On the Activities of Glucoamylase Chemically Fixed to Cyanogen Bromide-activated Cellulose

N-Carbamyl-D-amino acid amidohydrolase (DCase), produced with recombinant Escherichia coli cells using a cloned gene from Agrobacterium sp. strain KNK712, has been immobilized for use in the production of D-amino acids. The porous polymers, Duolite A-568 and Chitopearl 3003, were much better than other resins for the activity and stability of the adsorbed enzyme. The activity of DCase expressed on Duolite A-568 and Chitopearl 3003 amounted to 96 units/g-wet-resin and 91 units/g-wet-resin, respectively. DCase immobilized on Duolite A-568 was found to be most stable at about pH 7, and it was further stabilized by reductants such as dithiothreitol, L-cysteine, cysteamine, and sodium hydrosulfite. The stability during the repeated batch reactions was greatly improved when dithiothreitol was in the reaction mixture, and the higher crosslinking degree with glutaraldehyde also stabilized the immobilized enzyme. After 14 times repeated reactions, the remaining activity of the immobilized enzyme cross-linked with 0.1% and 0.2% of glutaraldehyde, and 0.2% of glutaraldehyde with dithiothreitol in the reaction mixture was 12%, 18%, and 63%, respectively. DCase produced with Pseudomonas sp. strain KNK003A and Pseudomonas sp. strain KNK505, which are thermotolerant soil bacteria, and that with Agrobacterium sp. strain KNK712 were also immobilized on Duolite A-568. The stability of the enzymes of thermotolerant bacteria during reactions was superior to that of Agrobacterium sp. strain KNK712, though the activity was lower than that of strain KNK712.     

Purification, Characterization of GH11 Endo-β-1,4-xylanase from Thermotolerant Streptomyces sp. SWU10 and Overexpression in Pichia pastoris KM71H

We have previously described two forms of an endo-β-1,4-xylanase (XynSW2A and XynSW2B) synthesized by thermotolerant Streptomyces sp. SWU10. Here, we describe another xylanolytic enzyme, designated XynSW1. The enzyme was purified to homogeneity from 2 L of culture filtrate. Its apparent molecular mass was 24 kDa. The optimal pH and temperature were pH 5.0 and 40 °C, respectively. The enzyme was stable in a wide pH ranges (pH 1–11), more than 80 % of initial activity remained at pH 2–11 after 16 h of incubation at 4 °C and stable up to 50 °C for 1 h. Xylobiose and xylotriose were the major xylooligosaccharides released from oat spelt xylan by the action of XynSW1, indicating of endo-type xylanase. The complete xynSW1 gene contains 1,011 bp in length and encode a polypeptide of 336 with 41 amino acids of signal peptide. The amino acid sequence analysis revealed that it belongs to glycoside hydrolase family 11 (GH11). The mature xynSW1 gene without signal peptide sequence was overexpressed in Pichia pastoris KM71H. The recombinant XynSW1 protein showed higher molecular mass due to the differences in glycosylation levels at the six N-glycosylation sites in the amino acid sequence and exhibited better physicochemical properties than those of the native enzyme including higher optimal temperature (60 °C), and specific activity, but lower optimal pH (4.0). Because of their stability in a wide pH ranges, both of native and recombinant enzymes of XynSW1, may have potential application in several industries including food, textile, biofuel, and also waste treatment.     

Identification of Des-methyl-DIF-1 Methyltransferase in Dictyostelium purpureum

For the production of D-amino acids using stable N-carbamyl-D-amino acid amidohydrolase (DCase) in an immobilized form, the DCase gene of Agrobacterium sp. KNK712 was mutagenized to increase its enzymatic thermostability. In a search for thermostability-related amino acid sites besides the two known sites of DCase, i.e., the 57th and 203rd amino acids, the new mutant enzyme found, in which the 236th amino acid, valine, had been changed to alanine, showed a 10°C increase in thermostability. These known three thermostability-related amino acids were changed to other amino acids by the PCR technique, and it was proved that the thermostability of the DCase increased when the 57th amino acid of DCase, histidine, was changed to leucine, the 203rd amino acid, proline, to asparagine, glutamate, alanine, isoleucine, histidine, or threonine, and the 236th amino acid, valine, to threonine or serine, in addition to the known mutations.     

Characterization of thermotolerant Acetobacter pasteurianus strains and their quinoprotein alcohol dehydrogenases

We isolated several thermotolerant Acetobacter species of which MSU10 strain, identified as Acetobacter pasteurianus, could grow well on agar plates at 41°C, tolerate to 1.5% acetic acid or 4% ethanol at 39°C, similarly seen with A. pasteurianus SKU1108 previously isolated. The MSU10 strain showed higher acetic acid productivity in a medium containing 6% ethanol at 37°C than SKU1108 while SKU1108 strain could accumulate more acetic acid in a medium supplemented with 4–5% ethanol at the same temperature. The fermentation ability at 37°C of these thermotolerant strains was superior to that of mesophilic A. pasteurianus IFO3191 strain having weak growth and very delayed acetic acid production at 37°C even at 4% ethanol. Alcohol dehydrogenases (ADHs) were purified from MSU10, SKU1108, and IFO3191 strains, and their properties were compared related to the thermotolerance. ADH of the thermotolerant strains had a little higher optimal temperature and heat stability than that of mesophilic IFO3191. More critically, ADHs from MSU10 and SKU1108 strains exhibited a higher resistance to ethanol and acetic acid than IFO3191 enzyme at elevated temperature. Furthermore, in this study, the ADH genes were cloned, and the amino acid sequences of ADH subunit I, subunit II, and subunit III were compared. The difference in the amino acid residues could be seen, seemingly related to the thermotolerance, between MSU10 or SKU1108 ADH and IFO 3191 ADH.     

Genome sequence of Acremonium strictum AAJ6 strain isolated from the Cerrado biome in Brazil and CAZymes expression in thermotolerant industrial yeast for ethanol production

Increased demand for biofuels promotes the search for new biomass-degrading fungi. Acremonium strictum is an environmentally widespread filamentous fungi found on plant debris; that secretes lignocellulose-degrading enzymes. A recently isolated A. strictum strain, AAJ6; native to the Brazilian Cerrado biome was evaluated for its capacity to degrade lignocellulosic substrates. In this study, whole-genome sequencing of AAJ6 was performed and 775 CAZy domains were identified which correlated to those of A. strictum strain DS1bioAY4a and other lignocellulolytic fungi; suggesting AAJ6 is a high CAZyme producer. We expressed the glycoside hydrolase families GH74 and GH3 from plasmid or genome-integrated to evaluate the ethanol production from cellulosic substrates in Brazilian industrial Saccharomyces cerevisiae strains (PE-2 and SA-1) evolved for thermotolerance (AMY12 and AMY35). Those expressing the genome-integrated enzymes showed the highest β-glucosidase activity and growth in medium with cellobiose at 40°C. The strain AGY005 (integrated cassettes) showed 19, 23 and 46% higher ethanol production in SHF, pSSF (partial hydrolysis SSF) and SSF processes, respectively, using Avicel, and ∼50% more ethanol using pre-treated sugarcane bagasse, compared to the strain with a plasmid-based expression. These results indicate the improved performance of thermotolerant industrial strains with genome-integrated CAZymes in the SSF process for 2G ethanol.     

Global Analysis of the Genes Involved in the Thermotolerance Mechanism of Thermotolerant Acetobacter tropicalis SKU1100

Acetobacter tropicalis SKU1100 is a thermotolerant acetic acid bacterium that grows even at 42 °C, a much higher temperature than the limit for the growth of mesophilic strains. To elucidate the mechanism underlying the thermotolerance of this strain, we attempted to identify the genes essential for growth at high temperature by transposon (Tn10) mutagenesis followed by gene or genome analysis. Among the 4,000 Tn10-inserted mutants obtained, 32 exhibited a growth phenotype comparable to that of the parent strain at 30 °C but not at higher temperatures. We identified the insertion site of Tn10 on the chromosomes of all the mutant strains by TAIL (Thermal Asymmetric Interlaced)-PCR, and found 24 genes responsible for thermotolerance. The results also revealed a partial overlap between the genes required for thermotolerance and those required for acetic acid resistance. In addition, the origin and role of these thermotolerant genes are discussed.     

Immobilization of glucansucrase for the production of gluco-oligosaccharides from Leuconostoc mesenteroides

Glucansucrase from Leuconostoc mesenteroides was immobilized in 1?% (w/v) with sodium alginate to produce oligosaccharides. Glucansucrase gave three activity bands of approx. 240, 178, and 165?kDa after periodic acid-Schiff staining with sucrose. The immobilized enzyme had 40?% activity after ten batch reactions at 30?°C and 75?% activity after a month of storage at 4?°C, which is six times more stable than the free enzyme. Immobilized enzyme was more stable at lower (3.5?4.5) and higher (6.5?7.0) pH ranges and higher temperatures (35?40?°C) compared with the free enzyme. Immobilized and free glucansucrase were employed in the acceptor reaction with maltose and each produced gluco-oligosaccharide ranging from trisaccharides to homologous pentasaccharides.     

Hybrid Selection of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Yeasts for Thermotolerance and Fermentation Activity

Molecular genetic screening of Saccharomyces yeasts, isolated from natural sources in the regions of the world with a hot climate (Africa, South America, Southeast and Central Asia) was used for the search of thermotolerant S. cerevisiae strains. Based on physiological tests, four strains were selected that could grow at high temperatures (42 and 43°C) and had good fermentation activity: 7962-4B, 3529-7B, 52922-4-1-1A- 1C, and 87-2421.1-2A. Hybrids of monosporic culture of distiller’s race XII (XII₇-2) with the thermotolerant strains were obtained. Unlike the strain XII₇-2, which is unable to grow at above 39°C, all hybrids showed good growth at 42°C. Two of the six hybrids analyzed, H2-1 (87-2421.1-2A × XII₇-2) and H3-2 (7962-4B × XII₇-2), showed higher fermentation activity than the parental strains. According to the results obtained, inter-strain hybridization is an efficient method of obtaining S. cerevisiae strains, which combine thermotolerance with high efficiency of alcoholic fermentation.     

Thermotolerance and trehalose accumulation induced by heat shock in yeast cells of Candida albicans

Candida albicans yeast cells growing exponentially on glucose are extremely sensitive to severe heat shock treatments (52.5°C for 5 min). When these cultures were subjected to a mild temperature preincubation (42°C), they became thermotolerant and displayed higher resistance to further heat stress. The intracellular content of trehalose was very low in exponential cells, but underwent a marked increase upon non-lethal heat exposure. The accumulation of trehalose is likely due to heat-induced activation of the trehalose-6-phosphate synthase complex, whereas the external trehalase remained practically unmodified. After a temperature reversion shift (from 42°C to 28°C), the pool of trehalose was rapidly mobilized without any concomitant change in trehalase activity. These results support an important role of trehalose in the mechanism of acquired thermotolerance in C. albicans and seem to exclude the external trehalase as a key enzyme in this process.     

Adaptation of the yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> to heat shock

The adaptive response of the yeast Yarrowia lipolytica to heat shock has been studied. Experiments showed that, after 10 min of incubation at 45°C, the survival rate of Yarrowia lipolytica cells was less than 0.1%. Stationary-phase yeast cells were found to be more thermotolerant than exponential-phase cells. A 60-min preincubation of cells at 37°C or pretreatment with low concentrations of H₂O₂ (0.5 mM) or menadione (0.05 mM) made them more tolerant to heat and to oxidative stress (120 mM hydrogen peroxide). The pH dependence of yeast thermotolerance has also been studied. The adaptation of yeast cells to heat shock and oxidative stress was found to be associated with a decrease in the intracellular level of cAMP and an increase in the activity of antioxidant enzymes (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase).     

Effect of amino acid analogs on the development of thermotolerance and on thermotolerant cells

Exposure of HA-1 Chinese hamster fibroblasts to amino acid analogs has been shown to have a heat-sensitizing effect as well as inducing the heat shock response (Li and Laszlo, 1985a). In this study, we have examined the effect of amino acid analogs on the development of thermotolerance after a brief heat shock or exposure to sodium arsenite and the effect of amino acid analogs on cells that are already thermotolerant. Exposure of HA-1 cells to amino acid analogs inhibited the development of thermotolerance following a mild heat shock or treatment with sodium arsenite. However, cells that were already thermotolerant were resistant to the sensitizing action of amino acid analogs. The refractoriness of thermotolerant cells to amino acid analog treatment developed in parallel with thermotolerance. The uptake of the arginine analog, canavanine, and its incorporation into proteins was not altered in the thermotolerant cells. Furthermore, another biological consequence of exposure to amino acid analogs, sensitization to ionizing radiation, also was not altered in the thermotolerant cells. The inhibition of the development of thermotolerance by amino acid analogs and the refractoriness of thermotolerant cells to the heat-sensitizing action of amino acid analogs lend further support the role of heat-shock proteins in the phenomenon of thermotolerance. © 1993 Wiley-Liss, Inc.     

A simple method of cellulase immobilization on a modified silica support

Two types of cellulase (Robillarda sp. Y-20 and Trichoderma reesei) were immobilized on Aminosilica-1 by physical adsorption. Enzymes were quickly immobilized and stable on the support. Specific activities of two types of immobilized cellulase were 0.24 U/mg support with Robillarda cellulase and 0.15 U/mg support with the Trichoderma one as CMCase. The pH-activity curves of both cellulase shifted slightly to lower pH on immobilization. Both immobilized cellulases showed essentially the same pH stabilities as their free forms. However, the immobilized enzymes were less stable than the free forms at temperatures higher than 50°C.     

Immobilization of phytase on epoxy-activated Sepabead EC-EP for the hydrolysis of soymilk phytate

In this work, an active phytase concentrated extract from soybean sprout was immobilized on a polymethacrylate-based polymer Sepabead EC-EP which is activated with epoxy groups. The immobilized enzyme exhibited an activity of 0.1 U/g of carrier and activity yield of 64.7%. The optimum temperature and pH for the activity of both free and immobilized enzymes were found as 60 °C and pH 5.0, respectively. The immobilized enzyme was more stable than free enzyme in the range of pH 3.0–8.0 and more than 70% of the original activity was recovered. Both the enzymes completely retained nearly about 84% of their original activity at 65 °C. The K_m and V_max values were measured as 5 mM and 0.63 U/mg for free enzyme and 12.5 mM and 0.71 U/mg for immobilized enzyme, respectively. Free and immobilized soybean sprout phytase enzymes were also used in the biodegradation of soymilk phytate. The immobilized enzyme hydrolysed 92.5% of soymilk phytate in 7 h at 60 °C, as compared with 98% hydrolysis observed for the native enzyme over the same period of time. The immobilization procedure on Sepabead EC-EP is very cheap and also easy to carry out, and the features of the immobilized enzyme are very attractive that the potential for practical application is considerable.     

Purification,characterization, and overexpression of an endo-1,4-β-mannanase from thermotolerant <Emphasis Type="Italic">Bacillus</Emphasis> sp. SWU60

Endo-β-1,4-mannanases are important catalytic agents in several industries. The enzymes randomly cleave the β-1,4-linkage in the mannan backbone and release short β-1,4-mannooligosaccharides and mannose. In the present study, mannanase (ManS2) from thermotolerant Bacillus sp. SWU60 was purified, characterized, and its gene was cloned and overexpressed in Escherichia coli. ManS2 was purified from culture filtrate (300 ml) by using hydrophobic, ion-exchange, and size-exclusive liquid chromatography. The apparent molecular mass was 38 kDa. Optimal pH and temperature for enzyme activity were 6.0 and 60?°C, respectively. The enzyme was stable up to 60?°C for 1 h and at pH 5–9 at 4?°C for 16 h. Its enzyme activity was inhibited by Hg²⁺. The full-length mans2 gene was 1,008 bp, encoding a protein of 336 amino acids. Amino acid sequence analysis revealed that it belonged to glycoside hydrolase family 26. Konjac glucomannan was a favorable substrate for recombinant ManS2 (rManS2). rManS2 also degraded galactomannan from locust bean gum, indicating its potential for production of glucomanno- and galactomanno-oligosaccharides. Both native and recombinant ManS2 from Bacillus sp. SWU60 can be applied in several industries especially food and feed.     

Effects of high-temperature stress and heat shock on two root maggots, Bradysia odoriphaga and Bradysia difformis (Diptera: Sciaridae)

Bradysia odoriphaga and B. difformis (Diptera: Sciaridae) are devastating pests of vegetables, ornamentals and edible mushrooms. In Chinese chive fields, the two Bradysia species occur with similar regularities: outbreaks in spring and autumn, and population decreases in summer. Temperature may be an important factor restricting their population abundance in summer. Here, we performed a life-table study under constant high temperatures and assessed the tolerance of two Bradysia species to heat shock. Life parameters of the Bradysia species indicated slow developmental rates, and low survival rates and fecundity, when the temperature was higher than 30 °C. At 34 °C, individuals were unable to reach the adult stages from eggs. Moreover, temperatures above 36 °C showed lethal effects, decreasing their survival rates. The median lethal time (LT50) values of 4th instar B. odoriphaga and B. difformis larvae were 46.82 and 32.97 h, respectively, while the values at 38 °C were 2.12 and 1.51 h, respectively. The 4th instar larvae and pupae possessed higher thermotolerance levels than adults and eggs, indicating sensitivities to heat stress. Moreover, B. odoriphaga was more thermotolerant than B. difformis. Thus, weak thermotolerance levels may restrict their occurrences during the period of summer heat, and the difference in thermotolerance levels between the two species may be related to their regional distributions.     

Immobilization of chitosan-modified invertase on alginate-coated chitin support via polyelectrolyte complex formation

Saccharomyces cerevisiae invertase was chemically modified with chitosan and further immobilized on sodium alginate-coated chitin support. The yield of immobilized protein was determined as 85% and the enzyme retained 97% of the initial chitosan-invertase activity. The optimum temperature for invertase was increased by 10 °C and its thermostability was enhanced by about 9 °C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was four-fold more resistant to thermal treatment at 65 °C than the native counterpart. The biocatalyst prepared retained 80% of the original catalytic activity after 50 h under continuous operational regime in a packed bed reactor.     

Improving the thermostability of <Emphasis Type="Italic">N</Emphasis>-carbamyl-<Emphasis Type="SmallCaps">d</Emphasis>-amino acid amidohydrolase by error-prone PCR

To facilitate the easier production of d-amino acids using N-carbamyl-d-amino acid amidohydrolase (DCase) in an immobilized form, we improved the enzymatic thermostability of highly soluble DCase-M3 of Ralstonia pickettii using directed mutagenesis. Six novel mutation sites were identified in this study, apart from several thermostability-related amino acid sites reported previously. The most thermostable mutant, in which the 12th amino acid had been changed from glutamine to leucine, showed a 7 °C increase in thermostability. Comparative characterization of the parental and mutant DCases showed that although there was a slight reduction in the oxidative stability of the mutants, their kinetic properties and high solubility were not affected. The mutated enzymes are expected to be applied to the development of a fully enzymatic process for the industrial production of d-amino acids.     

Thermal and Operational Characteristics of Glutaryl-7-aminocephalosporanic acid Acylase Immobilized on Silica Gel Modified by Epoxide Silanization

Summary In this study, an investigation was performed into the thermal and operational characteristics of glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase (EC 3.5.1.-) immobilized on silica gel that had been modified by epoxide silanization. The pH values for the optimum activity of free and immobilized GL-7-ACA acylase were almost the same. However, the pH-dependent activity profile for the immobilized GL-7-ACA acylase is considerably expanded. Both free and immobilized enzymes generally had the highest activity at 50 °C. In thermodynamic studies, it was found that immobilization using epoxide silanization made GL-7-ACA acylase thermodynamically stable. In the results of repeated batch production of 7-ACA, 89.0 and 83.5% of the 7-ACA produced at the initial cycle were maintained after 20 times of recycle at 25 °C and 30 °C, respectively. Hence it was suggested that mass production of 7-ACA at 25 °C using immobilized GL-7-ACA acylase by epoxide silanization would be possible on a large scale.     

Investigation on the immobilization of Pseudomonas isoamylase onto polysaccharide matrices

This study examined Pseudomonas isoamylase immobilized onto polysaccharide matrices, among which included agarose, cellulose, and raw corn starch. For chemical binding of polysaccharides activated with tosyl chloride, a high specific activity of 23144?U/g-starch was obtained as compared with matrices of cellulose and agarose with 3229?U/g-cellulose and 84?U/g-agarose, respectively. For raw corn starch, isoamylase desorption occurred when the immobilized enzyme by physical adsorption was subjected to 0.05?M acetate buffer with pH?5.2 at 40?°C; this is despite the considerable affinity between the enzyme and the matrix. In contrast, no detectable activity leached from the matrix for chemical binding, regardless of whether maltose, i.e. an affinity species to isoamylase, was added. For immobilized starch-isoamylase, its optimal activity performance was obtained in broader pH?ranges of 3.5–5.5 and 5?°C higher than those of the free enzymes. More specifically, the free enzyme's activity markedly decreased within five hours while the immobilized starch-isoamylase exhibited a fairly stable behavior over a three day incubation period at 40?°C. After 175 days of storage at 4?°C, the residues of relative activity of 75% and 45% were obtained with respect to immobilized and free isoamylases, respectively.     

Decolorization of Alcohol Distillery Wastewater by Thermotolerant White Rot Fungi

To determine a thermotolerant fungus strain for decolorization of alcohol distillery wastewater (ADW), 38 fungus strains were studied. Capacity for ligninolytic enzyme production was examined at 35 and ⁴³C on agar media containing 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and MnCl₂. At 43°C, four Pycnoporus coccineus strains showed a higher potential for ADW decolorization both on agar media and in liquid media. Immobilized mycelia on polyurethane foam removed about threefold more total phenol than did free mycelia under conditions of shaking at 43°C. Moreover, immobilized mycelia removed nearly 50% more color than did free mycelia.