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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2016, Vol. 36 Issue (11): 30-38    DOI: 10.13523/j.cb.20161105
    
Saturated Mutation Effects on Catalytic Efficiency and Product Specificity of Starch Binding Site N623 of Cyclodextrin Glucanotransferase from Geobacillus sp. CHB1
GUO Yong hua1,2, CHEN Ji chen1, CAI Hai song1, CHEN Long jun1, LIN Xin jian1,2
1. Soil and Fertilizer Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350003, China;
2. College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China
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Abstract  

In order to improve catalytic efficiency and product specificity of cyclodextrin glucanotransferase (CGTase) from Geobacillus sp. CHB1, acid sequences and simulation structure model were analyzed, found out that the 623th amino acid residues of starch binding sites II probably affected its catalytic efficiency. Using overlapping PCR method, 19 kinds of mtuants on the 623th amino acid residues (N623) of starch binding sites II of CGTase were built. The mutant CGTase genes were respectively linked with pET-28a(+)-ompA and expressed in Escherichia coli BL21(DE3). The recombinant pure enzyme was used to transform soluble starch into cyclodextrins (CDs). HPLC analysis results show that, compared to wild-type CGTase, mutant N623T increases catalytic efficiency of CGTase, the total cyclization activity increased 58.6%, α-cyclization activity increased 64%, β-cyclization activity increased 80.5%, while γ-cyclization activity was reduced by 35.3%. In terms of product specificity, compared to wild-type CGTase, the total starch conversion rate by mutant N623T increased from 11.3% to 39.7%, of which α-cyclodextrin, γ-cyclodextrin proportion reduced to 32.8% and 7.7%, β-cyclodextrin increased to 59.5%. The possible mechanism was that, compared to wild-type CGTase, mutant N623T. Threonine residue in place of asparagine caused conformation of starch binding site II was changed, the conformation optimized substrate acting direction, in favor of the reaction is carried out, thereby improving the catalytic efficiency of the enzyme.

Key words： Product specificity      Saturated mutation      Cyclodextrin glucanotransferase      Catalytic efficiency     
Received: 28 April 2016      Published: 25 November 2016
ZTFLH:  Q812  
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GUO Yong hua, CHEN Ji chen, CAI Hai song, CHEN Long jun, LIN Xin jian. Saturated Mutation Effects on Catalytic Efficiency and Product Specificity of Starch Binding Site N623 of Cyclodextrin Glucanotransferase from Geobacillus sp. CHB1. China Biotechnology, 2016, 36(11): 30-38.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20161105     OR     https://manu60.magtech.com.cn/biotech/Y2016/V36/I11/30

[1] Loftsson T, Duchene D. Cyclodextrins and their pharmaceutical applications. International Journal of Pharmaceutics, 2007, 329(1-2):1-11.
[2] Uitdehaag J C M, Veen B A V D, Dijkhuizen L, et al. Catalytic mechanism and product specificity of cyclodextrin glycosyltransferase, a prototypical transglycosylase from the α-amylase family. Enzyme & Microbial Technology, 2002, 30(01):295-304.
[3] Leemhuis H, Kelly R M, Dijkhuizen L. Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications. Applied Microbiology & Biotechnology, 2009, 85(4):823-835.
[4] Christiansen C, Hachem M A,Jane[AK?]k K Š, et al. The carbohydrate-binding module family 20-diversity, structure, and function. Febs Journal, 2009, 276(18):5006-5029.
[5] Dalmia B K, Schütte K, Nikolov Z L. Domain E of Bacillus macerans cyclodextrin glucanotransferase:An independent starch-binding domain. Biotechnology & Bioengineering, 1995, 47(47):575-584.
[6] Lawson C L, Montfort R, Van, B Strokopytov, et al. Nucleotide Sequence and X-ray structure of cyclodextrin glycosyltransferase from Bacillus circulans Strain 251 in a maltose-dependent crystal Form. Journal of Molecular Biology, 1994, 236(2):590-600.
[7] Sanoja-Romina R, Oviedo N, Sánchez S. Microbial starch-binding domain. Current Opinion in Microbiology, 2005, 8(3):260-267.
[8] Kimura K, Kataoka S, Nakamura A, et al. Functions of the COOH-terminal region of cyclodextrin glucanotransferase of alkalophilic ja:math sp.#1011:relation to catalyzing activity and pH stability. Biochemical & Biophysical Research Communications, 1989, 161(3):1273-1279.
[9] Chang H Y, Irwin P M, Nikolov Z L. Effects of mutations in the starch-binding domain of Bacillus macerans cyclodextrin glycosyltransferase. Journal of Biotechnology, 1998, 65(2-3):191-202.
[10] Veen B A V D, Uitdehaag J C M, Dijkstra B W, et al. Engineering of cyclodextrin glycosyltransferase reaction and product specificity. Biochimica Et Biophysica Acta, 2000, 1543(2):336-360.
[11] Studier F. Protein production by auto-induction in high-density shaking cultures. Protein Expression & Purification, 2005, 41(1):207-234.
[12] Lejeune A, Sakaguchi K, Imanaka T. A spectrophotometric assay for the cyclization activity of cyclomaltohexaose (α-cyclodextrin) glucanotransferase. Analytical Biochemistry, 1989, 181(1):6-11.
[13] Mäkelä M, Korpela T, Laakso S. Colorimetric determination of β-cyclodextrin:two assay modifications based on molecular complexation of phenolphatalein. Journal of Biochemical & Biophysical Methods, 1987, 14(2):85-92.
[14] Kato T,Horikoshi K. Colorimetric determination of.gamma.-cyclodextrin. Analytical Chemistry, 2002, 56(9):1738-1740.
[15] Bradford M M. A rapid method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 1976, 72(s1-2):248-254.
[16] Arnold K, Bordoli L, Kopp J T. The SWISS-MODEL workspace:a web-based environment for protein structure homology modelling. Bioinformatics, 2006, 22(2):195-201(7).
[17] Kopp J, Schwede T. The SWISS-MODEL repository of annotated three-dimensional protein structure homology models. Nucleic Acids Research, 2004, 32(22):230-234.
[18] Zhang Y. I-TASSER server for protein 3D structure prediction. Bmc Bioinformatics, 2008, 9(3):297-315.
[19] 杨冬, 田靖斐, 陈晟, 等. 亚位点-7处突变对碱性芽胞杆菌CGT酶产物特异性的影响. 生物工程学报, 2012, 28(2):191-202. Yang D, Tian J F, Chen S, et al. Effect of mutating subsite-7 on product specificity of cyclodextrin glucanotransferase from alkalophilic Bacillus clarkia. Chinese Journal of Biotechnology, 2012, 28(2):191-202.
[20] Giardina T, Gunning A P, Juge N, et al. Both binding sites of the starch-binding domain of Aspergillus niger glucoamylase are essential for inducing a conformational change in amylose. Journal of Molecular Biology, 2001, 313(5):1149-1159.
[21] Penninga D, Veen B A, Van Der, Knegtel R M, et al. The raw starch binding domain of cyclodextrin glycosyltransferase from Bacillus circulans strain 251. Journal of Biological Chemistry, 1996, 271(51):32777-32784.
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