Purification and properties of the catalytic domain of the thermostable pullulanase type II from Thermococcus hydrothermalis

A pullulanase type II was produced in Escherichia coli using the relevant gene from Thermococcus hydrothermalis. This protein was purified and its pullulanolytic and amylolytic activities were characterised. The optimum temperature and Ca²⁺ concentration for each activity were identical (105 °C and 0.09 mM), whereas the optimum pH (pH_pullulan 5.75, pH_amylose 5) and the influence of Ca²⁺ ions on the kinetic parameters were different. Further analyses revealed that this enzyme exhibits an endo-processive-like action and specifically cleaves -1,6 bonds in pullulan.     

Microbial amylolytic enzymes

Starch-degrading, amylolytic enzymes are widely distributed among microbes. Several activities are required to hydrolyze starch to its glucose units. These enzymes include alpha-amylase, beta-amylase, glucoamylase, alpha-glucosidase, pullulan-degrading enzymes, exoacting enzymes yielding alpha-type endproducts, and cyclodextrin glycosyltransferase. Properties of these enzymes vary and are somewhat linked to the environmental circumstances of the producing organisms. Features of the enzymes, their action patterns, physicochemical properties, occurrence, genetics, and results obtained from cloning of the genes are described. Among all the amylolytic enzymes, the genetics of alpha-amylase in Bacillus subtilis are best known. Alpha-Amylase production in B. subtilis is regulated by several genetic elements, many of which have synergistic effects. Genes encoding enzymes from all the amylolytic enzyme groups dealt with here have been cloned, and the sequences have been found to contain some highly conserved regions thought to be essential for their action and/or structure. Glucoamylase appears usually in several forms, which seem to be the results of a variety of mechanisms, including heterogeneous glycosylation, limited proteolysis, multiple modes of mRNA splicing, and the presence of several structural genes.     

Characterization of yeast amylolytic enzymes by HPLC maltooligosaccharides determination

Summary A simple method for determination of starch hydrolysis degree by measurement of maltooligosaccharides using HPLC on SGX C-18 column with deionised water as mobile phase was presented. Separation of seven oligosaccharides in an order from glucose to maltoheptaose illustrated the action of two enzyme systems taking part of starch hydrolysis and following fermentation to ethanol.     

Production of amylolytic enzymes by Bacillus amyloliquefaciens in pure culture and in co-culture with Zymomonas mobilis

Mixed cultures of Bacillus amyloliquefaciens MIR-41 and Zymomonas mobilis Flo-B3 showed a 2.5 fold increase in -amylase production, and a 20 times fold decrease in ethanol production compared with pure cultures. Enhanced -amylase production by B. amyloliquefaciens in mixed cultures after 24 h could be attributed to the lack of repression in the synthesis of -amylase by ethanol and protease inhibition by the pH of the culture medium.     

Extremely thermostable amylolytic enzyme from the archaebacterium Pyrococcus furiosus

Abstract One of the most thermostable and thermoactive enzymes ever described has been characterized from a hyperthermophilic archaebacterium Pyrococcus furiosus . The enzyme system of this bacterium was capable of hydrolyzing starch forming a mixture of various oligosaccharides. Unlike the amylases from aerobic bacteria this enzyme does not require metal ions for activity or stability. The enzyme is catalytically active over a very broad temperature range, namely between 40°C and 140°C. The half life of this peculiar enzyme during autoclaving at 120°C is 2 h.     

Iteration model of starch hydrolysis by amylolytic enzymes.

An elaborate computer program to simulate the process of starch hydrolysis by amylolytic enzymes was been developed. It is based on the Monte Carlo method and iteration kinetic model, which predict productive and non-productive amylase complexes with substrates. It describes both multienzymatic and multisubstrate reactions simulating the "real" concentrations of all components versus the time of the depolymerization reaction the number of substrates, intermediate products, and final products are limited only by computer memory. In this work, it is assumed that the "proper" substrate for amylases is the glucoside linkages in starch molecules. Dynamic changes of substrate during the simulation adequately influence the increase or decrease of reaction velocity, as well as the kinetics of depolymerization. The presented kinetic model, can be adapted to describe most enzymatic degradations of a polymer. This computer program has been tested on experimental data obtained for alpha- and beta-amylases.     

High-affinity amylolytic enzymes produced by the mould Trichoderma harzianum

Summary The apparent substrate constants of the amylolytic enzymes produced by the mould Trichoderma harzianum CBS 354.33 were measured. The value for -amylase was 64 mg starch·1^-1 which is very low as compared with those of other -amylases. The substrate constant for glucoamylase was 78 mg starch·l^-1. Both enzymes were sensitive to Acarbose; 50% inhibition was observed at 2.5 mg·l^-1 (-amylase) and 0.10 mg·l^-1 (glucoamylase).     

Production of extracellular debranching activity by amylolytic yeasts

Summary The production of extracellular pullulan-degrading enzymes by several amylolytic yeast strains was studied. The highest activity was obtained with species of Endomycopsis, Lipomyces, Filobasidium, Leucosporidium, and Trichosporon, and also with some amylase-hyperproducing mutants. The most active strains are potentially valuable partners for intergeneric protoplast fusion.     

The thy pol-2 intein of Thermococcus hydrothermalis is an isoschizomer of PI-TliI and PI-TfuII endonucleases

Thy Pol-2 intein, from Thermococcus hydrothermalis, belongs to the same allelic family as Tli Pol-2 (PI-TliI), Tfu Pol-2 (PI-TfuII) and TspTY Pol-3 mini-intein, all inserted at the pol-c site of archaeal DNA polymerase genes. This new intein was cloned, expressed in Escherichia coli and purified. The intein is a specific endonuclease (PI-ThyI) which cleaves the inteinless sequence of the Thy DNA pol gene. Moreover, PI-TliI, PI-TfuII and PI-ThyI are very similar endonucleases which cleave DNA in the same optimal conditions at 70°C yielding similar 3′-hydroxyl overhangs of 4 bp and the reaction is subject to product inhibition. The three enzymes are able to cleave the three DNA sequences spanning the pol-c site and a 24 bp consensus cleavage site was defined for the three isoschizomers. However, the exact size of the minimal cleavage site depends both on the substrate sequence and the endonuclease. The inability of the isoschizomers to cleave the inteinless DNA polymerase gene from Pyrococcus spp. KOD is due to point substitutions on the 5′ side of the pol-c site, suggesting that the absence of inteins of this allelic family in DNA polymerase genes from Pyrococcus spp. can be linked to small differences in the target site sequence.     

Cloning of an alpha-glucosidase gene from Thermococcus hydrothermalis by functional complementation of a Saccharomyces cerevisiae mal11 mutant strain.

alpha-Glucosidase is found in methanogenic and thermophilic archaea and also in eukaryotes and bacteria. The gene encoding the enzyme was cloned from Thermococcus hydrothermalis by complementation of a Saccharomyces cerevisiae deficiency maltase mutant strain. The gDNA clone isolated encodes an open reading frame corresponding to a protein of 242 amino acids. The protein shows 42% identity to a Pyrococcus horikoshii unknown ORF but no similarities were obtained with polysaccharidase sequences.     

Thermal stabilization of amylolytic enzymes by covalent coupling to soluble polysaccharides

The immobilization of pullulanase and beta-amylase on soluble polysaccharides (dextrans and amylose) has been carried out. The method used for coupling the enzymes to the carbohydrate support involves limited periodate oxidation of the polysaccharide followed by reductive alkylation with sodium cyanoborohydride or borohydride. The influence of the degree of functionalization of the carbohydrate, the incubation time, the nature of the reducing agent and, for the dextrans studied, their molecular weight, on the properties of the conjugate were studied. We have observed an apparent correlation between the molecular weight of the glycoprotein conjugates formed and their thermal stability, resistance to urea denaturation and their kinetic parameters. By selecting the proper experimental conditions leading to conjugates with maximum thermal stabilities, it has also been shown that beta-amylase conjugates can hydrolyze starch at a temperature 20 degrees C higher than the corresponding value for the native enzyme. This result demonstrates that conjugation may result in modified enzymes leaving a high operational stability at elevated temperatures. We suggest that the immobilization method presented in this article represents an approach to the stabilization of enzymes employed at an industrial level, which may be of general application.     

Isolation and characterization of Schwanniomyces alluvius amylolytic enzymes

The extracellular amylolytic enzymes of Schwanniomyces alluvius were studied to determine future optimization of this yeast for the production of industrial ethanol from starch. Both alpha-amylase and glucoamylase were isolated and purified. alpha-Amylase had an optimum pH of 6.3 and was stable from pH 4.5 to 7.5. The optimum temperature for the enzyme was 40 degrees C, but it was quickly inactivated at temperatures above 40 degrees C. The Km for soluble starch was 0.364 mg/ml. The molecular weight was calculated to be 61,900 +/- 700. alpha-Amylase was capable of releasing glucose from starch, but not from pullulan. Glucoamylase had an optimum pH of 5.0 and was stable from pH 4.0 to greater than 8.0. The optimum temperature for the enzyme was 50 degrees C, and although less heat sensitive than alpha-amylase, it was quickly inactivated at 60 degrees C. Km values were 12.67 mg/ml for soluble starch and 0.72 mM for maltose. The molecular weight was calculated to be 155,000 +/- 3,000. Glucoamylase released only glucose from both soluble starch and pullulan. S. alluvius is one of the very few yeasts to possess both alpha-amylase and glucoamylase as well as some fermentative capacity to produce ethanol.     

Enzyme hydrolysis of cassava peels: treatment by amylolytic and cellulolytic enzymes

Cassava peels provide a cheap non-food biomass waste that can be hydrolyzed to simple sugars as a useful feedstock. Unlike most crop wastes, they have high starch content as well as lignocellulose. In this study, an enzymatic treatment of cassava peels by various concentrations of amylase and glucoamylase is considered. Steam explosion pre-treatments reduced rate and yield of hydrolysis. Milled peels suspended at 10% w/v yielded a maximum reducing sugar of 0.41?g (as glucose) per gram of peels. HPLC analysis showed that levels of soluble oligosaccharides remained low throughout. A pretreatment with amylase at 95?°C slightly increased rates although final yield was the same. Additional treatment with cellulolytic enzymes increases the total hydrolysis yield to 0.61?g (as glucose) per gram of peels representing 91% of the carbohydrate in cassava peels.     

Production of long-chain isomaltooligosaccharides from maltotriose using the thermostable amylomaltase and transglucosidase enzymes

Amylomaltase and transglucosidase were combined to produce long-chain isomaltooligosaccharides (IMOs). IMOs are effective prebiotics that stimulate the growth of healthy bacteria in human intestines and thus promote better overall health. In this study, the p17bAMY amylomaltase was expressed from its gene, which had been directly isolated from soil samples, while transglucosidase was purchased and purified by a gel-filtration column. Crude amylomaltase was purified by heat treatment, Q-, and phenyl-sepharose column. The purified amylomaltase had a molecular weight of 57 kDa. Specificity on the substrates of the amylomaltase was also studied and it was found that this enzyme was able to catalyze transglucosylation activity using substrates G₂ to G₇. However, G₃ was the most preferred substrate for the enzyme. Here, K _m-G3 and k _cat/K _m were 23 mM and 1.72 × 10⁸ mM/min, respectively. Amylomaltase and transglucosidase were tested both alone and in combination on a G₃ substrate to study the efficient process for the IMOs production. The obtained products from the enzymatic reactions were monitored using the TLC analytical method and a densitometer. The amylomaltase led to products containing linear maltooligosaccharides, while the transglucosidase produced short-chain IMOs. Interestingly, when amylomaltase and transglucosidase were used in combination, long-chain IMOs with sizes larger than IMO₄ were observed under the determined condition.