2 ( EC ) 2D2

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1 Journal of Huazhong Agricultural University Vol. 29 No. 2 Apr. 2010, , PCR lac4,p ET2 28a ( + ),BL21, ( ) U/ ml Ni2Resin HP,SDS2PA GE, 122 ku, 43, min, p H 7. 0, p H ,Ca 2 + Zn 2 + Fe 2 + Co 2 + Mg 2 + Mn 2 +, ( ) U/ mg, Km mmol/ L,vmax mmol/ (min mg) ; ; ; Q A (2010) ( EC ) 2D2,,,, ;p ET228a ( + ), p H, D H5 BL21 ;DNA, p H, Omega ;pmd182t [1 ] Ta KaRa ; Fermentas [2 ] ; ;Ni2Resin HP ; ;, ; [3 ],, ( Kl uy verom yces l actis) l ac4, p ET228a ( + ), Ni2Resin HP ( GenBank M84410),, 5 S al, 5, X ho ( 5 A TA GTCGACA GA T2 : ; :2009Ο06Ο28 3 (2004C12010) 3 3. E2mail : hzau. edu. cn,,1982,. :. E2mail : hzau. edu. cn T4 DNA T aq DNA DNA [2 ]2 / GTCT T GCCT TA T TCCT GA 3, 5

2 A TACTCGA GT TA T TCAAAA GCGA GA TCAAAC 3 ), DNA, T aq DNA ON P G 1 mol ON P PCR ( :94 4 min ;94 1 min, 43 p H (U) 50 1 min,72 3 min,35 ;72 10 min), pmd182t 1) p H ON P G S al X ho pmd182t2l ac4, p H (0. 1 mol/ L Na2 HPO42 Na H2 PO4,p H 6. 0 p H 6. 3 p H 6. 6 p H 3 kb l ac p H 7. 3 p H 7. 6 p H 8. 0 ; 0. 1 mol/ L p ET228a ( + ) 2,p H 9. 0), p ET228a2l ac4. 2, 37, p H, p H p ET228a2l ac4 BL21, LB, 2) p H r/ min ; 1 % 0. 1 mol/ L ON P G, LB,,, D600 nm ( 2. 5 h) ; IP T G ( mmol/ L, ), 4. 5 h ; r/ min 1 min, PBS (137 mmol/ L NaCl,2. 7 3) p H p H 3. 0 mmol/ L KCl, 10 mmol/ L Na2 HPO4, 2 mmol/ L mol/ L 2 ;p H KH2 PO4,p H 7. 4), ; r/ min 0. 1 mol/ L ; p H 20 min, SDS2PA GE, mol/ L 2 IP T G, 60 min, p ET228a ( + ) p H 7. 0, 43 p H 7. 0,,p H Ni2Resin H P 4) min,5 000 r/ min 1 min min, ; 3 PBS 43 p H min,, 3 ; 2,, ( mmol/ L ;5 % ;1 PBS ) 5) min,,sds2pa GE p H 7. 0, SDS2PA GE (, PBS K + Cu 2 + Cd 2 + Fe 3 + Ca 2 + Zn 2 + Fe 2 + Co h, Mg 2 + Mn 2 + ) 2 mmol/ L,, Bradford Assay,, [ 4 ] 6) Km vmax 43 p H ml 0. 9 ml 0. 4 % 2, ON P G ( D2 (ON P G),43 5 min, mmol/ L), 1 ml 1 mol/ L Na2 CO3,, Lineweaver2Burk 420 nm, Km vmax

2 : 177 ( 321 2 3), 2. 1494 DNA PCR, 117. 6 ku, 3 kb DNA ( 1), (3 078 bp), pmd182 T S al X ho,, 3 kb DNA ( 2), ( 322) ( 321) 2, pmd182t M. DNA DNA marker ; 1. lac4 PCR PCR product of lac4. 1 PCR Fig.

3 2 : 177 ( ), DNA PCR, ku, 3 kb DNA ( 1), (3 078 bp), pmd182 T S al X ho,, 3 kb DNA ( 2), ( 322) ( 321) 2, pmd182t M. DNA DNA marker ; 1. lac4 PCR PCR product of lac4. 1 PCR Fig. 1 Gel electrophoresis analysis of PCR product of lac4 gene IP T G p ET228a ( + ), 122 ku ( ), l ac4 BL21, 41, 4. 5 ku, 122 ku, 2, Ni2Resin H P, SDS2PA GE, 122 ku ( 32P2LAC),, Bradford Assay, g/ ml, ( ) U/ ml, ( ) U/ mg M. DNA DNA marker ; 1. pmd182t2lac4 S al and X ho pmd182 T2lac4 digested wit h S al and X ho ; 2. p ET228a2lac4 S al and X ho p ET228a2lac4 digested wit h S al and X ho. 2 Fig. 2 Identification of recombinant plasmid with restrict dige stion 2 p ET228a2l ac4 BL21, IP T G, M. Protein marker P2LAC. Ni 2 + Purified recombi2 nant lactase by Ni 2 + 2chelating chromatography ; 1 3. IP T G Supernantant,precipi2 tant,total protein of recombinant strain with IPTG inducement ; 4. IP T G Total protein of recombinant strain wit hout IP T G; 5 6. IP T G IP T G BL21/ p ET228a ( + ) Total protein of BL21/ p ET2 28a ( + ) recombinant strain induced with IP TG and without IPT G,respectively. 3 SDS2PAGE Fig. 3 SDS2PAGE analysis of recombinant lactase 1) p H p H SDS2PA GE, p H,

4 p H 7. 0, p H 7. 0 ( 4) p H %, p H 3. 0 p H 11. 0, ( 4) Co 2 + Mg 2 + Mn ph ph, % % Fig. 4 The ph optimum and ph stability % % % %, K + graph of the recombinant lactase,cd 2 + Fe 3 + 2),, % %, Cu 2 +,41 45, 29 51,, % 43 ( 5) 6 Fig. 6 The thermostability graph of the recombinant lactase 0, 37, 30 min, ) ( 7) (C K),Ca 2 + Zn 2 + Fe CK; 2. Cu 2 + ; 3. Cd 2 + ; 4. Fe 3 + ; 5. K + ; 6. Ca 2 + ; 7. Zn 2 + ; 8. Fe 2 + ; 9. Co 2 + ; 10. Mg 2 + ; 11. Mn Fig. 5 The temperature optimum graph Fig. 7 Effect of metallic ions on activity of lactase of the recombinant lactase 4) Km vmax, ,, ( 6) min,, 50 %, 43 50, ( 8), Y =,43 2 min 30 %, X , R 2 = , Km = min 50 2 min mmol/ L, vmax = mmol/ (min mg)

5 2 : Fig. 8 The curve of 1/ v to 1/ [ S ], 5 L,6 mg/ ml, U/ ml [11 ], U/ ml, 0. 66,, mg/ ml [12 ] ( A s pergill us ni ger) [ ] ( Kl uy verom yces l actis) [ ],,, p H 7. 0, p H (p H ) ( Paenibacill us) ( B acill us,, stearot hermop hil us) ( T hermotoga m a2 riti m a) ( Py rococcu f uriosus) ( B acill us ci rcul ans) ( L acto2 p H,, bacill us aci dop hil us) ( R hi z obi um me2 liloti) ( Fl avobacteri um) ( ) U/ ml,,,,, U/ ml,, U/ ml [5 ] U/ ml, U/ ml [6 ] ( ) U/ ml,, ( bgab) 100 U/ mg [ 7 ], U/ mg [8 ] ( ) U/ mg,,,,, [9 ], [10 ], ( A s pergill us ni ger) ( B acill us ci rcul ans) ( A s pergill us can di d us ) ( A licyclobacill us aci docal dari us), [ 1 ] DOMIN GU ES L, TEIXEIRA J A, PEN T TILA M,et al. Con2 struction of a flocculent S accharomyces cerevisiae strain secre2 ting high levels of A s pergillus ni ger 2galactosidase [J ]. Appl Microbiol Biotechnol,2002,58 : [ 2 ] T HU H N,BARBARA S,STANIMIRA K,et al. Characteriza2 tion and molecular cloning of a heterodimeric 2galactosidase from t he probiotic strain L actobacillus aci dophilus R22 [ J ].

6 FEMS Microbiol Lett,2007,269 : [ 3 ],,. [ 10 ] TOSH O R,BRUNO M,OLL E H,et al. The methylotrophic [J ].,2008,48 (7) : [ 4 ] BRADFORD M M. A rapid and sensitive met hod for t he quan2 titation of microgram quantities of protein utilizing the princi2 ple of protein2dye binding [ J ]. Anal Biochem, 1976, 72 : [ 5 ],,,. [J ].,2003,11 (1) : [ 6 ],,,. Paenibacillus sp. K1 2 [J ]., 2008,43 (6) : [ 7 ],,,. 2 [J ].,2008, 36 (10) : [ 8 ] MADEL IN E L, PA TRICK V,L IAM F,et al. Purification and some characteristics of a recombinant dimeric Rhizobium me2 liloti 2galactosidase expressed in Escherichia coli [J ]. Enzyme and Microbial Technology,2001,28 : [ 9 ],,,. 21,42 [J ].,2008,27 (5) : yeast Pichia pastoris as a host for the expression and produc2 tion of thermostable xylanase from the bacterium R hodother2 mus marinus[j ]. FEMS Yeast Research,2005,5 : [ 11 ],,. [J ].,2005,45 (2) : [ 12 ]. [ D]. :,2007. [ 13 ] DOMIN GU ES L,ONN ELA M L, TEIXEIRA J A,et al. Con2 struction of a flocculent brewer s yeast strain secreting A s per2 gillus ni ger 2galactosidase [ J ]. Appl Microbiol Biotechnol, 2000,54 : [ 14 ] MANU EL B, EST HER R B, ESPERANZA C M,et al. Engi2 neered autolytic yeast st rains secreting Kluy verom yces lactis 2 galactosidase for production of heterologous proteins in lactose media[j ]. Journal of Biotechnology,2004,109 : [ 15 ] BECERRA M, DIAZ P S, CERDAN E, et al. Heterologous Kluy verom yces lactis 2galactosidase secretion by S accharo2 myces cerevisiae Letters,2001,23 : super2secreting mutants [ J ]. Biotechnology Expression and Enzymatic Properties of Lactase Gene from Kl uyveromyces l actis in Escherichia coli XU Shun2qing CH EN Xing2zhou CU I L uo2sheng L IAN G Yun2xiang ZHAN G Zhong2ming S t ate Key L aboratory of A g ricult ural M icrobiolog y, H uaz hong A g ricult ural Uni versit y, W uhan , Chi na Abstract The lactase gene ( l ac4) was amplified f rom t he genome of Kl uy verom yces l actis through polymerase chain reaction ( PCR) and t hen cloned into Escherichi a coli expression vector p ET228a ( + ), and t he l ac4 gene was successf ully expressed in Escherichi a coli 21,enzyme activity reached ( ) U/ ml. The recombinant lactase was p urified wit h affinity chromatograp hy technology,t hen ana2 lyzed by SDS2PA GE,it s molecular weight was approximate 122 ku. The optimal reaction conditions of lactase such as temperate,p H were 43 and 7. 0,it s half2life was 30 min at 37. It was very stable on t he condition of p H 4. 0 to10. 0,and can be greatly activated by Ca 2 +,Zn 2 +,Fe 2 +,Co 2 +,Mg 2 +,Mn 2 +,it s specific activity was ( ) U/ mg,t he response constant Km was mmol/ L,the maximum reaction velocity vmax was mmol/ (min mg). Key words Kl uy verom yces l actis ; lactase ; gene expression ; enzymatic properties ( : )

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