flavonoid carbon glycoside compounds such as isoorientin, orientin, vitexin and isovitexin; and can catalyze the mutual conversion between flavonoid carbon glycoside compounds such as isoorientin, orientin, vitexin and isovitexin.

[0008] The genes corresponding to the series of enzyme compositions were heterologously expressed in Escherichia coli, and proteins W974-1c-Glic1B, 2A / 3A, 2B / 3B, and P581a c-Glic1B, 2A / 3A were obtained through purification.

[0009] Taking luteolin as substrate, under the co-catalysis of W974-1 c-Glic1B, 2A / 3A, and 2B / 3B, three substances, isoorientin, orientin, and luteolin, can be generated; under the co-catalysis of W974-1 c-Glic1B and 2A / 3A, two substances, isoorientin and luteolin, can be generated; under the co-catalysis of W974-1 c-Glic1B and 2B / 3B, two substances, orientin and luteolin, can be generated. With isoorientin as substrate, under the co-catalysis of W974-1 c-Glic1B, 2A / 3A, 2B / 3B, three substances, namely isoorientin, orientin, and luteolin, can be generated; under the co-catalysis of P581a c-Glic1B, 2A / 3A, W974-1 c-Glic2B / 3B, three substances, namely isoorientin, orientin, and luteolin, can also be generated. With orientin as substrate, under the co-catalysis of W974-1 c-Glic1B, 2A / 3A, 2B / 3B, three substances, namely isoorientin, orientin, and luteolin, can be generated; under the co-catalysis of P581a c-Glic1B, 2A / 3A, W974-1 c-Glic2B / 3B, three substances, namely isoorientin, orientin, and luteolin, can also be generated. The above enzyme-catalyzed reactions were subjected to HPLC detection, and the reaction products such as isoorientin and orientin were successfully identified.

[0010] Taking apigenin-7-O-glucoside as substrate, under the co-catalysis of W974-1 c-Glic1B, 2A / 3A, and 2B / 3B, three substances, isovitexin, vitexin, and apigenin, can be generated; under the co-catalysis of W974-1 c-Glic1B and 2A / 3A, two substances, isovitexin and apigenin, can be generated; under the co-catalysis of W974-1 c-Glic1B and 2B / 3B, two substances, vitexin and apigenin, can be generated. Taking isovitexin as substrate, under the co-catalysis of W974-1 c-Glic1B, 2A / 3A, and 2B / 3B, three substances, isovitexin, vitexin, and apigenin can be generated. Taking vitexin as substrate, under the co-catalysis of W974-1 c-Glic1B, 2A / 3A, and 2B / 3B, three substances, isovitexin, vitexin, and apigenin can be generated. Taking vitexin as substrate, under the co-catalysis of W974-1 c-Glic1B, 2A / 3A, and 2B / 3B, three substances, isovitexin, vitexin, and apigenin can be generated. The above enzyme-catalyzed reactions were subjected to HPLC detection, and the reaction products such as isovitexin and vitexin were successfully identified.

[0011] Using isoschaftoside as substrate, Apigenin-6-C-arabinoside and Apigenin-8-C-glucoside were generated under the co-catalysis of P581a c-Glic1B, 2A / 3A, and W974-1 c-Glic2B / 3B. The above enzyme-catalyzed reactions were detected by LC-MS, and the reaction products such as Apigenin-6-C-arabinoside and Apigenin-8-C-glucoside were successfully identified.

[0012] Using luteolin and apigenin as substrates, five substances, namely, isoorientin, orientin, isovitexin, vitexin, and luteolin, can be generated under the co-catalysis of W974-1 c-Glic1B, 2A / 3A, and 2B / 3B; three substances, namely, isoorientin, isovitexin, and luteolin, can be generated under the co-catalysis of W974-1 c-Glic1B and 2A / 3A; three substances, namely, orientin, vitexin, and luteolin, can be generated under the co-catalysis of W974-1 c-Glic1B and 2B / 3B. With orientin and apigenin as substrates, under the co-catalysis of W974-1 c-Glic1B, 2A / 3A, and 2B / 3B, five substances, namely, isoorientin, orientin, isovitexin, vitexin, and luteolin, can be generated; under the co-catalysis of W974-1 c-Glic1B and 2B / 3B, three substances, namely, orientin, vitexin, and luteolin, can be generated. The above enzyme-catalyzed reactions were tested by HPLC, and the reaction products, namely, isoorientin, orientin, isovitexin, and vitexin, were successfully identified.

[0013] Using apigenin-7-O-glucoside and luteolin as substrates, five substances, namely isovitexin, vitexin, isoorientin, orientin and apigenin, can be generated under the co-catalysis of W974-1 c-Glic1B, 2A / 3A and 2B / 3B; three substances, namely isovitexin, isoorientin and apigenin, can be generated under the co-catalysis of W974-1 c-Glic1B and 2A / 3A; three substances, namely vitexin, isoorientin and apigenin, can be generated under the co-catalysis of W974-1 c-Glic1B and 2B / 3B. With vitexin and luteolin as substrates, under the co-catalysis of W974-1 c-Glic1B, 2A / 3A, and 2B / 3B, five substances, namely, isovitexin, vitexin, isoorientin, orientin, and luteolin, can be generated; under the co-catalysis of W974-1 c-Glic1B and 2B / 3B, three substances, namely, vitexin, orientin, and luteolin, can be generated. The above enzyme-catalyzed reactions were tested by HPLC, and the reaction products, namely, isoorientin, orientin, isovitexin, and vitexin, were successfully identified.

[0014] The method for producing different types of flavonoid carbon glycoside compounds using an enzyme composition provided by the present invention has the characteristics of simple steps, low cost, high efficiency, economy and environmental protection, and can be used for the purposes of medicine, food, feed, chemical raw material manufacturing and research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The HPLC chromatogram of W974-1 c-Glic1B, 2A / 3A, 2B / 3B conversion of luteolin and its time-conversion efficiency curve

[0016] like Figure 1 As shown, A is the HPLC chromatogram of luteolin conversion, where a is the chromatogram of luteolin detected before conversion; b is the chromatogram detected after 0.75h of conversion; c is the chromatogram detected after 2h of conversion; d is the chromatogram of the reference substances luteolin, isoorientin, orientin, and luteolin, 1 is the chromatographic peak of luteolin, 2 is the chromatographic peak of isoorientin, 3 is the chromatographic peak of orientin, and 4 is the chromatographic peak of luteolin. B is the time-conversion efficiency curve of luteolin conversion, where 1 is the curve of luteolin, 2 is the curve of orientin, 3 is the curve of isoorientin, and 4 is the curve of luteolin.

[0017] Figure 2 This is the HPLC chromatogram of W974-1 c-Glic1B, 2A / 3A, W974-1 c-Glic1B, 2B / 3B conversion of luteolin

[0018] like Figure 2 As shown, A is the HPLC chromatogram of luteolin transformed by W974-1 c-Glic1B and 2A / 3A, where a is the chromatogram of luteolin detected before transformation; b is the chromatogram detected 3 hours after transformation; c is the chromatogram of reference substances luteolin, isoorientin, and luteolin, 1 is the chromatogram peak of luteolin, 2 is the chromatogram peak of isoorientin, and 3 is the chromatogram peak of luteolin. B is the HPLC chromatogram of luteolin transformed by W974-1 c-Glic1B and 2B / 3B, where a is the chromatogram of luteolin detected before transformation; b is the chromatogram detected 2 hours after transformation; c is the chromatogram of reference substances luteolin, orientin, and luteolin, 1 is the chromatogram peak of luteolin, 2 is the chromatogram peak of orientin, and 3 is the chromatogram peak of luteolin.

[0019] Figure 3 The HPLC chromatogram and time-conversion efficiency curve of W974-1 c-Glic1B, 2A / 3A, 2B / 3B conversion to isoorientin

[0020] like Figure 3As shown, A is the HPLC chromatogram of the conversion of isoorientin, where a is the chromatogram of isoorientin detected before conversion; b is the chromatogram detected 2 hours after conversion, 1 is the chromatogram peak of isoorientin, 2 is the chromatogram peak of orientin, and 3 is the chromatogram peak of luteolin; c is the chromatogram of the reference substance orientin; d is the chromatogram of the reference substance isoorientin; e is the chromatogram of the reference substance luteolin. B is the time-conversion efficiency curve of the conversion of isoorientin, where 1 is the curve of isoorientin, 2 is the curve of luteolin, and 3 is the curve of orientin.

[0021] Figure 4 The HPLC chromatogram and time-conversion efficiency curve of W974-1 c-Glic1B, 2A / 3A, 2B / 3B into orientin

[0022] like Figure 4 As shown, A is the HPLC chromatogram of orientin conversion, where a is the chromatogram of orientin detected before conversion; b is the chromatogram detected 2h after conversion, 1 is the chromatogram peak of orientin, 2 is the chromatogram peak of isoorientin, and 3 is the chromatogram peak of luteolin; c is the chromatogram of the reference substance orientin; d is the chromatogram of the reference substance isoorientin; e is the chromatogram of the reference substance luteolin. B is the time-conversion efficiency curve of orientin conversion, where 1 is the curve of orientin, 2 is the curve of luteolin, and 3 is the curve of isoorientin.

[0023] Figure 5 This is the HPLC chromatogram of the conversion of W974-1 c-Glic1B, 2A / 3A, 2B / 3B to apigenin-7-O-glucoside and its time-conversion efficiency curve

[0024] like Figure 5 As shown, A is the HPLC chromatogram of the conversion of apigenin-7-O-glucoside, where a is the chromatogram of apigenin-7-O-glucoside detected before conversion; b is the chromatogram detected after 0.75h of conversion; c is the chromatogram detected after 2h of conversion; d is the chromatogram of the reference substances apigenin-7-O-glucoside, isovitexin, vitexin, and apigenin, 1 is the chromatographic peak of apigenin-7-O-glucoside, 2 is the chromatographic peak of isovitexin, 3 is the chromatographic peak of vitexin, and 4 is the chromatographic peak of apigenin. B is the time-conversion efficiency curve of the conversion of apigenin-7-O-glucoside, where 1 is the curve of apigenin-7-O-glucoside, 2 is the curve of apigenin, 3 is the curve of vitexin, and 4 is the curve of isovitexin.

[0025] Figure 6 This is the HPLC chromatogram of the conversion of W974-1 c-Glic1B, 2A / 3A, and W974-1 c-Glic1B, 2B / 3B to apigenin-7-O-glucoside

[0026] like Figure 6 As shown, A is the HPLC chromatogram of apigenin-7-O-glucoside converted by W974-1 c-Glic1B and 2A / 3A, wherein a is the chromatogram of apigenin-7-O-glucoside detected before conversion; b is the chromatogram detected 1 hour after conversion, c is the chromatogram of reference substances apigenin-7-O-glucoside, isovitexin, and apigenin, 1 is the chromatographic peak of apigenin-7-O-glucoside, 2 is the chromatographic peak of isovitexin, and 3 is the chromatographic peak of apigenin. B is the HPLC chromatogram of apigenin-7-O-glucoside converted by W974-1 c-Glic1B, 2B / 3B, where a is the chromatogram of apigenin-7-O-glucoside detected before conversion; b is the chromatogram detected 4 hours after conversion, c is the chromatogram of reference substances apigenin-7-O-glucoside, vitexin, and apigenin, 1 is the chromatographic peak of apigenin-7-O-glucoside, 2 is the chromatographic peak of vitexin, and 3 is the chromatographic peak of apigenin. C is the HPLC chromatogram of apigenin-7-O-glucoside converted by P581a c-Glic1B, 2A / 3A, wherein a is the chromatogram of apigenin-7-O-glucoside detected before conversion; b is the chromatogram detected 1 h after conversion, c is the chromatograms of reference substances apigenin-7-O-glucoside, isovitexin, and apigenin, 1 is the chromatographic peak of apigenin-7-O-glucoside, 2 is the chromatographic peak of isovitexin, and 3 is the chromatographic peak of apigenin.

[0027] Figure 7 The HPLC chromatogram of W974-1 c-Glic1B, 2A / 3A, 2B / 3B conversion of isovitexin and its time-conversion efficiency curve

[0028] like Figure 7 As shown, A is the HPLC chromatogram of the conversion of isovitexin, where a is the chromatogram of isovitexin detected before conversion; b is the chromatogram detected 4 hours after conversion, 1 is the chromatogram peak of isovitexin, 2 is the chromatogram peak of vitexin, and 3 is the chromatogram peak of apigenin; c is the chromatogram of the reference substance vitexin; d is the chromatogram of the reference substance isovitexin; e is the chromatogram of the reference substance apigenin. B is the time-conversion efficiency curve of the conversion of isovitexin, where 1 is the curve of isovitexin, 2 is the curve of apigenin, and 3 is the curve of vitexin.

[0029] Figure 8 The HPLC chromatogram of W974-1 c-Glic1B, 2A / 3A, 2B / 3B conversion of vitexin and its time-conversion efficiency curve

[0030] like Figure 8As shown, A is the HPLC chromatogram of vitexin conversion, where a is the chromatogram of vitexin detected before conversion; b is the chromatogram detected 4 hours after conversion, 1 is the chromatogram peak of vitexin, 2 is the chromatogram peak of isovitexin, and 3 is the chromatogram peak of apigenin; c is the chromatogram of the reference substance vitexin; d is the chromatogram of the reference substance isovitexin; e is the chromatogram of the reference substance apigenin. B is the time-conversion efficiency curve of vitexin conversion, where 1 is the curve of vitexin, 2 is the curve of apigenin, and 3 is the curve of isovitexin.

[0031] Fig. 9 The HPLC chromatogram and mass spectrum of P581a c-Glic1B, 2A / 3A, and W974-1 c-Glic2B / 3B conversion to isoschaftoside

[0032] like Fig. 9 As shown, A is the HPLC chromatogram of the conversion of isoschaftoside, where a is the chromatogram of isoschaftoside detected before conversion, and 1 is the chromatogram peak of isoschaftoside; b is the chromatogram detected 20 hours after conversion, where 1 is the chromatogram peak of isoschaftoside, 2 is the chromatogram peak of Apigenin-8-C-glucoside, and 3 is the chromatogram peak of Apigenin-6-C-arabinoside. B is the identification mass spectrum of 2 and 3. As shown in the figure, 2 is identified as Apigenin-8-C-glucoside and 3 is Apigenin-6-C-arabinoside by mass spectrometry.

[0033] Fig.10 This is the HPLC chromatogram of the conversion of P581a c-Glic1B, 2A / 3A, and W974-1 c-Glic2B / 3B to isoorientin and orientin

[0034] like Fig.10 As shown, A is the HPLC chromatogram of isoorientin converted by P581a c-Glic1B, 2A / 3A, and W974-1 c-Glic2B / 3B, wherein a is the chromatogram of the reference substances isoorientin, orientin, and luteolin, 1 is the chromatographic peak of orientin, 2 is the chromatographic peak of isoorientin, and 3 is the chromatographic peak of luteolin; b is the chromatogram of isoorientin before conversion; and c is the chromatogram after 8 hours of conversion. B is the HPLC chromatogram of orientin converted by P581a c-Glic1B, 2A / 3A, and W974-1 c-Glic2B / 3B, where a is the chromatogram of the reference substances isoorientin, orientin, and luteolin, 1 is the chromatographic peak of orientin, 2 is the chromatographic peak of isoorientin, and 3 is the chromatographic peak of luteolin; b is the chromatogram of orientin before conversion; and c is the chromatogram after 8 hours of conversion.

[0035] Fig.11This is the HPLC chromatogram of the conversion of W974-1 c-Glic1B, 2A / 3A, and 2B / 3B to luteolin in the presence of apigenin.

[0036] like Fig.11 As shown, a is the chromatogram before conversion of luteolin; b is the chromatogram after 0.5h of conversion; c is the chromatogram after 1.5h of conversion; d is the chromatogram of reference substances isoorientin, orientin, vitexin, isovitexin, luteolin, luteolin, and apigenin, 1 is the chromatographic peak of luteolin, 2 is the chromatographic peak of isoorientin, 3 is the chromatographic peak of orientin, 4 is the chromatographic peak of vitexin, 5 is the chromatographic peak of isovitexin, 6 is the chromatographic peak of luteolin, and 7 is the chromatographic peak of apigenin.

[0037] Fig.12 This is the HPLC chromatogram of the conversion of W974-1 c-Glic1B, 2A / 3A, and W974-1 c-Glic1B, 2B / 3B to luteolin in the presence of apigenin.

[0038] like Fig.12 As shown, A is the HPLC chromatogram of the conversion of luteolin by W974-1 c-Glic1B, 2A / 3A in the presence of apigenin, wherein a is the chromatogram before the conversion of luteolin; b is the chromatogram after 1.5 h of conversion; c is the chromatogram after 4 h of conversion; d is the chromatogram of the reference substances isoorientin, isovitexin, luteolin, luteolin, and apigenin, 1 is the chromatographic peak of luteolin, 2 is the chromatographic peak of isoorientin, 3 is the chromatographic peak of isovitexin, 4 is the chromatographic peak of luteolin, and 5 is the chromatographic peak of apigenin. B is the HPLC chromatogram of the conversion of luteolin by W974-1 c-Glic1B, 2B / 3B in the presence of apigenin, wherein a is the chromatogram before the conversion of luteolin; b is the chromatogram after 1.5 h of conversion; c is the chromatogram after 4 h of conversion; d is the chromatogram of the reference substances orientin, vitexin, luteolin, luteolin, and apigenin, 1 is the chromatographic peak of luteolin, 2 is the chromatographic peak of orientin, 3 is the chromatographic peak of vitexin, 4 is the chromatographic peak of luteolin, and 5 is the chromatographic peak of apigenin.

[0039] Fig.13 This is the HPLC chromatogram of W974-1 c-Glic1B, 2A / 3A, 2B / 3B transforming orientin in the presence of apigenin

[0040] like Fig.13 As shown, a is the chromatogram of orientin before conversion; b is the chromatogram after 2 hours of conversion; c is the chromatogram of the reference substances isoorientin, orientin, vitexin, isovitexin, luteolin, and apigenin, 1 is the chromatographic peak of orientin, 2 is the chromatographic peak of isoorientin, 3 is the chromatographic peak of vitexin, 4 is the chromatographic peak of isovitexin, 5 is the chromatographic peak of luteolin, and 6 is the chromatographic peak of apigenin.

[0041] Fig.14 This is the HPLC chromatogram of the conversion of W974-1 c-Glic1B, 2A / 3A, and 2B / 3B to apigenin-7-O-glucoside in the presence of luteolin.

[0042] like Fig.14 As shown, a is the chromatogram of apigenin-7-O-glucoside before conversion; b is the chromatogram after 0.5h of conversion; c is the chromatogram of the reference substances isoorientin, orientin, vitexin, isovitexin, apigenin-7-O-glucoside, luteolin, and apigenin, 1 is the chromatographic peak of apigenin-7-O-glucoside, 2 is the chromatographic peak of isoorientin, 3 is the chromatographic peak of orientin, 4 is the chromatographic peak of vitexin, 5 is the chromatographic peak of isovitexin, 6 is the chromatographic peak of luteolin, and 7 is the chromatographic peak of apigenin.

[0043] Fig.15 HPLC chromatogram of the conversion of W974-1 c-Glic1B, 2A / 3A, and W974-1 c-Glic1B, 2B / 3B to apigenin-7-O-glucoside in the presence of luteolin

[0044] like Fig.15 As shown, A is the HPLC chromatogram of the conversion of apigenin-7-O-glucoside by W974-1 c-Glic1B, 2A / 3A in the presence of luteolin, wherein a is the chromatogram before the conversion of apigenin-7-O-glucoside; b is the chromatogram after 1.5 h of conversion; c is the chromatogram after 4 h of conversion; d is the chromatogram of the reference substances isoorientin, isovitexin, apigenin-7-O-glucoside, luteolin, and apigenin, 1 is the chromatographic peak of apigenin-7-O-glucoside, 2 is the chromatographic peak of isoorientin, 3 is the chromatographic peak of isovitexin, 4 is the chromatographic peak of luteolin, and 5 is the chromatographic peak of apigenin. B is the HPLC chromatogram of the conversion of apigenin-7-O-glucoside by W974-1 c-Glic1B, 2B / 3B in the presence of luteolin, wherein a is the chromatogram before the conversion of apigenin-7-O-glucoside; b is the chromatogram after 1.5 h of conversion; c is the chromatogram after 4 h of conversion; d is the chromatogram of the reference substances orientin, vitexin, apigenin-7-O-glucoside, luteolin, and apigenin, 1 is the chromatographic peak of apigenin-7-O-glucoside, 2 is the chromatographic peak of orientin, 3 is the chromatographic peak of vitexin, 4 is the chromatographic peak of luteolin, and 5 is the chromatographic peak of apigenin.

[0045] Fig.16 This is the HPLC chromatogram of W974-1 c-Glic1B, 2A / 3A, 2B / 3B converting vitexin in the presence of luteolin.

[0046] like Fig.16 As shown, a is the chromatogram before conversion of vitexin; b is the chromatogram after 2 hours of conversion; c is the chromatogram of the reference substances isoorientin, orientin, vitexin, isovitexin, luteolin, and apigenin, 1 is the chromatographic peak of vitexin, 2 is the chromatographic peak of isovitexin, 3 is the chromatographic peak of orientin, 4 is the chromatographic peak of isoorientin, 5 is the chromatographic peak of luteolin, and 6 is the chromatographic peak of apigenin. DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0048] Example 1

[0049] Obtaining genes of W974-1 c-Glic1B, 2A / 3A, 2B / 3B, P581a c-Glic1B, 2A / 3A contained in the series enzyme composition of the present invention and expressing and purifying the corresponding proteins

[0050] A: The genes corresponding to the eight proteins were obtained by screening after full-genome sequencing of two strains of bacteria, Enterococcus avium 2020-W974-1 (deposit number: CGMCC No.22234) and Enterococcus gallinarum 2016-P581a (deposit number: CGMCCNo.22235). The genes corresponding to W974-1 c-Glic1B, 2A, 3A, 2B, 3B, and P581a c-Glic1B, 2A, and 3A were synthesized to construct the pET-28a recombinant expression plasmid of the eight genes. After successful construction, the recombinant plasmid was introduced into Stbl3 chemically competent Escherichia coli by heat shock method, and the positive clones were screened by kanamycin-containing plates for colony PCR verification, and the positive transformants were picked for plasmid extraction and the extracted plasmids were sequenced by Sanger method. The correctly sequenced plasmid was transformed into E. coli BL21 (DE3) by heat stimulation, and then the E. coli containing the plasmid was inoculated into Luria-Bertani (LB) medium containing kanamycin and cultured at 37°C. When OD600 reached 0.6-0.8, isopropyl β-D-1-thiogalactoside (IPTG) was added to induce the culture for more than 16 hours. The culture solution was centrifuged at 4000rpm for 30min and the cells were collected and resuspended in a binding buffer (50mM Tris-HCl pH 8.0, 0.5M NaCl) containing 1mM phenylmethylsulfonyl fluoride (PMSF). For W974-1 c-Glic2A, 3A, 2B, 3B, P581a c-Glic2A, 3A, before lysis, the combinations were W974-1 c-Glic2A / 3A, W974-1 c-Glic2B / 3B, and P581a c-Glic2A / 3A. The cells were broken by ultrasound on ice, and the broken cells were removed by high-speed centrifugation (22000 rpm, 4°C, 45 min), and the supernatant was taken for protein purification.

[0051] B: The supernatant was pumped into the HiTrap His affinity chromatography column by a peristaltic pump to adsorb the protein on the chromatography column, and the waste liquid was discarded. After washing with a buffer (50mM Tris-HCl pH 8.0, 0.5M NaCl, 20mM imidazole) to remove impurities, the bound protein was gradient eluted using an elution buffer (50mM Tris-HCl pH 8.0, 0.5M NaCl, 1M imidazole), and the eluate was collected for SDS-PAGE electrophoresis to determine the protein content and molecular weight. The eluate with a higher protein content was collected and dialyzed for 12 hours using a dialysis membrane and an anion exchange binding column buffer (50mM Tris-HCl pH 8.0, 0.1M NaCl, 1mM DTT) to remove imidazole and used for the next ion exchange purification. The dialyzed sample was pumped into the HiTrap Q anion exchange column by a peristaltic pump, and the protein was eluted using a NaCl gradient (50mM Tris-HCl pH 8.5, 1M NaCl, 1mM DTT). The eluate was collected, and the protein purity and molecular weight were determined by the UV absorption curve and SDS-PAGE electrophoresis during protein elution. The eluate with higher protein purity was collected and concentrated by an ultrafiltration tube. The concentrated protein solution was further purified by size exclusion chromatography in 20mM Tris-HCl pH 7.4, 150mM NaCl, 1mM DTT buffer, and the protein purity was confirmed by UV absorption curve and SDS-PAGE electrophoresis. The corresponding protein was collected and concentrated by an ultrafiltration tube to obtain a uniform protein sample, which was the purified protein W974-1 c-Glic1B, 2A / 3A, 2B / 3B, P581a c-Glic1B, 2A / 3A.

[0052] Example 2

[0053] Biotransformation of luteolin and apigenin-7-O-glucoside by W974-1 c-Glic1B, 2A / 3A, 2B / 3B

[0054] A: The biotransformation reaction was carried out in 450 μL 1× PBS, pH 7.4, containing 1 mM MnCl 2 , 1mM NAD + , 10mM DTT and 0.1mM luteolin or apigenin-7-O-glucoside, the concentration of each enzyme was 20μM, and the reaction was carried out at 37℃. 50μL of the reaction solution was taken out at 0, 0.5, 0.75, 1, 2, 4, 8, 12 and 20h of the reaction and three volumes of methanol were added to terminate the reaction. The denatured protein was removed by centrifugation at 14000rpm for 2min at 4℃, and 150μL of the supernatant was filtered through a 0.22μM filter membrane for HPLC analysis.

[0055] B: If Figure 1 and Figure 5 As shown, luteolin was converted into isoorientin, orientin and luteolin by the enzyme composition; apigenin-7-O-glucoside was converted into isovitexin, vitexin and apigenin by the enzyme composition.

[0056] Example 3

[0057] Biotransformation of luteolin and apigenin-7-O-glucoside by W974-1 c-Glic1B, 2A / 3A, W974-1 c-Glic1B, 2B / 3B

[0058] A: The biotransformation reaction was carried out in 100 μL 1× PBS, pH 7.4, containing 1 mM MnCl 2 , 1mM NAD + , 10mM DTT and 0.1mM luteolin or apigenin-7-O-glucoside, the concentration of each enzyme was 20μM, reacted at 37℃, 50μL of the reaction solution was taken out at 0h, 1h, 2h or 3h of the reaction, and three times the volume of methanol was added to terminate the reaction, and mixed. Centrifuged at 4℃, 14000rpm for 2min to remove denatured proteins, and the supernatant was filtered through a 0.22μM filter membrane and 150μL was taken for high performance liquid chromatography analysis.

[0059] B: If Figure 2 and Figure 6 As shown, luteolin was converted into isoorientin and luteolin by W974-1 c-Glic1B, 2A / 3A, and into orientin and luteolin by W974-1 c-Glic1B, 2B / 3B; apigenin-7-O-glucoside was converted into isovitexin and apigenin by W974-1 c-Glic1B, 2A / 3A, and into vitexin and apigenin by W974-1 c-Glic1B, 2B / 3B.

[0060] Example 4

[0061] W974-1 Biotransformation of c-Glic1B, 2A / 3A, 2B / 3B isoorientin, orientin, vitexin, and isovitexin

[0062] A: The biotransformation reaction was carried out in 350 μL 1× PBS, pH 7.4, containing 1 mM MnCl 2 , 1mM NAD +, 10mM DTT and 0.1mM isoorientin or orientin or vitexin or isoorientin, the concentration of each enzyme is 20μM, react at 37℃, take out 50μL of the reaction solution at 0, 2, 4, 6, 8, 12 and 20h of the reaction and add three times the volume of methanol to terminate the reaction. Centrifuge at 4℃, 14000rpm for 2min to remove denatured proteins, and filter the supernatant through a 0.22μM filter membrane and take 150μL for high performance liquid chromatography analysis.

[0063] B: If Figure 3-4 and Figure 7-8 As shown, isoorientin is converted into orientin and luteolin by the enzyme composition; orientin is converted into isoorientin and luteolin by the enzyme composition; vitexin is converted into isovitexin and apigenin by the enzyme composition; isovitexin is converted into vitexin and apigenin by the enzyme composition.

[0064] Example 5

[0065] Biotransformation of isoschaftoside, isoorientin, and orientin by P581a c-Glic1B, 2A / 3A, and W974-1 c-Glic2B / 3B

[0066] A: The biotransformation reaction was carried out in 100 μL 1× PBS, pH 7.4, containing 1 mM MnCl 2 , 1mM NAD + , 10mM DTT and 0.1mM isoschaftoside or isoorientin or orientin, the concentration of each enzyme is 20μM, react at 37℃, take out 50μL of the reaction solution at 0, 8 or 20h of the reaction and add three times the volume of methanol to terminate the reaction. Centrifuge at 4℃, 14000rpm for 2min to remove denatured proteins, and filter the supernatant through a 0.22μM filter membrane and take 150μL for high performance liquid chromatography analysis.

[0067] B: If Figure 9-10 As shown, isoschaftoside is converted into Apigenin-8-C-glucoside and Apigenin-6-C-arabinoside by the enzyme composition, and the products are identified as Apigenin-8-C-glucoside and Apigenin-6-C-arabinoside by mass spectrometry. Isorientin is converted into orientin and luteolin by the enzyme composition; orientin is converted into isorientin and luteolin by the enzyme composition.

[0068] Example 6

[0069] Biotransformation of luteolin by W974-1 c-Glic1B, 2A / 3A, 2B / 3B, W974-1 c-Glic1B, 2A / 3A, and W974-1 c-Glic1B, 2B / 3B in the presence of apigenin

[0070] A: The biotransformation reaction was carried out in 200 μL 1× PBS, pH 7.4, containing 1 mM MnCl 2 , 1mM NAD + , 10mM DTT and 0.1mM luteolin and apigenin, the concentration of each enzyme was 20μM, reacted at 37℃, 50μL of the reaction solution was taken out at 0, 0.5h, 1.5h, and 4h of the reaction, and three volumes of methanol were added to terminate the reaction. Centrifuged at 4℃, 14000rpm for 2min to remove denatured proteins, and 150μL of the supernatant was filtered through a 0.22μM filter membrane and used for HPLC analysis.

[0071] B: If Figure 11-12 As shown, in the presence of apigenin, W974-1 c-Glic1B, 2A / 3A, 2B / 3B converted luteolin into isoorientin, orientin, vitexin, isovitexin and luteolin; W974-1 c-Glic1B, 2A / 3A converted luteolin into isoorientin, isovitexin and luteolin; W974-1 c-Glic1B, 2B / 3B converted luteolin into orientin, vitexin and luteolin.

[0072] Example 7

[0073] Biotransformation of apigenin-7-O-glucoside by W974-1 c-Glic1B, 2A / 3A, 2B / 3B, W974-1 c-Glic1B, 2A / 3A, and W974-1 c-Glic1B, 2B / 3B in the presence of luteolin

[0074] A: The biotransformation reaction was carried out in 200 μL 1× PBS, pH 7.4, containing 1 mM MnCl 2 , 1mM NAD + , 10mM DTT and 0.1mM apigenin-7-O-glucoside and luteolin, the concentration of each enzyme was 20μM, reacted at 37℃, 50μL of the reaction solution was taken out at 0, 0.5h, 1.5h, and 4h of the reaction, and three volumes of methanol were added to terminate the reaction. Centrifuge at 4℃, 14000rpm for 2min to remove denatured proteins, and filter the supernatant through a 0.22μM filter membrane and take 150μL for HPLC analysis.

[0075] B: If Figure 14-15As shown, in the presence of luteolin, W974-1 c-Glic1B, 2A / 3A, 2B / 3B converted apigenin-7-O-glucoside into isoorientin, orientin, vitexin, isovitexin and luteolin; W974-1 c-Glic1B, 2A / 3A converted apigenin-7-O-glucoside into isoorientin, isovitexin and luteolin; W974-1 c-Glic1B, 2B / 3B converted apigenin-7-O-glucoside into orientin, vitexin and luteolin.

[0076] Example 8

[0077] Biotransformation of orientin by W974-1 c-Glic1B, 2A / 3A, 2B / 3B in the presence of apigenin; and biotransformation of vitexin by W974-1 c-Glic1B, 2A / 3A, 2B / 3B in the presence of luteolin

[0078] A: The biotransformation reaction was carried out in 100 μL 1× PBS, pH 7.4, containing 1 mM MnCl 2 , 1mM NAD + , 10mM DTT and 0.1mM orientin and apigenin or vitexin and luteolin, the concentration of each enzyme was 20μM, reacted at 37℃, 50μL of the reaction solution was taken out at 0 and 2h of the reaction, and three volumes of methanol were added to terminate the reaction. The denatured protein was removed by centrifugation at 4℃, 14000rpm for 2min, and the supernatant was filtered through a 0.22μM filter membrane and 150μL was taken for high performance liquid chromatography analysis.

[0079] B: If Fig.13 and 16 As shown, in the presence of apigenin, orientin was converted into isoorientin, vitexin, isovitexin and luteolin by W974-1 c-Glic1B, 2A / 3A, 2B / 3B. In the presence of luteolin, vitexin was converted into isoorientin, orientin, isovitexin and apigenin by W974-1 c-Glic1B, 2A / 3A, 2B / 3B.

Claims

1. A series of enzyme compositions capable of isomerizing flavonoid oxygen glycoside compounds and flavonoid carbon glycoside compounds into different types of flavonoid carbon glycoside compounds, characterized in that The series of enzyme compositions are derived from Enterococcus avium 2020-W974-1 and Enterococcus gallinarum 2016-P581a.

2. The series of enzyme compositions according to claim 1, wherein the series of enzyme compositions are W974-1 c-Glic1B, 2A / 3A, 2B / 3B (enzyme composition 1); W974-1 c-Glic1B, 2A / 3A (enzyme composition 2); W974-1 c-Glic1B, 2B / 3B (enzyme composition 3); P581a c-Glic1B, 2A / 3A, W974-1 c-Glic2B / 3B (enzyme composition 4).

3. The enzyme composition according to claim 1 or 2, characterized in that Flavonoid oxygen glycoside compounds can be isomerized into flavonoid carbon glycoside compounds.

4. The enzyme composition according to claim 1 or 2, characterized in that Flavonoid glycoside compounds can be isomerized into different types of flavonoid glycoside compounds.

5. The enzyme composition according to claim 3, characterized in that The flavonoid oxygen glycoside compounds can be luteolin and apigenin-7-O-glucoside.

6. The enzyme composition according to any one of claims 3-4, characterized in that The flavonoid carbon glycoside compound can be isoorientin, orientin, vitexin and isovitexin.

7. The use of the series of enzyme compositions according to claim 1, characterized in that The method can be used for preparing flavonoid carbon glycoside compounds.

8. The use of the series of enzyme compositions according to claim 7, characterized in that The method can utilize flavonoid oxygen glycoside compounds or flavonoid carbon glycoside compounds to prepare different types of flavonoid carbon glycoside compounds.

9. Use of the series of enzyme compositions according to claim 7 or 8, characterized in that It can utilize flavonoid oxygen glycoside compounds such as luteolin and apigenin-7-O-glucoside to prepare flavonoid carbon glycoside compounds such as isoorientin, orientin, vitexin and isovitexin.

10. Use of the series of enzyme compositions according to claim 7 or 8, characterized in that The invention can prepare different types of flavonoid carbon glycoside compounds by utilizing the mutual conversion between flavonoid carbon glycoside compounds such as orientin, orientin, vitexin and isovitexin.

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