Identification of acceptor substrate binding subsites +2 +3 in the amylomaltase from Thermus thermophilus HB8

Glycoside hydrolase family 77 (GH77) belongs to the α-amylase superfamily (Clan H) together with GH13 and GH70. GH77 enzymes are amylomaltases or 4-α-glucanotransferases, involved in maltose metabolism in microorganisms and in starch biosynthesis in plants. Here we characterized the amylomaltase from the hyperthermophilic bacterium Thermus...

Deglycosylation of ovalbumin prohibits formation of a heat-stable conformer

To study the influence of the carbohydrate-moiety of ovalbumin on the formation of the heat-stable conformer S-ovalbumin, ovalbumin is deglycosylated with PNGase-F under native conditions. Although the enzymatic deglycosylation procedure resulted in a complete loss of the ability to bind to Concavalin A column-material, only in about 50% the...

Three-way stabilization of the covalent intermediate in amylomaltase, an alpha-like transglycosylase

Amylomaltases are glycosyl hydrolases belonging to glycoside hydrolase family 77 that are capable of the synthesis of large cyclic glucans and the disproportionation of oligosaccharides. Using protein crystallography, we have generated a flip book movie of the amylomaltase catalytic cycle in atomic detail. The structures include a covalent...

The novel antifungal agent PLD-118 is neither metabolized by liver microsomes nor inhibits cytochrome P450 in vitro

PLD-118 is a novel, oral antifungal drug, under development for the treatment of Candida infections. Possible metabolism of PLD-118 by rat, dog and human S9 liver homogenates and inhibition of human cytochrome P450 (CYP) enzymes were investigated. PLD-118 (10 and 100 μm) incubated for 0-60 min with S9 fractions and NADPH was determined by HPLC,...

Effects of succinylation on the structure and thermostability of lysozyme

The influence of succinylation on lysozyme is studied using circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. The spectroscopic data reveal that at room temperature the structures of succinylated lysozyme and native lysozyme are similar. However, the calorimetric results show that the thermal stability of...

Mutational analysis of the role of calcium ions in the Lactobacillus reuteri strain 121 fructosyltransferase (levansucrase and inulosucrase) enzymes

Bacterial fructosyltransferase enzymes belonging to glycoside hydrolase family 68 (GH68) are not known to require a metal cofactor. Here, we show that Ca2+ ions play an important structural role in the Lactobacillus reuteri 121 levansucrase (Lev) and inulosucrase (Inu) enzymes. Analysis of the Bacillus subtilis Lev 3D structure [Meng, G. and...

Amylomaltase of Pyrobaculum aerophilum IM2 produces thermoreversible starch gels

Amylomaltases are 4-alpha-glucanotransferases (EC 2.4.1.25) of glycoside hydrolase family 77 that transfer alpha-1,4-linked glucans to another acceptor, which can be the 4-OH group of an alpha-1,4-linked glucan or glucose. The amylomaltase-encoding gene (PAE1209) from the hyperthermophilic archaeon Pyrobaculum aerophilum IM2 was cloned and...

Stabilization versus inhibition of TAFIa by competitive inhibitors in vitro

Two competitive inhibitors of TAFIa (activated thrombin-activable fibrinolysis inhibitor), 2-guanidinoethyl-mercaptosuccinic acid and potato tuber carboxypeptidase inhibitor, variably affect fibrinolysis of clotted human plasma. Depending on their concentration, the inhibitors shortened, prolonged, or had no effect on lysis in vitro. The...

A novel, inducible, citral lyase purified from spores of Penicillium digitatum

A novel lyase, combining hydratase and aldolase activity, that converts citral into methylheptenone and acetaldehyde, was purified from spores of Penicillium digitatum. Remarkably, citral lyase activity was induced 118-fold by incubating nongerminating spores with the substrate, citral. This cofactor independent hydratase/aldolase, was purified...

Properties and applications of starch-converting enzymes of the alpha-amylase family

Starch is a major storage product of many economically important crops such as wheat, rice, maize, tapioca, and potato. A large-scale starch processing industry has emerged in the last century. In the past decades, we have seen a shift from the acid hydrolysis of starch to the use of starch-converting enzymes in the production of maltodextrin,...

Improvement of enantioselectivity by immobilized imprinting of epoxide hydrolase from Rhodotorula glutinis

The yeast Rhodotorula glutinis contains an enantioselective, membrane-associated epoxide hydrolase (EH). Partially purified EH was immobilized in a two-step procedure. In the first step, the proteins were derivatized with itaconic anhydride. In the second step, the derivatized proteins were co-polymerized with ethylene glycol dimethacrylate in...

Differential maintenance of cytochrome P450 enzymes in cultured precision-cut human liver slices

The maintenance of the major hepatic cytochrome P450 (CYP) enzymes has been studied in precision-cut human liver slices cultured for up to 72 h in supplemented RPMI 1640 medium. The relative apoprotein levels of 11 CYP enzymes were determined using a panel of antipeptide antibodes. In addition, 7-ethoxyresorufin O-deethylase, tolbutamide...

The effect of storage of whole potatoes of three cultivars on the patatin and protease inhibitor content; a study using capillary electrophoresis and MALDI-TOF mass spectrometry

The content and biological activity of patatin and the protease inhibitors of molecular size 20-22 kDa present in whole potato tubers were investigated as a function of storage time. The amount of buffer-extractable protein decreased gradually during storage of whole potatoes of the cultivars Bintje and Desiree for 47 weeks whereas, for Elkana,...

Heat-induced conformational changes of patatin, the major potato tuber protein

This paper presents a first structural characterization of isolated patatin, the major potato tuber protein, at ambient and elevated temperatures. Isolated patatin at room temperature is a highly structured molecule at both secondary and tertiary levels. It is estimated from far-ultraviolet circular dichroism data that about 33% of the residues...

Optimization and stability of glucoamylase production by recombinant strains of Aspergillus niger in chemostat culture

When grown on a medium containing 5 g maltodextrin L-1, Aspergillus niger transformant N402[pAB6-10]B1, which has an additional 20 copies of the glucoamylase (glaA) gene, produced 320 ± 8 mg (mean ± S.E.) glucoamylase (GAM) L-1 in batch culture and 373 ± 9 mg GAM L-1 in maltodextrin- limited chemostat culture at a dilution rate of 0.13 h-1....

Isolation of α-amylase on crosslinked starch

The isolation and purification of α-amylase from an industrial enzyme sample is described using crosslinked starch powder as an affinity adsorbent. The process was studied with regard to the stability of the adsorbent, the stability of the enzyme, and the capacity of the adsorbent for the enzyme. The adsorbent was used in a repeated adsorption...