alpha-CYCLODEXTRIN (JECFA Food Additives Series 48)

The stability of a biocatalyst is an important factor in industrial applications. Cyclodextrin glucanotransferases are available from both mesophiles and extremophiles (Table ) allowing selection of a CGTase with an appropriate thermostability. Highly stable CGTases, however, display greater hydrolytic activity on starch than their less stable counterparts (Kelly et al. ). This may result in lower CD yields as hydrolytic products stimulate the degradation of CDs in the coupling reaction. The stability of enzymes with otherwise beneficial properties can be enhanced via protein engineering (Eijsink et al. ), but there is only one report were mutagenesis was used to improve the temperature stability of a CGTase. The stability of Bacillus circulans 251 CGTase was raised by engineering a salt bridge on the surface of the B-domain (Leemhuis et al. ). Other site-directed mutagenesis and directed evolution studies have revealed that engineering of CGTases for reaction specificity, generally, delivers variants with reduced thermostability (Kelly et al. ).

Response surface methodology to evaluation the …

06/10/2005 · Doctoral Thesis

and studies on optimization of CGTase production from B

Closely related to the chemical modification method is the immobilization of enzymes onto particles to facilitate the recovery of the expensive biocatalyst from product streams for reuse of the expensive biocatalyst. In addition, immobilization usually stabilizes the biocatalyst under industrial settings. A small number of reports have described the immobilization of CGTases covalently to supports, such as Eupergit C (Martin et al. ) and glyoxyl-agarose (Ferrarotti et al. ), or by entrapment in sodium alginate beads (Arya and Srivastava ). Covalent immobilization is, generally, more favorable as the biocatalyst is not leaking away, but unfortunately, this typically reduces the activity of CGTase to below 10% due to the inaccessibility of a large portion of the immobilized enzyme for the polymeric substrate. One report describes an alternative strategy to facilitate the recovery of biocatalyst, namely, entrapping bacterial cells that display CGTases on the surface in polyvinyl-cryogel beads (Martins et al. ). Chemical modification processes can, therefore, provide a means of improving the application range of CGTases in industry.

The objective of the thesis was to get a deeper genetic and ..

Cyclodextrin glycosyltransferase (CGTase) is a distinctive enzyme that has the capability of producing cyclodextrin (CD) from starch. The CD as the product of CGTase has numerous applications in various industries such as foods, cosmetics and toiletries, textiles and agrochemistry. Therefore, CGTase is considered as an industrially important enzyme and its production improvement is very crucial. So,essential efforts to increase its activity are desirable. CGTase production has never been investigated in Generally Regarded as Safe (GRAS) organism, Lactococcus lactis despite its advantages. The CGTase biosynthesis by recombinant Lactococcuslac tis NZ:NSP:CGT using different carbon sources ((corn starch), potato (dextrin from starch), tapioca starch and several soluble potato starches) and nitrogen sources (yeast extract, meat extract, peptone from meat, peptone from soymeal and peptone from casein) was carried out in batch cultivation using 250 mL shake-flask. Statistical optimization was performed using artificial neural network technique in order to optimize the culture condition (temperature) and medium compositions (carbon and nitrogen sources concentrations) to achieve maximum CGTase production. The experimental data from the aforementioned fermentation experiments were analyzed in order to obtain the kinetic parameter values and establish the basis of a kinetic model. The optimum parameters obtained were used to run batch fermentation in a 2L stirred tank bioreactor. The best carbon source leading to maximum CGTase biosynthesis was determined as Nacalai Tesque GR soluble potato starch. The maximum CGTase activity and productivity obtained by this carbon source were 7.99 U/mL and 1 U/mL.h, respectively. Yeast extract (Merck) was selected as the best nitrogen source due to its highest CGTase activity (9.88 U/mL) and productivity (0.99 U/mL.h) obtained. In screening stage of CGTase fermentation, carbon source concentration, nitrogen source concentration and temperature were recognized as three significant fermentation parameters. The optimum values for these parameters were determined through statistical optimization as 20°C for temperature and 3.82 and 5.67% (w/v) of soluble starch and yeast extract concentrations, respectively. The maximum CGTase activity obtained using the optimum values was 22.09 U/mL, which was closed to the predicted value (24.17 U/mL). The models used in this study were based on unstructured model equations including logistic and Luedeking-Piret, which were suitable to explain the growth, substrate consumption and CGTase production by L. lactis NZ:NSP:CGT in batch cultivation. According to the results, CGTase production is a growth-associated process. Production of CGTase in 2L stirred tank bioreactor (15.36 U/mL) was lower than shake-flask, which shows the essential optimization studies in bioreactor scale.

of stevioside to improve the edulcorant quality by lower substitution using cornstarch hydrolyzate and CGTase Li, Sha et al
The recombinant alpha-CGTase was purified to homogeneity through either nickel affinity chromatography or a combination of ion-exchange and ..

Research On Kinetics And Fermentation ..

Biocatalysis is, generally, regarded as an environment friendly technology. Nevertheless, CD synthesis may become even more environmental friendly if CGTase variants can be obtained, either via screening or protein engineering, that effectively convert native starch granules into specific CDs. The long standing aim of CGTase engineering is to construct variants producing a single type of CD. We feel that the current developments in the understanding of CGTase structure/function and advances in CGTase protein engineering will allow the creation of such a desirable catalyst in the coming years, employing combinatorial site-saturation mutagenesis. Moreover, new insights may lead to the design of CGTases forming high amounts of CDs consisting of 9 or more glucose monomers or CGTase variants that are capable of glycosylating, a wide variety of molecules bearing a hydroxyl function.

Amiri, Azin (2014) Improvement of cyclodextrin glycosyltransferase biosynthesis by recombinant Lactococcus lactis NZ:NSP:CGT. Masters thesis, Universiti Putra …

Engineering of cyclodextrin glucanotransferases and …

In a study on the differential effects of cyclodextrins on human erythrocytes , -, -, and -cyclodextrin were incubated at increasing concentrations with erythrocytes for 30 min. The cyclodextrins induced a change in the shape, from discocyte to spherocyte, but with -cyclodextrin haemolysis occurred before the change was complete. -Cyclodextrin, at a concentration of 1 mmol/L led to a significant release of cholesterol and protein from the erythrocytes, whereas -cyclodextrin at this dose led to release of phospholipid and not of cholesterol or protein (Ohtani et al., 1989).The effects of the cyclodextrins on P388 murine leukaemia cells were examined by exposing the cells to increasing concentrations of cyclodextrins for 48 h in a medium containing 10% fetal calf serum. Initial cytotoxicity was elicited at 11 mmol/L -cyclodextrin, 2.5 mmol/L -cyclodextrin, and 55 mmol/L -cyclodextrin (Leroy-Lechat et al., 1994).In a study of the effects of cyclodextrins on the luminescence of an suspension, the concentrations of natural and chemically modified cyclodextrins required to reduce the luminescence by 20% and 50% were determined as an indication of their cytotoxicity. - and -Cyclodextrin interfered minimally with the bacterial luminescence and consequently were essentially non-toxic (Bär & Ulitzur, 1994).In a human cell suspension culture system that exhibits ciliogenesis, the ciliary beat frequency remained stable to -cyclodextrin at 10% (w/v) and -cyclodextrin at 2% (w/v) after 30 min of exposure. At 5–10% (w/v), however, -cyclodextrin caused mild to severe inhibition after 45 min. The effect was partially reversible (Agu et al., 2000).In a study to examine whether cyclodextrins act as carriers for peptides, the digestion and absorption of two model peptides, glycosylated calcitonin and octreotide, was examined in a human colorectal carcinoma cell line (Caco-2) and in rats . Some evidence of enhancement of absorption was seen but not with -cyclodextrin at 1% (w/v). In contrast, -cyclodextrin enhanced absorption in both systems. To examine the effect of cyclodextrins on the integrity of tight junctions between cell monolayers, similar tests were performed with PEG-4000. -Cyclodextrin had a greater effect on permeation than -cyclodextrin. The results suggest that the absorption-enhancing effect of cyclodextrins depends on the compounds and the cyclodextrin tested (Haeberlin et al., 1996). In another study with the Caco-2 cell culture system, the effect of -cyclodextrin was examined on the permeability of the membrane to mannitol. Enhancement was observed only at a concentration of 5%, and no effect was seen at 0.1 or 1% (McAllister & Taylor, 1999). In a further study with the Caco-2 cell culture system, the effect of cyclodextrins on absorption of PEG-4000 was examined. No effect was seen at concentrations up to 5% (w/v), in contrast to the previous study. It was concluded that -cyclodextrin does not open tight junctions of Caco-2 monolayers (Hovgaard & Brondsted, 1995). In order to examine the effects of - and -cyclodextrin on intestinal absorption, the absorption of sulfanilic acid, a non-absorbed drug, was studied in ligated loops of rat small intestine . The experiments were performed in the presence or absence of -acetyl-L-cysteine which removes the mucus that covers the intestinal mucosa. After perfusion with 10 mmol/l of - and -cyclodextrin, absorption of sulfanilic acid was not enhanced. However, when -cyclodextrin was given in combination with a-acetyl-L-cysteine, there was a significant increase in the absorption of sulfanilic acid. -Cyclodextrin in combination with -acetyl-L-cysteine had no effect. Analysis of the perfusate after removal of the mucous layer indicated that -cyclodextrin promoted the release of phospholipids, while -cyclodextrin promoted the release of cholesterol. The increased permeability was considered to occur via transcellular rather than paracellular pathways (Nakanishi et al., 1992). The gene coding for cyclodextrin-glycosyl transferase (CGTase) is derived from a strain of the M5a1 (formally M5a1 is a gram-negative rod, faculative anaerobe which belongs to the family Enterobacteriaceae and is found in the faecal microflora of 30–40% of persons. M5a1 is considered to be non-pathogenic; it has a history of safe use and a negligible capacity to persist in humans. After the gene coding for CGTase had been isolated from this strain and characterized, it was cloned in pHE3, isolated again, and inserted in the expression vector pJF118EH. PJF11EH is derived from PBR322, a widely used vector which is considered to be safe. The cloned DNA fragment of contained only the CGTase gene, as demonstrated by sequence analysis (Bender, 1977; Henneke et al., 1982; Fürste et al., 1986). A strain derived from K12 was used as the host for the CGTase expression and secretion vector. K12 is considered to be a non-pathogenic strain and is used for production of enzymes for food use. For the production of CGTase, the recombinant strain was cultured in a standard medium. The pH of the culture broth was adjusted by the addition of food-grade ammonia or phosphoric acid. The was grown to a certain density, after which enzyme production was initiated by addition of isopropyl thiogalactoside and the supernatant filtered and concentrated to the crude CGTase preparation. A CGTase preparation derived from K12 but differing in the source organism of the gene coding for CGTase () was considered previously by the Committee (Annex 1, reference 137). In a 3-month study in rats receiving the enzyme in the diet, there was no evidence of treatment-related effects. In a study of mutagenicity in strains TA1535, TA1537, TA98, and TA100, CGTase had no mutagenic activity. -Cyclodextrin is used as a carrier for flavours, colours, and sweeteners in foods such as dry mixes, baked goods, and instant teas and coffee, as a stabilizer for flavours, colours, vitamins, and polyunsaturated fatty acids in dry mixes and dietary supplements (

Therefore, these organisms secrete several starch degrading enzymes of which cyclodextrin glycosyltransferase (CGTase) is an example. Zie: Summary in "thesis"

Modifying enzyme activity and selectivity by immobilization ..

The gene coding for cyclodextrin-glycosyl transferase (CGTase) is derived from a strain of the M5a1 (formally M5a1 is a gram-negative rod, faculative anaerobe which belongs to the family Enterobacteriaceae and is found in the faecal microflora of 30–40% of persons. M5a1 is considered to be non-pathogenic; it has a history of safe use and a negligible capacity to persist in humans. After the gene coding for CGTase had been isolated from this strain and characterized, it was cloned in pHE3, isolated again, and inserted in the expression vector pJF118EH. PJF11EH is derived from PBR322, a widely used vector which is considered to be safe. The cloned DNA fragment of contained only the CGTase gene, as demonstrated by sequence analysis (Bender, 1977; Henneke et al., 1982; Fürste et al., 1986). A strain derived from K12 was used as the host for the CGTase expression and secretion vector. K12 is considered to be a non-pathogenic strain and is used for production of enzymes for food use. For the production of CGTase, the recombinant strain was cultured in a standard medium. The pH of the culture broth was adjusted by the addition of food-grade ammonia or phosphoric acid. The was grown to a certain density, after which enzyme production was initiated by addition of isopropyl thiogalactoside and the supernatant filtered and concentrated to the crude CGTase preparation.