Study on Purif ication of Invertase from Yeast

Transcription

1 Journal of Food Science and Biotechnology 26 No. 4 Vol. J ul : (2007) ,, (,, ) : SDS 3,50 % g Mono Q, 3, 5. 6,SDS2PA GE 60 kd Km mol/ L 3, SDS SDS,, : ; ;; :Q ; TS : A Study on Purif ication of Invertase from Yeast L I Nan, ZHUAN G Su2xing, DIN G Yi ( School of Life Sciences, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing , China) Abstract :This paper used t hree met hods to extract invertase f rom yeast, which included in t his manuscript, t hree different ext ractio n met hod breaking cell s by adding met hylbenzene, f ro st grinding, and adding SDS for ext racting invertase f ro m yeast were investigated. Then t he p urified invertase was o btained by p recipitatatio n wit h 50 % et hyl alco hol and Mo no Q chromatograp hy. The molecular weight of t he enzyme was 60kD by SDS2PA GE, and the p I was 5. 6 by isoelect ric focusing. The invertase followed t ypical Michaelis2Menten Kinetics wit h apparent Km of mol/ L. The result s showed all t hree met hods had bot h advantages and disadvantages. The invertase ext racted by adding SDS and f ro st grinding had much more total activit y t han t hat of ext racted by adding met hylbenzene. A highest total invertase activit y was fo und in t he SDS ext ractio n p rocess, and it was a co nvenient and eco no mical met ho d for commerical production of invertase f rom yeast. Key words : yeast ;invertase ;extraction ;p urification ( Sucrase, EC ) ( Invertase) 21,2, D2 D2, , [1 ],,,, : : (19822),,,. : (19542),,,,. edu. cn

2 84 26, [2 ] [ 128 ], SDS 3,SDS,,, , ; MonoQ (10/ 10) Phast Gel IEF 329, Amersham 1. 2 (SDS),Serva ;,Amersham ; UV22201,Shimadzu ; Waters 650, Wa2 ters ; Phast System,Amersham ;, Hitachi ;,Labconco ;,Bio2Rad ;,Orion 1. 3 [ 9 ] 015 ml, 013 ml p H mol/ L NaAc2HAc, 012 ml 011 mol/ L,37 15 min,01125 ml 1 mol/ L NaO H DNS [10 ] 540 nm, DNS 1 ma 1 (U) [11 ] 114 Lowry [10 ],, 115 SDS2PAGE [ 12 ], 12 %,5 % 116 Phast System Amersham g,20 ml,214 g NaAc 415 ml, 10 min,37 69 h 418 ml 4 mol/ L HAc 15 ml, p H 415, r/ min 30 min,, g,20 ml,,25 min, 15 min (), 25 min, 3 h,, 1 mol/ L HAc p H 510,40 30 min, r/ min 15 min,, SDS 10 g,60 ml 013 mmol/ L SDS,40 12 h, r/ min 15 min,,4 118 SDS 30 %,4, r/ min 15 min,, 50 % 4 1 h, r/ min 15 min,,,4 SDS, 50 % (p H mol/ L Tris2 HCl ),4 [ 13 ] 119 MonoQ SDS, MonoQ (10/ 10) ( p H mol/ L Tris2HCl ), 0 1 mol/ L 60 min NaCl (p H mol/ L Tris2HCl ) 015 ml/ min, 3 ml, 280 nm,,, ,

3 4 : 85 1, SDS,, 1 3 Tab. 1 Comparing the total activities of invertse from yeast with three different extraction methods / U SDS SDS 2, 013 mmol/ L 019 mmol/ L 115 mmol/ L 30 mmol/ L SDS, 013 mmol/ L SDS,,, 2 SDS Tab. 2 Comparing the extraction eff iciency of invertase at SDS / (mmol/ L) different SDS concentration / mg / U / SDS SDS 50 %,, MonoQ, (1 2),,, MonoQ,, SDS 3 SDS,50 % 8615 % MonoQ, SDS , 4 1 MonoQ Fig. 1 MonoQ chromatography of invertase extracted by frost grinding 2 MonoQ SDS Fig. 2 MonoQ chromatography of invertase isolated by SDS extraction method 3 SDS Tab. 3 A summary of purif ication of invertase isolated by / mg / U / frost grinding and SDS extraction method SDS / % MonoQ SDS Tab. 4 Purif ication results of MonoQ chromatography of in2 vertase extracted by frost grinding and SDS extractoin method / mg / U / SDS

4 86 26 (3),, 3 Fig. 3 Isoelectric point determination of invertase from yeast on PhastGel ( ph3-9) 214 Km 5 20 mmol/ L p H min Km mol/ L (4) 4 Lineweaver2Burk Fig. 4 Lineweaver2Burk plot of the invertase (3), SDS2PA GE 60 kd (5) 3, SDS 3 (1), 5 SDS2PAGE Fig. 5 Molecule Weight determination of invertase from yeast by SDS2PAGE SDS, ,, SDS, (2),SDS,,, SDS SDS 013 mmol/ L,, 3,SDS,2,,3,SDS, SDS Km,,01013 mol/ L (2) [7-8 ] 516,[5 ], SDS2 PA GE, 60 kd, [5-6 ] 4,3,, SDS

5 4 : 87,,, ( References) : [1 ],. [J ].,1997, 18 (2) : CH EN Bing, L IN Xuan. Studies on hydrolysis of sucrose by sucrase[j ]. Journal of Zhanjiang Normal College, 1997, 18 (2) : (in Chinese) [2 ],,. [J ].,2002, 1 (23) : SUN Guo2zhi, FEN G Hui2yong, XU Qin2min. Studies on extracting method of invertase f rom yeast[j ]. Science and Tech2 nology of Food Industry, 2002, 1 (23) : (in Chinese). [3 ],. [J ].,1999, 15 (1) : FAN Ye2peng, DU Peng. The kinetics of activation of yeast invertase by ascorbic acid[j ]. Chinese Journal of Biochemistry and Molecular Biology, 1999, 15 (1) : (in Chinese) [4 ],. [J ].,2002,21 (3) : XU Pei2ya, Q IU Le2quan. Studies on method development of invertase Ion exchange chromatogram purification[j ]. Labo2 ratory Research and Exploration, 2002,21 (3) : (in Chinese) [ 5 ] Belcarz A, Ginalska G, Lobarzewski J, et al. The novel non2glycosylated invertase f rom Candida utilis[j ]. Biochimica et Biophysica Acta, 2002, 1594 : [6 ] Francisco P Ch vez, L uis Rodriguez, Joaqu n D az, et al. Purification and characterization of an invertase f rom Candi da utilis :comparision with natural and recombinant yeast invertases[j ]. Journal of Biotechnology, 1997 : [7 ],,. [J ].,1996,22 (1) : TAN G Yun2ming, L I Nan, CH EN Ding2f u. Isolation, purification, and some properties of invertase f rom sweet [ J ]. Patato Acta Phytophysiologica Sinica, 1996,22 (1) : (in Chinese) [8 ]. [J ].,1995,37 (8) : TAN G Yun2ming. Isolation, purification, and some properties of invertase f rom leaves of mandarin orange[j ]. Acta Botan2 ica Sinica, 1995, 37 (8) : (in Chinese) [9 ],,. Km [J ].,1999,17 (4) : XU Hua, L U Shan2hua, SUN Ai2min. Determination of Nanjing,1999,17 (4) : Km of invertase from yeast [ J ]. Acta Universitatis Medicinalis [10 ]. [ M ]. :,1980 :22-24, [11 ],. [J ].,1999,18 (3) : GUAN Bin, DIN G You2fang. The modification of the DNS method for the determination of reducing sugar[j ]. Journal of WuXi University of Light Industry, 1999,18 (3) : (in Chinese) [ 12 ] Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T41[J ]. Nature,1970,227 : [13 ],,. [J ]. :,2004,23 (2) :1-4. ZH EN G Xue2ling, L I Li2min, YAO Hui2yuan. The preparation, f ractionation, purification and composition analysis of wheat bran water2soluble pentosans[j ]. Journal of Wuxi University of Light Industry,2004,23 (2) :1-4. (in Chinese) ( :,)