1
22
N. Atanasova et al. / Process Biochemistry 46 (2011) 116–122
15.12 mg ml 1) and 7-fold higher quantity of ␥-CD (3.54 mg ml−1).
Moreover, the starch conversion by the recombinant CGTase is 35%
14] compared to 47% by CGTase presented in this work. The ␥-CD
−
(
[7] Hirano K, Ishihara T, Ogasawara S, Maeda H, Abe K, Nakajima T, et al. Molecular
cloning and characterization of a novel ␥-CGTase from alkalophilic Bacillus sp.
Appl Microbiol Biotechnol 2006;70:193–201.
[
[
8] Biwer A, Antranikian G, Heinzle E. Enzymatic production of cyclodextrins. Appl
Microbiol Biotechnol 2002;59:609–17.
yield achieved by CGTase from alkaliphilic Bacillus sp. 7–12 [17]
was 13.9% compared to 18.7% by the studied CGTase. Significantly
higher CD yield by the present CGTase was also established in com-
parison to that reached by CGTase from alkaliphilic Bacillus clausii
[
9] Alves-Prado HF, Gomes E, da Silva R. Purification and characterization of a cyclo-
maltodextrin glucanotransferase from Paenibacillus campinasensis H69-3. Appl
Biochem Biotechnol 2007;136-140:41–55.
[10] Yim DG, Sato YH, Park YK. Production of cyclodextrin from starch by
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11] Alves-Prado HF, Carneiro AAJ, Pavezzi FC, Gomes E, Boscolo M, Franco CML, et
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starches as substrates. Appl Biochem Biotechnol 2008;146:3–13.
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step purification of cyclodextrin glycosyltransferase from alkalophilic Bacillus
firmus by ion exchange chromatography. Biochemical Eng J 2008;39:510–5.
[
11], namely 2.1-fold and 3.9-fold higher quantity of - and ␥-CD,
respectively. It should be also noted the relatively high quantity of
the formed ␥-CD without any reaction manipulations which is of
an industrial interest. Moreover, B. pseudalcaliphilus 20RF CGTase
could be effectively used in alkaline reaction medium forming only
[
[

- and ␥-CDs with a production ratio 80%:20% (Table 6). Another
advantage of the novel CGTase is its ability to degrade raw starches
and thus the cost involved in gelatinization of the starch can be
avoided. Reports on the application of raw starch for cyclodextrin
production are few [25].
[13] Charoensakdi R, Murakami S, Aoki K, Rimphanitchayakit V, Limpaseni T.
Cloning and expression of cyclodextrin glycosyltransferase gene from Paeni-
bacillus sp. T16 isolated from hot spring soil in Northern Thailand. J Biochem
Mol Biol 2007;40:333–40.
[14] Rahman K, Illias RM, Hassan O, Mahmood NAN, Rashid NAA. Molecular cloning
of a cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. TS1-1
and characterization of the recombinant enzyme. Enzyme Microbial Technol
4
. Conclusion
2006;39:74–84.
[
[
[
15] Ong RM, Goh KM, Mahadi NM, Hassan O, Rahman RNZ, Illias RM. Cloning,
extracellular expression and characterization of a predominant -CGTase from
Bacillus sp. G1 in E. coli. J Ind Microbiol Biotechnol 2008;35:1705–14.
16] Cao X, Jin Z, Chen F, Wang X. Purification and properties of cyclodextrin
glucanotransferase from an alkalophilic Bacillus sp. 7–12. J Food Biochem
2004;28:463–75.
In this study, the purification and characterization of a novel
CGTase from B. pseudalcaliphilus 20RF was reported. The purifica-
tion to homogeneity of the enzyme was achieved by ultrafiltration
and starch adsorption with a recovery of 63% activity and 18-fold
purification. The purified enzyme was monomer and its molecular
weight was estimated to be 70 kDa. The applied purification proce-
dure is easily feasible under industrial conditions. In comparison to
other CGTases obtained from alkaliphiles, the present CGTase from
B. pseudalcaliphilus 20RF could be effectively used for conversion of
raw starch into cyclodextrins in a wide pH range, from 5.0 to 10.0
17] Cao X, Jin Z, Wang X, Chen F. A novel cyclodextrin glycosyl transferase from
an alkalophilic Bacillus species: purification and characterization. Food Res Int
2005;38:309–14.
[18] Doukyu N, Kuwahara H, Aono R. Isolation of Paenibacillus illinoisensis that
produces cyclodextrin glucanotransferase resistant to organic solvents. Biosci
Biotechnol Biochem 2003;67:334–40.
[
19] Atanasova N, Petrova P, Ivanova V, Yankov D, Vassileva A, Tonkova A. Isola-
tion of novel alkaliphilic Bacillus strains for cyclodextrin glucanotransferase
production. Appl Biochem Biotechnol 2008;149:155–67.
20] Laemmli UK. Clevage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 1970;227:680–5.
21] Kaneko T, Kato T, Nakamura N, Horokoshi K. Spectrophotometric determination
of cyclization activity of -cyclodextrin-forming cyclomaltodextrin glucan-
otransferase. J Jpn Soc Starch Sci 1987;34:45–8.
22] Bradford MM. A rapid and sensitive method for the quantitation of micro-
gram quantities of protein utilizing the principle of protein-dye binding. Anal
Biochem 1976;72:248–54.
23] Akimura K, Yagi T, Yamamoto S. Purification and properties of Bacillus coagulans
cyclomaltodextrin glucanotransferase. J Ferment Bioeng 1991;71:322–8.
24] Fujita Y, Tsubouchi H, Inagi Y, Tomita K, Ozaki A, Nakamura K. Purification and
properties of cyclodextrin glycosyltransferase from Bacillus sp. AL-6. J Ferment
Bioeng 1990;70:150–4.
25] Gawande BN, Goel A, Patkar AY, Nene SN. Purification and properties of a novel
raw starch degrading cyclomaltodextrin glucanotransferase from Bacillus fir-
mus. Appl Microbiol Biotechnol 1999;51:504–9.
◦
and temperatures 60–70 C. One of the most important features of
[
the present enzyme was its high activity at pH 6.0 and 9.0 (two pH
optima). Moreover, the enzyme was more heat resistant after pre-
[
◦
treatment in alkaline pH 9.0 at high temperatures 65–70 C, than
at pH 6.0 under the same conditions. The enzyme showed a sig-
nificant stability in the presence of 15 mM various metal ions and
[
◦
reagents after 30 min incubation at 25 C.
[
[
The purified CGTase from B. pseudalcaliphilus 20RF could be
used for an efficient cyclodextrin production without any additives
which is of an industrial interest. The achieved high conversion of
an insoluble raw commercial corn starch into cyclodextrins (47%)
with predominant formation of -CD (80%) and ␥-CD (18.7%) and
[
◦
negligible quantity of ␣-CD (1.3%) after 24 h of reaction at 60 C, pH
6

2
.0 and 47-50% conversion with production of only two types CDs,
- and ␥-CD (80%:20%) in alkaline pH 9.0, makes B. pseudalcaliphilus
0RF CGTase industrially desired for cyclodextrin manufacture.
[26] Martins RF, Hatti-Kaul R. A new cyclodextrin glycosyltransferase from an
alkalophilic Bacillus agaradhaerens isolate: purification and characterization.
Enzyme Microb Technol 2002;30:116–24.
[
27] Sabioni JG, Park YK. Cyclodextrin glycosyltransferase production by alkalophilic
Bacillus lentus. Rev Microbiol 1992;23:128–32.
Acknowledgements
[28] Sian HK, Said M, Hassan O, Kamaruddin K, Ismail AF, Rahman RA, et al. Purifica-
tion and characterization of cyclodextrin glucanotransferase from alkalophilic
Bacillus sp. G1. Proc Biochem 2005;40:1101–11.
This work was supported by the National Scientific Foundation
of Bulgarian Ministry of Education and Science (the grant WUH
[
29] Sin KA, Nakamura A, Kobayashi K, Masaki H, Uozumi T. Cloning and sequenc-
ing of a cyclodextrin glucanotransferase gene from Bacillus ohbensis and its
expression in Escherichia coli. Appl Microbiol Biotechnol 1991;35:600–5.
30] Higuti IH, Grande SW, Sacco R, Jose do Nascimento A. Isolation of
alkalophilic CGTase-producing bacteria and characterization of cyclodextrin-
glycosyltransferase. Braz Arch Biol Technol 2003;46:183–6.
3
4
01/07), program BG051PO001-3.3.04/32 and a bilateral grant P-
3/07 between the Bulgarian and Czech Academies of Sciences.
[
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