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Food Chem ; 305: 125441, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31494499


Samples of granular corn starch were treated with 1,4-α-glucan branching enzyme (GBE) for 20 h using three different methods. These GBE modification methods all increased glycosidic linkage ratio, cyclic glucan content, and proportion of short chains while reducing weight mean molecular weight. The in vitro digestion rates of the modified starches were suppressed. Among these methods, a novel two-stage modification method comprising a 10-h GBE treatment, gelatinization, and a second 10-h GBE treatment, produced samples with the lowest in vitro digestibility. The rapidly digestible starch content was 34.2% lower than that of the control and 18.0% lower than that of the product of one-stage modification with the same duration. Fine structure characterization showed that more cluster structures were proved during the two-stage modification. This two-stage method suppressed the digestibility of corn starch and increased the substrate concentration, showing great potential for the industrial processing of slowly-digestible starchy foods.

Int J Biol Macromol ; 136: 460-468, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31207329


Inhibition of cyclodextrin glycosyltransferases (CGTases) by their product cyclodextrins limits the efficiency of cyclodextrin production. In an effort to produce variants with good activity but reduced product inhibition, six mutants were constructed at position 603 of the CGTase from Bacillus circulans STB01, which exhibits mixed-type product inhibition. In a kinetic analysis, N603I showed reduced noncompetitive inhibition while N603K, N603H and N603R showed increased noncompetitive inhibition. Unexpectedly, N603E and N603D exhibited reductions in both competitive and noncompetitive product inhibition. Noncompetitive product inhibition is closely related to the interaction between the cyclodextrin and the enzyme in maltose binding site 2 (MBS2). Structural models led to the suggestion that there is increased interaction between maltose binding sites 1 and 2 in mutants N603E and N603D, which may have led to the unexpected results. N603D exhibited a 23.9% greater cyclodextrin yield per gram of enzyme than the wild-type, suggesting it has potential for industrial use. Further reductions in product inhibition may be gained through studies of maltose binding site interactions.

Int J Biol Macromol ; 115: 1194-1201, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29733932


The limits that cyclodextrin products impose on their industrial production from starch by cyclodextrin glycosyltransferases (CGTases) are a severe problem. In this paper, mutants at residue Leu600 of the ß-CGTase from Bacillus circulans STB01 were constructed in an effort to decrease the product inhibition exhibited by ß-cyclodextrin. A kinetic analysis of the inhibition of the wild-type and mutant ß-CGTases by ß-cyclodextrin revealed that the mutations do not alter the inhibition mode, which is mixed-type. However, the values of the inhibition constants (Ki and Ki') of the mutants L600I, L600E and L600R are higher than those of the wild-type enzyme, weakening the product inhibition. The mutant L600Y only exhibited a decrease in noncompetitive inhibition, with the value of Ki' increasing by 40%. Comparison of the Km' values and the 3D model structures of the wild-type and mutant CGTases suggests that this decrease in product inhibition is related to a decrease in binding affinity between the product cyclodextrin and the enzyme.

Bacillus/enzimologia , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Mutação , Ciclização , Ciclodextrinas/metabolismo , Ciclodextrinas/farmacologia , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/química , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica