The role of carboxylic acid groups in the action of glucoamylase I

Gray, C.J.; Jolley, Michael E.

doi:10.1016/0014-5793(73)80560-5

Cited by 15 publications

(5 citation statements)

References 22 publications

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“…However, both maltose and acarbose were unable to prevent the initial decrease in activity during the treatment with cadxxtiimide. A similar trend was reported by others (17,27,34) and it is suggested that exposed and readily reacting non-catalytic carboxyl groups were modified first while the catalytic grou~s) reacted in the second phase leading to almost complete inactivation. Still, the second-order rate constants for the latter phase were not unusually low, consistent with the suggestion that the rearrangement to N-acylurea is promoted for partially buried O-acylisourea intermediates (38,56).…”

Section: Discussionsupporting

confidence: 87%

“…The time course for the inactivation of glucoamylase GI from A. niger by EAC, EDC and CMC at pH 4.7 in the second type of process is shown in Figure 1. EAC was clearly more reactive than the CMC and EDC which both had been employed in previous studies of fungal glucoamylases (17,27,30,34). Under identical conditions glucoamylase G2 lost the activity at 2.3-fold the rate found for GI (data not shown).…”

Section: Inactivation Of Glucoamylase With Carbodiiinidesmentioning

confidence: 64%

“…36). Among the many carbohydrases studied several reports described the use of carbodiimides in characterization of fungal glucoamylases (17,27,30,34,42). In the present study attempts have been made to distinguish different categories of functionally important carboxyl groups in A. niger glucoamylase and a procedure enabling specific labelling of 2-4 critical residues has been established.…”

Section: Discussionmentioning

confidence: 99%

“…Chemical modifications have suggested that carboxyl groups in the different carbohydrases, a-amylase (33), 13-amylase (28), glucoamylase (17), pullulanase (39), ct-glucosidase (32,61), trehalase (55) and sucrase-isomaltase (7) participate in the catalytic hydrolysis of starch and related compounds. The reaction between Gl and G2 glucoamylase (EC 3.2.…”

Section: Introductionmentioning

confidence: 99%

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Chemical modification of carboxyl groups in glucoamylase from Aspergillus niger

Svensson

Møller

Clarke

1988

Carlsberg Res. Commun.

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Glucoamylases GI and G2 from Aspergillus niger lost the catalytic activity by prolonged treatment with 1 -ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC). A tolal of 23 and 16 carboxyl groups were substituted out of 71 and 54 present in the GI and G2 enzyme, respectively. The kinetics and the pH-dependence of the inactivation were compatible with the existence of one essential carboxyl group with a pK~ 5.5. The inhibitor acarbose (a pseudotetrasaccharide) and the substrate maltose prevented EAC-modification of 4 and 2 carboxyl groups, respectively, with considerable retention of enzyme activity. Following removal of these fig,ands, differenlial labelling of critical carboxyl residue(s) was achieved by means of [3H]EAC.Glucoamylase G2 with 12 EAC groups incorporated, prepared in the presence of acarbose, displayed a two-fold increase of K~ and a slight reduction of V,~ for hydrolysis of starch relative to the unmodified enzyme. Both K~ and Vm~ for hydrolysis of maltose were almost unaffected. In addition, maltose and three inhibitors perturbed the UV absorbance spectrum of this derivative. An altered shape of the acarbose-induced difference spectrum implied substitution of a carboxyl group near a binding tryptophanyl residue at a distance from the catalytic site. Efficient protection by acarbose against only the second part of the biphasic course of inactivation similarly reflected that residues playing different roles in the mechanism of action reacted with EAC.In separate experiments, evidence was obtained for a preferred reaction of ethylenimine with enzymatically important carboxyl groups in glucoamylase.

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Section: Discussionsupporting

confidence: 87%

Section: Inactivation Of Glucoamylase With Carbodiiinidesmentioning

confidence: 64%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Chemical modification of carboxyl groups in glucoamylase from Aspergillus niger

Svensson

Møller

Clarke

1988

Carlsberg Res. Commun.

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“…One essential carboxyl group is located in subsite 2 near subsite 3 (51). Glucoamylase from A. niger also has one or more essential carboxyl groups (52), as does enzyme from A. niger (46) and A. saitoi (53), in which the carboxyl group was also located in subsite 2 or 3. Further studies of the A. niger enzymes has identified four carboxylic acid in the active center: Asp 153, Asp 176, Glu 179, and Glu 180 (54).…”

mentioning

confidence: 99%

Mutagenesis of the active site of glucoamylase from Aspergillus awamori

Sierks

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MATERIALS AND METHODS Enzymes and reagents Plasmid purification, subcloning, and sequencing Construction of vector for mutagenesis Expression and purification of mutant enzymes Determination of kinetic parameters RESULTS DISCUSSION REFERENCES SECTION 2. MUTAGENESIS OF ACTIVE SITE CARBOXYLIC ACID RESIDUES OF Aspergillus avamori GLUCOAMYLASE 58 INTRODUCTION 59 MATERIALS AND METHODS 62 Enzymes and reagents 62 Cassette mutagenesis 62 RESULTS 64 DISCUSSION 69 REFERENCES 74 f ill SECTION 3. MUTAGENESIS OF Aspergillus avamori GLUCOAMYLASE RESIDUES BASED ON FUNCTIONAL HOMOLOGIES 76

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Chemical stabilization of glucoamylase from Aspergillus niger against thermal inactivation

Lenders

Crichton

1988

Biotech & Bioengineering

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The applicability of crosslinking an enzyme to an oxidized polysaccharide by reductive alkylation to enhance thermostability has been investigated for glucoamylase from Aspergillus niger. Direct covalent coupling of the enzyme to periodate-oxidized dextran in the presence of NaBH(3)CN results in a conjugate which has thermal properties similar to those of the native enzyme. Our working hypothesis postulates that enhancement of thermostability will result from rigidification of the protein's conformation subsequent to the formation of multiple covalent bonds between the protein and the support. On the basis of the known characteristics of glucoamylase from Aspergillus niger, it would seem necessary to introduce additional amino groups in the polypeptide chain of the protein. The incorporation of new amino groups was performed in two phases. First, the glycosidic part of glucoamylase was oxidized by periodate and the resulting aldehyde groups were reductively aminated by a diaminoalkane and NaBH(3)CIM. Secondly, additional amino groups were introduced on carboxyl functions into the previously aminated glucoamylase by a diaminoalkane and a water-soluble carbodiimide in the presence of maltose to protect the active site. The final derivative was then coupled to periodate-oxidized dextran T-70 in the presence of NaBH(3)CN. Starting with native glucoamylase, three successive operations give rise to a conjugate which retained 27% of the initial activity when measured with soluble starch and 39% when measured with maltopentaose. Using substrates of various sizes, it was observed that steric hindrance at the active site may result from covalent coupling to dextran T-70. It was demonstrated in heat inactivation experiments that the thermostability of the conjugate was in all cases superior to that of the native enzymes. Finally, it was observed that the operational stability of the conjugate was at least twice that of native glucoamylase at 70 degrees C on 18% maltodextrin. Additional experiments rule out the possibility that thermosta-bilization of the complex is due to other reasons than the increase in the amino content of the protein prior to crosslinking. Neither chemical modification, reticulation nor change in the net charge of the protein resulted in a derivative of glucoamylase which presented enhanced thermostability after conjugation. We conclude that for enzymes which have a low content of available amino groups, the thermostabilization method proposed previously by the present authors may still be applicable if additional amino groups are introduced into the protein prior to its crosslinking to an oxidized polysaccharide. This new example reinforces the generality of this method of stabilization.

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The role of carboxylic acid groups in the action of glucoamylase I

Cited by 15 publications

References 22 publications

Chemical modification of carboxyl groups in glucoamylase from Aspergillus niger

Chemical modification of carboxyl groups in glucoamylase from Aspergillus niger

Mutagenesis of the active site of glucoamylase from Aspergillus awamori

Chemical stabilization of glucoamylase from Aspergillus niger against thermal inactivation

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