DOI:10.16774/j.cnki.issn.1674-2214.2015.02.001 2015 2 4 44 2,, (, 310014) : pet28b-valg BL21(DE3), 10~40 C ph 5~11,, ph 30 C 8.0 1 mmol/l Ca 2+ Mg 2+ Mn 2+ Co 2+, Cu 2+ Zn 2+, 8 μmol/l 2 μmol/l A, K mb 35.275 μmol/l,v m 0.153 μmol/(l min), A (UDPG), UDPG : ; ; :Q55 :A :1674-2214(2015)02-0001-06 Studies on purification and characteristics of glycosyltransferase from an engineering strain CHEN Xiaolong, LIAN Zhenjing, FAN Yongxian (College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310014, China) Abstract: The recombinant plasmid pet28b -ValG was functionally expressed in E.coli BL21 (DE3) and the overexpressed glycosyltransferase was purified sequentially by nickel affinity chromatography, ultra-filtration desalination and freeze-drying. Then the purified enzyme was characterized and it showed good stability at 10-40 C and ph 5-11. The optimum reaction temperature and ph were 30 C and 8.0, respectively. The metal ions, such as Ca 2+, Mg 2+, Mn 2+ and Co 2+, activated the enzyme activity, while Cu 2+ and Zn 2+ inhibited the enzyme activity instead. The maximum enzyme activity was obtained at substrate and product concentrations of 8 μmol/l and 2 μmol/l, respectively. When sufficient validoxylamine A was added as substrate, the Lineweaver-Burk plot showed that the K mb and V m were 35.275 μmol/l and 0.153 μmol/(l min), respectively. The affinity of validoxylamine A was much better than that of the uridine diphosphate glucose (UDPG) to the glycosyltransferase, which made it necessary to study the supply of UDPG in the process of the whole-cell catalysis. Keywords: glycosyltransferase; purification; enzymatic properties :2014-12-15 : (2012C12003-1) : (1970 ),,,,,E-mail: richard_chen@zjut.edu.cn. 1
44, min, 50 mmol/l ph 8.0, Tris-HCl 3, A B C D E F G H 8, A HCl, 0.05 g/ml,, 200 W, 1 s, 1 s, A A, 3 min, 8 4 C 12000 r/min [1], A, 10 ml;, A A, 5 (ph 8.0,25 mmol/l Tris-HCl), Ni-NTA ; ValG [2] A,, A, 10,, 1 ml/min, 5 mmol/l A A, (ph 8.0,25 mmol/l Tris -HCl,500 mmol/l, NaCl) 250 mmol/l (ph 8.0,25 [3] His-tag, A Millipore ;,, 4 C 5000 r/min, 25, mmol/l Tris-HCl(pH 8.0),,, -20 C 3 μl 50 mmol/l Tris-HCl (ph 8.0) 1 50 mmol/l ph 8.0 Tris- 30 min, Ni-NTA 1.6 cm 20 cm mmol/l Tris-HCl,500 mmol/l NaCl), 1 ml/min,,2 μl 25 mmol/l Tris-HCl(pH 8.0) 1.1 (50 μg/ml),2 μl 1.1.1 pet28b-valg BL21(DE3) (UDPG) ( 8.20 μmol/l),3 μl A (10.44 μmol/l) 10 μl, 30 C 20 min, 100 C 1.1.2 6 min 5 μl HPLC ph :, ; 1 μmol A U Bio-Rad ; 1.2.2 SDS-PAGE Sigma ; 1 ml 12000 r/min 5 ;HPLC min, 1 ml, 20 μl LC-20AT;PCR Bio -Rad S1000 2, 8 min 1.2, 10 μl, 65 V, 1.2.1,4 C 10000 r/min 5 120 V, 2
2,, : 1.2.3 mmol/l Tris-HCl(pH 8.0),2 μl 25 mmol/l 3 μl 50 mmol/l Tris-HCl (ph 8.0) Tris-HCl (ph 8.0) (50,2 μl 25 mmol/l Tris-HCl(pH 8.0) μg/ml),2 μl UDPG ( 8.20 μmol/l), (50 μg/ml),2 μl 3 μl A ( A (UDPG) ( 8.20 μmol/l),3 μl A (10.44 μmol/l) 10 μl, 20 min, 100 C 6 min 5 μl mmol/l Tris-HCl(pH 8.0),2 μl 25 mmol/l 1.2.4 Tris-HCl (ph 8.0) (50 10~60 C, 5 C 30 min, 10 μl 2 μl A ( 10.44 μmol/l),1 1.2.5 ph 3.62 4.82 6.83 μmol/l), 50 mmol/l ph 3 4 5 6 7 8 9 10 (ph 3~4(Na 2 HPO 4 - ) 1.2.9 ph 5~7(Na 2 HPO 4 -NaH 2 PO 4 ) ph 8~9(Tris-HCl), UDPG ph 10~11(Na 2 CO 3 -NaHCO 3 ), 50 13.67 10.25 6.83 μmol/l, A mmol/l, ph ), 1.2.6 ph 50 mmol/l ph 3 4 5 6 7 8 9 10 11, 30 min,10 μl, 5 μl A 2.1 1.2.7 (KCl CaCl 2 1 2 MgCl 2 MnCl 2 CrCl 3 CoCl 2 ZnCl 2 FeCl 2 NiSO 4 Cu- SO 4 AlCl 3 ) 10 mmol/l K + Ca 2+ Mg 2+ Mn 2+ Cr 3+ Co 2+ Zn 2+ Fe 2+ Ni 2+ Cu 2+ Al 3+, 10 μl : 3 μl 50 mmol/l Tris- HCl (ph 8.0),2 μl 25 mmol/l Tris-HCl (ph 8.0) (50 μg/ml), 2 μl UDPG ( 8.20 μmol/l),2 μl A (10.44 μmol/l),1 μl 10 mmol/l ( 1.49 2.98 5.96 7.45 8.95 10.44 11.93 14.91 17.89 μmol/l),, 10~60 C, 5 C 5 μl A 10 μl : 3 μl 50 μg/ml),2 μl UDPG ( 8.20 μmol/l),,, 5 μl μl A ( A 0.08 0.20 0.40 0.80 1.21 1.61 2.01 5 μl A 3.58 4.47 5.96 7.45 8.95 μmol/l, 10 μl, 30 C 20 min,, 5 μl A 100 C 6 min, 5 μl,, A 28.33 μmol/l, UDPG 3.41 4.78 6.83 10.25 13.66 μmol/l, 2 1 mmol/l), 5 1 μl A 1 1.2.8, 10 μl : 3 μl 50 50~80 min, 3
44 6 5 4 3 2 1 M ValG 45 kda 2,,80~110 min, 5 mmol/l, 135~150 min, 250 mmol/l, 10 C,,,, 60 C 8%,, ; 2, 10~40 C, 45 C, 2 [5] Marker, 2.3 ph, ph (100%) SDS-PAGE,BandScan, ph ValG 32%, ph 45 kd,, ph (47 kd),, 4,,,,250 mmol/l, ValG 1862.5 U 2191.2 U, ValG 85%, [4] 2.2 (100%), ( 4 ph, ), 4, ph 8~9, ph 4~8,, ph, ph 3,,, 3 ph 10, 3, ph, ph 11, 30 C, 10~30 C, 9%, ph 8~10,, 4 62.64%, DTG (, DXD ) [6],, 35 C,, 3
2,, : (100%),,DTG, ph,,, 6 7, ph ph, ph 5~11, 80%,,, ph 4, [7] 2.4 (100%), 1 mmol/l,, 5 6 5 5,1 mmol/l Al 3+ Cr 3+ Fe 2+, Ni 2+,Cu 2+ Zn 2+ 90%, K + Ca 2+ Mg 2+ Mn 2+ Co 2+, 150%, ( ( ); 7, 1.5 Mg 2+ ) 234% μmol/l,, 2 μmol/l,,, 167%,,,, 7 μmol/l, [8] 2.5 2.6 A, (100%), :,, 8 9,, 8 A,, 7 6, 1.49~8 μmol/l,,1 mmol/l Cu 2+ Zn 2+ 8 μmol/l,, 18 μmol/l, 5
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