Studies on purification and characteristics of glycosyltransferase from an engineering strain

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1 DOI: /j.cnki.issn ,, (, ) : pet28b-valg BL21(DE3), 10~40 C ph 5~11,, ph 30 C mmol/l Ca 2+ Mg 2+ Mn 2+ Co 2+, Cu 2+ Zn 2+, 8 μmol/l 2 μmol/l A, K mb μmol/l,v m μmol/(l min), A (UDPG), UDPG : ; ; :Q55 :A : (2015) 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 , 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 C and ph 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 μmol/l and 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 : : (2012C ) : (1970 ),,,,, richard_chen@zjut.edu.cn. 1

2 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 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 pet28b-valg BL21(DE3) (UDPG) ( 8.20 μmol/l),3 μl A (10.44 μmol/l) 10 μl, 30 C 20 min, 100 C min 5 μl HPLC ph :, ; 1 μmol A U Bio-Rad ; SDS-PAGE Sigma ; 1 ml 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 r/min V, 2

3 2,, : 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 Tris-HCl (ph 8.0) (50 10~60 C, 5 C 30 min, 10 μl 2 μl A ( μmol/l), ph μmol/l), 50 mmol/l ph (ph 3~4(Na 2 HPO 4 - ) 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 ), μmol/l, A mmol/l, ph ), ph 50 mmol/l ph , 30 min,10 μl, 5 μl A (KCl CaCl 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 ( 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 μl A μmol/l, 10 μl, 30 C 20 min,, 5 μl A 100 C 6 min, 5 μl,, A μmol/l, UDPG μmol/l, 2 1 mmol/l), 5 1 μl A , 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.

4 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 U 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,, %, DTG (, DXD ) [6],, 35 C,, 3

5 2,, : (100%),,DTG, ph,,, 6 7, ph ph, ph 5~11, 80%,,, ph 4, [7] 2.4 (100%), 1 mmol/l,, ,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] 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

6 44 8,,, : UDPG, A, A, A,, UDPG, UDPG,, [4] LIEBL W, FEIL R, GABELSBERGER J, et al. Purification and, characterization of a novel thermostable 4-alpha-glucanotransferase of Thermotoga maritima cloned in Escherichia coli[j]. Eu-, :1/V=1/V m (1+ K mb /[B] +K ma /[A]), A A,B ropean Journal of Biochemistry, 1992, 207(1): UDPG; [5] RYU S I, PARK C S, CHA J, et al. A novel trehalose-synthesizing glycosyltransferase from Pyrococcus horikoshii: molecular A, 9, cloning and characterization[j]. Biochemical and Biophysical :1/V=1/V m (1+K mb / Research Communications, 2005, 329(2): [B]),1/C (UDPG) 1/V y=230.43x+ [6] BAI Linquan, LI Lei, XU Hui, et al. Functional analysis of the ,R 2 =0.9932, validamycin biosynthetic gene cluster and engineered produc UDPG K mb μmol/l, tion of validoxylamine A[J]. Chemistry and Biology, 2006, 13 (4): V m μmol/(l min), [7] LAIRSON L L, HENRISSAT B, DAVIES G J, et al. Glycosyltransferases: structures, functions, and mechanisms [J]. Annual 8 A K ma μmol/l, Review of Biochemistry, 2008, 77(1): 521., A, [8] MORIWAKI C, COSTA G L, PAZZETTO R, et al. Production UDPG,, and characterization of a new cyclodextrin glycosyltransferase from Bacillus firmus isolated from Brazilian soil[j]. Process Biochemistry, 2007, 42(10): ,UDPG,,, DTG, 6 9 : [1]. [D]. :,2012. [2] SINGH D, SEO M J, KWON H J, et al. Genetic localization and heterologous expression of validamycin biosynthetic gene cluster isolated from Streptomyces hygroscopicus var. limoneus KCCM (IFO 12704)[J]. Gene, 2006, 376(1): [3] LOUGHRAN S T, LOUGHRAN N B, RYAN B J, et al. Modified His-tag fusion vector for enhanced protein purification by immobilized metal affinity chromatography[j]. Analytical Biochemistry, 2006, 355(1): A ( : )

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