Inactivation of glucosyltransferase ofAspergillus niger with subtilisin

Summary Treatment of amyloglucosidase ofAspergillus niger with subtilisin, soluble or immobilized, resulted in an almost complete inactivation of glucosyltransferase. Amyloglucosidase preparations treated with subtilisin converted starch to glucose in high yields, thus confirming this procedure to be a simple and efficient way of removing glucosyltransferase from amyloglucosidase.     

Comparison of the action of glucoamylase and glucosyltransferase on D-glucose, maltose, and malto-oligosaccharides.

The action patterns of glucoamylase (amyloglucosidase) and glucosyltransferase (transglucosylase) on D-[1-14C]glucose, [1-14C]maltose, and [1-14C]malto-oligosaccharides (labeled at position 1 of the D-glucose group at the reducing end) have been investigated by paper-chromatographic and oligosaccharide-mapping techniques. Under the conditions of the experiments, the extent of conversion of D-glucose and of maltose into new oligosaccharides was 2.2 and 1.9% with glucoamylase, and 5.7 and 33% with glucosyltransferase. The major oligosaccharides produced by both enzymes were isomaltose (6-O-alpha-D-glucopyranosyl-alpha-D-glucose), panose (O-alpha-D-glucopyranosyl (1 leads to 6)-O-alpha-D-glucopyranosyl-(1 leads to 4)-alpha-D-glucose), and nigerose (3-O-alpha-D-glucopyranosyl-alpha-D-glucose). The glucosyltransferase also synthesized oligosaccharides from malto-oligosaccharides of higher molecular weight to yield compounds having alpha-(1 leads to 6)-linked D-glucosyl groups at the non-reducing ends. Glucoamylase exhibited little, if any, such activity on malto-oligosaccharides.     

Use of purified amyloglucosidase for the specific determination of total carbohydrate content of rat liver homogenate in a single step.

In prior studies, we employed a crude amyloglucosidase preparation, in conjunction with glucose oxidase reagent, to determine total carbohydrate in liver and muscle homogenates by a two-step procedure. Glycogen content was determined by subtracting tissue glucose (determined separately). By use of a purified amyloglucosidase, we have now verified the accuracy of two-step assay of rat liver homogenate with crude amyloglucosidase. The reliability of glucose oxidase detection of glucose in amyloglucosidase hydrolysates was established by comparing results with those obtained with hexokinase/glucose 6-phosphate dehydrogenase reagent. The availability of purified amyloglucosidase made it feasible to assay total carbohydrate content of homogenate in a single step, by incubating aliquots in the presence of both amyloglucosidase and glucose oxidase reagents. Results obtained with this one-step assay agreed with those of two-step analysis. To our knowledge, this is the first report of direct enzymic assay of total carbohydrate in rat liver homogenate, in a single step.     

Characterization of a protein:glucosyltransferase activity in human platelets

Human platelets exhibited significant glucosyltransferase activity, that transfer [14C]glucose from UDP-Glc to an endogenous protein acceptor. The enzyme protein:glucosyltransferase responsible for the catalysis was characterized and compared with glycogen:glucosyltransferase. We describe a partial separation of both activities, the ratio of protein:glucosyltransferase/glycogen:glucosyltransferase varied from 7:1 in a crude homogenate of platelets to 36:1 in the Sephadex G-100 column. This procedure failed to separate the protein:glucosyltransferase from its endogenous acceptor. Glucosylation of protein demonstrated dependence with respect to time and both protein and UDP-Glc concentration, and was saturated by very low concentration of donor and acceptor substrates. It was inhibited 76% by 5 mM Mn2+ concentration and was activated 23 and 11% by 5 mM concentrations of Ca2+ and Mg2+, respectively. With respect to glycogen:glucosyltransferase, when the effect of time, protein, and substrate concentration were determined under identical conditions, it did not show the same dependence. At 5 mM concentration, Mn2+, Ca2+, and Mg2+ were activators of the enzyme 43, 80, and 200%, respectively. On the basis of these characteristics, we conclude that the synthesis of glucoprotein and glycogen are catalyzed by two distinct enzymes. Addition of exogenous glycogen (range 0.002-1%) inhibited the protein:glucosyltransferase, whereas at 0.001-0.007% concentration it was acceptor substrate for glycogen:glucosyltransferase activity. At higher concentrations this activity was strongly inhibited. The concentration of glycogen in platelets could play a regulatory role in forming the glucoprotein and the glycogen molecules.     

Aqueous two-phase extraction for downstream processing of amyloglucosidase

A polymer/salt aqueous two-phase system has been successfully employed for separation and purification of amyloglucosidase. The effects of system pH, molecular weight of polymer and composition of the two-phase system on amyloglucosidase partition behaviour in polyethylene glycol (PEG 4000, 6000)/disodium hydrogen phosphate were investigated. Experimental data are explained based on Kim's theoretical model for the prediction of biomolecule partitioning in a PEG/salt system.     

Preparation and properties of immobilized amyloglucosidase

Amyloglucosidase was immobilized on a copolymer of methyl methacrylate and 2-dimethylaminoethyl methacrylate. The resulting immobilized amyloglucosidase has 19% of the soluble enzyme specific activity. The pH optimum of immobilized amyloglucosidase is shifted towards acidity by 1.9 units. The temperature optimum of immobilized enzyme is shifted upward by 5°C. The immobilized amyloglucosidase has the maximum stability at pH 4.6, whereas the soluble enzyme has maximum stability at pH 5.5. While soluble amyloglucosidase has a maximum thermal stability at 50°C, the stability of the immobilized amyloglucosidase steadily decreases with the increase in temperature.     

N-tris-(beta-D-galactopyranosyloxymethyl)glycine methylamide as an antigenic determinant of neoglycoproteins]

A chemical synthesis of N-tris (beta-D-galactopyranosyloxymethyl) glycine methylamide (trisgalactosylglycine) has been carried out. Trisgalactosylglicine derivatives of bovine serum albumin, ovalbumin and amyloglucosidase from Aspergillus have been obtained. The binding of trisgalactosylglycine residues to bovine serum albumin and ovalbumin was performed by the carbodiimide method; amyloglucosidase galactosylation was performed by using the reductive amination method. The latter technique seems to be the most mild one because it does not interfere with the peptide structure of the protein being analyzed. The antiserum specifically raised against the trisgalactosylglycine derivative of bovine serum albumin as well as the monospecific antibodies isolated from it can interact with both the antigen and the trisgalactosylglycine derivatives of ovalbumin and amyloglucosidase. Native proteins are not precipitated with this antiserum. This suggests that the trigalactosylglycine residues (bovine serum albumin, ovalbumin, amyloglucosidase) covalently bound to various proteins act as immunologic determinants regardless of the mode of their binding.     

Production of ethanol from raw cassava starch by a nonconventional fermentation method

Raw cassava root starch was transformed into ethanol in a one-step process of fermentation, in which are combined the conventional processes of liquefaction, saccharification, and fermentation to alcohol. Aspergillus awamori NRRL 3112 and Aspergillus niger were cultivated on wheat bran and used as Koji enzymes. Commercial A. niger amyloglucosidase was also used in this experiment. A raw cassava root homogenate–enzymes–yeast mixture fermented optimally at pH 3.5 and 30°C, for five days and produced ethanol. Alcohol yields from raw cassava roots were between 82.3 and 99.6%. Fungal Koji enzymes effectively decreased the viscosity of cassava root fermentation mashes during incubation. Commercial A. niger amyloglucosidase decreased the viscosity slightly. Reduction of viscosity of fermentation mashes was 40, 84, and 93% by commercial amyloglucosidase, A. awamori, and A. niger enzymes, respectively. The reduction of viscosity of fermentation mashes is probably due to the hydrolysis of pentosans by Koji enzymes.     

Regulation of protein secretion by mycelial culture of the mushroom Termitomyces clypeatus

Termitomyces clypeatus secreted a 24-kDa xylanase constitutively in xylan medium, but required a gluconeogenic amino acid or Krebs cycle acid for the secretion of a 56-kDa amyloglucosidase in dextrin medium. Aspartate, glutamate, succinate and fumarate all increased secretion of amyloglucosidase from 50% to >90% and enzyme production by 10-fold with little effect on xylanase production. Glutamate or succinate stimulated in vitro release of intracellular amyloglucosidase from washed mycelia in the presence of cycloheximide. Amyloglucosidase accumulated in the absence of glutamate was a high-molecular-mass protein that did not migrate in PAGE. Cellular regulation by the fungus of the secretion of amyloglucosidase is indicated.     

A one-step process for the production of single-cell protein and amyloglucosidase

Summary A new Rhizopus species was isolated from traditional Indonesian food, tempeh. The newly isolated species was similar in its morphological characteristics to Rhizopus oligosporus UQM 145F, but grew faster on potato-dextrose agar as well as in submerged culture. The new isolate was found to convert ground cassava tuber directly into single cell protein without pretreatment due to its high amyloglucosidase formation.From 100 g ground tuber, a dry biomass of 33.75 g containing 26.48% true protein together with 60 ml of highly active amyloglucosidase (282 units) was obtained in 12 h. The amyloglucosidase was recovered by ultrafiltration, releasing 26.226 millimol glucose/l/min from soluble starch. The crude enzyme exhibited a pH optimum between 4.6 and 5.0, a temperature optimum between 55 and 60° C and an apparent Km of 3.125 g/l. High substrate concentrations and ammonium sulphate are inhibitory to the enzyme.     

Biologically active polymer supports based on cellulosic derivatives: synthesis and kinetic study

Bioactive cellulose derivatives have been synthesised by coupling enzymes/antibiotics on carboxymethyl cellulose acid chloride and cellulose carbonate. The effect of pH and temperature on the enzymatic activity of amyloglucosidase immobilised on cellulose carbonate was studied. Michaelis-Menten kinetics have been obeyed to the first degree of approximation despite the restricted mobility of the attached enzyme on the polymer support. Lineweaver-Burk plots for the amyloglucosidase immobilized on carboxymethyl cellulose acid chloride at ambient pH with cellulose carbonate at pH 8 have also been plotted. The Michaelis-Menten constant for the immobilized amyloglucosidase on cellulose carbonate at pH 8 was 9.1 mM, and the activation energy for starch hydrolysis was 21.8 kcals/mole.     

Carbohydrate content and regulation following injection of different glycogenolytic enzymes.

The effect of injection of glycogenolytic enzymes on tissue glycogen, blood glucose and plasma insulin was studied in mice. No effects were observed following phosphorylase, whereas the hydrolytic enzymes, alpha-amylase and acid amyloglucosidase depressed liver glycogen. In addition acid amyloglucosidase induced a decrease in blood glucose, a slight elevation of plasma insulin and a marked increase in tolbutamide-stimulated insulin release. At the doses given none of the enzymes affected muscle glycogen. Amyloglucosidase pretreatment markedly enhanced insulin release induced by glibenclamide, leucine, isoleucine, lysine and glucose whereas insulin release stimulated by IPNA, ACTH, glucagon and "CCK-PZ" was unaffected. Injection of acid amyloglucosidase has a profound influence on carbohydrate content and regulation in mice. It is suggested that the dependence or independence of amyloglucosidase activity among the insulin secretagogues tested might reflect different or partially different mechanisms in the process of insulin secretion.     

Direct enzymatic procedure for the determination of liver glycogen

A method is proposed to measure glycogen content in liver homogenates without extraction and acid hydrolysis of tissue glycogen. Homogenates were treated with amyloglucosidase, which degrades glycogen to glucose, and the glucose was the determined enzymatically by the use of glucose oxidase and peroxidase. The method was shown to yield nearly complete (99%) recoveries of standard glycogen, while 5 hr of acid hydrolysis of standard glycogen were required to obtain comparable recoveries. When compared to an acid hydrolysis method for liver, amyloglucosidase degradation of rat liver glycogen and subsequent determination of glucose resulted in higher values for glycogen content. The amyloglucosidase, glucose oxidase: peroxidase method has the advantage of rapidity, whereas the traditional method consisting of extraction, precipitation, and acid hydrolysis is not only time consuming, but may also be subject to losses of glycogen in each step.     

A new procedure for the measurement of fungal and bacterial α-amylase

Summary A procedure for the measurement of fungal and bacterial -amylase in crude culture filtrates and commercial enzyme preparations is described. The procedure employs end-blocked (non-reducing end)p-nitrophenyl maltoheptaoside in the presence of amyloglucosidase and -glucosidase, and is absolutely specific for -amylase. The assay procedure is simple, reliable and accurate.     

Nucleotide sequence of a glucosyltransferase gene from Streptococcus sobrinus MFe28.

The complete nucleotide sequence was determined for the Streptococcus sobrinus MFe28 gtfI gene, which encodes a glucosyltransferase that produces an insoluble glucan product. A single open reading frame encodes a mature glucosyltransferase protein of 1,559 amino acids (Mr, 172,983) and a signal peptide of 38 amino acids. In the C-terminal one-third of the protein there are six repeating units containing 35 amino acids of partial homology and two repeating units containing 48 amino acids of complete homology. The functional role of these repeating units remains to be determined, although truncated forms of glucosyltransferase containing only the first two repeating units of partial homology maintained glucosyltransferase activity and the ability to bind glucan. Regions of homology with alpha-amylase and glycogen phosphorylase were identified in the glucosyltransferase protein and may represent regions involved in functionally similar domains.     

Co-immobilization of amyloglucosidase and pullulanase for enhanced starch hydrolysis

Summary Amyloglucosidase and pullulanase were co-immobilized using a hydrophilic polyurethane foam (Hypol^® 2002). The combined amyloglucosidase and pullulanase activity of the immobilized enzyme was 32.2% ± 1.7% relative to the non-immobilized enzyme. The co-immobilized enzymes were capable of using a variety of glycogen and starch substrates. Co-immobilization of amyloglucosidase and pullulanase increased the glucose yield 1.6-fold over immobilized amyloglucosidase alone. No decrease in activity was observed after 4 months storage for the co-immobilized enzymes. The results suggest that co-immobilization of amyloglucosidase and pullulanase in polyurethane foams is a potentially useful approach for commercial starch hydrolysis. Offprint requests to: K. B. Storey     

A radioassay of enzymes catalyzing the glucosylation and sulfation steps of glucosinolate biosynthesis in Brassica species

A new method for assaying the enzymes uridine diphosphoglucose (UDPglucose):thiohydroximate glucosyltransferase and 3'-phosphoadenosine-5'-phosphosulfate:desulfoglucosinolate sulfotransferase has been designed. The assay system is based on the separation of nonionic [14C]desulfobenzylglucosinolate from anionic [14C]UDPglucose and anionic [14C]benzylglucosinolate, respectively, by differential adsorption to DEAE-ion-exchange disks. The procedure eliminates elaborate chromatographic techniques. The method was used to measure both enzymes in several Brassica spp. In addition, sulfotransferase activity was monitored during partial purification from seedlings of Brassica napus (cv Westar).     

Preparative scale isolation of galactosylhydroxylysine.

A method is described for the isolation and purification of gram quantities of the hydroxylysine-monosaccharide from commercially available marine sponge. The procedure utilized alkaline hydrolysis followed by purification by ionexchange chromatography and gel filtration. Compositional analysis indicated that the final product contained only galactose, hydroxylysine, and HCl which were present in equimolar quantities and comprised 94% of the dry weight. This preparation has been utilized as a substrate for the assay of UDP-glucose:collagen glucosyltransferase (EC 2.4.1.66) of human platelets.     

Effect of fasting on islet lysosomal enzyme activities and the in vivo insulin response to different secretagogues

The effect of a 24 hr starvation period on islet lysosomal enzyme activities and the in vivo insulin response to glucose, glibenclamide and L-isopropyl-noradrenaline (L-IPNA) was studied in mice. It was observed that fasting induced a significant decrease of islet acid amyloglucosidase activity, whereas the activities of acid phosphatase, beta-N-acetyl-glucosaminidase, and beta-glucuronidase in islet tissue were unaffected by the fasting period studied. Starvation markedly reduced the acute insulin response to a maximal dose of glucose or glibenclamide. However, the insulin response to a maximal dose of L-IPNA was of similar magnitude in both fed and fasted animals. Pretreatment of fasted mice with purified fungal acid amyloglucosidase could restore the impaired insulin response to glucose to the normal level seen in fed mice. It is suggested that islet acid amyloglucosidase activity is of importance for glucose-stimulated insulin secretion, and that reduced levels of islet amyloglucosidase may contribute to the impairment of glucose-induced insulin release seen after fasting.     

Adsorption of amyloglucosidase on inorganic carriers

A simple method for immobilizing amyloglucosidase by adsorption on inorganic carriers is described. Amyloglucosidase was adsorbed on acid-activated molecular sieve and on alumina. The immobilized enzyme preparations exhibited 50–100% of the initial activity and possessed high temperature stability. A prolonged working life span was achieved, which could possibly satisfy requirements for industrial application.