Distinct sucrose isomerases catalyze trehalulose synthesis in whiteflies,Bemisia argentifolii,and Erwinia rhapontici

Isomaltulose [alpha-D-glucopyranosyl-(1,6)-D-fructofuranose] and trehalulose [alpha-D-glucopyranosyl-(1,1)-D-fructofuranose] are commercially valuable sucrose-substitutes that are produced in several microorganisms by the palI gene product, a sucrose isomerase. Trehalulose also occurs in the silverleaf whitefly, Bemisia argentifoli, as the major carbohydrate in the insect's honeydew. To determine if the enzyme that synthesizes trehalulose in whiteflies was similar to the well-characterized sucrose isomerase from microbial sources, the properties of the enzymes from whiteflies and the bacterium, Erwinia rhapontici, were compared. Partial purification of both enzymes showed that the enzyme from whiteflies was a 116 kD membrane-associated polypeptide, in contrast to the enzyme from E. rhapontici, which was soluble and 66 kD. The enzyme from E. rhapontici converted sucrose to isomaltulose and trehalulose in a 5:1 ratio, whereas the enzyme from whiteflies produced only trehalulose. Unlike the E. rhapontici enzyme, the whitefly enzyme did not convert isomaltulose to trehalulose, but both enzymes catalyzed the transfer of fructose to trehalulose using sucrose as the glucosyl donor. The results indicate that trehalulose synthase from whiteflies is structurally and functionally distinct from the sucrose isomerases described in bacteria. The whitefly enzyme is the first reported case of an enzyme that converts sucrose to exclusively trehalulose.     

Production of adenine arabinoside by gel-entrapped cells of Enterobacter aerogenes in water-organic cosolvent system

Summary Gel-entrapped whole cells of Enterobacter aerogenes, which has a transglycosylation activity, were used to produce adenine arabinoside from uracil arabinoside and adenine, in an appropriate water-organic cosolvent system. Cells of E. aerogenes entrapped with a hydrophilic photo-crosslinkable resin prepolymer, ENT-4000, or a urethane prepolymer, PU-6, had a high and stable transglycosylation activity. To improve the poor solubility in water of the substrate (adenine) and product (adenine arabinoside), dimethyl sulfoxide was selected as the cosolvent based on the criteria of operational stability of the immobilized biocatalyst and solubility of both substrate and product. Addition of 40% dimethyl sulfoxide to the reaction mixture permitted use of a high substrate concentration range which gave high productivity under homogeneous reaction conditions. The immobilized cells of E. aerogenes exhibited a markedly improved operational stability, retaining their initial level of activity during repeated use for at least 35 days at 60°C in 40% dimethyl sulfoxide. When the reaction was carried out with 150 mM uracil arabinoside and 50 mM adenine as the substrates, the yield of adenine arabinoside was maintained at 100% based on the molar ratio of adenine, throughout the reaction.Abbreviations used AraA adenine arabinoside - AraU uracil arabinoside     

Production of palatinose using<Emphasis Type="Italic"> Serratia plymuthica</Emphasis> cells immobilized in chitosan

In recent decades, the production of palatinose has aroused great interest since this structural isomer of sucrose has interesting potential. We describe a simple and effective method of immobilizing Serratia plymuthica cells in chitosan. The sucrose isomerase activity of immobilized preparations was enhanced many times by activation with fresh nutrient medium and subsequent drying. The preparations obtained were physically very stable with high enzyme activity and excellent operational stability. The effect of temperature, pH and substrate concentration on enzyme activity of the immobilized cells was investigated. Using immobilized cells, a complete conversion of sucrose (40% solution) into palatinose was achieved in 4 h in a "batch"-type enzyme reactor. The use of free or immobilized cells had no effect on the composition of the solution, in particular the sugar content. The palatinose content was 80% and that of trehalulose 7%.     

Repeated-batch culture of immobilizedGibberella fujikuroi B9 for gibberellic acid production: An optimization study

The performance of immobilized fungal cells on celite beads for the production of gibberrelic acid was investigated in flasks and 7-L stirred-tank reactor. Repeated incubations of immobilized fungal cells increased cell concentrations and volumetric productivity. The maximum volumetric productivity obtained in the immobilized-cell culture was 3-fold greater than that in suspended-cell culture. The concentration of cotton seed flour (CSF), amont the various nutrients supplied, most significantly influenced productivity and operational stability. Notably, insoluble components in CSF were found to be essential for production. CSF at 6 g/L with 60 g/L glucose was found to be optimal for gibberellic acid production and stable operation by preventing excessive cell growth.     

Continuous production of gluconic acid and sorbitol from Jerusalem artichoke and glucose using an oxidoreductase of Zymomonas mobilis and inulinase

Gluconic acid and sorbitol were simultaneously produced from glucose and Jerusalem artichoke using a glucose-fructose oxidoreductase of Zymomonas mobilis and inulinase. Inulinase was immobilized on chitin by cross-linking with glutaraldehyde. Cells of Z. mobilis permeabilized with toluene were coimmobilized with chitin-immobilized inulinase in alginate beads. The optimum amounts of both chitin-immobilized inulinase and permeabilized cells for coimmobilization were determined, and operational conditions were optimized. In a continuous stirred tank reactor operation, the maximum productivities for gluconic acid and sorbitol were about 19.2 and 21.3 g/L/h, respectively, at the dilution rate of 0.23 h(-1) and the substrate concentration of 20%, but operational stability was low because of the abrasion of the beads. As an approach to increase the operational stability, a recycle packed-bed reactor (RPBR) was employed. In RPBR operation, the maximum productivities for gluconic acid and sorbitol were found to be 23.4 and 26.0 g/L/h, respectively, at the dilution rate of 0.35 h(-1) and the substrate concentration of 20% when the recirculation rate was fixed at 900 mL/h. Coimmobilized enzymes were stable for 250 h in a recycle packed-bed reactor without any loss of activity, while half-life in a continuous stirred tank reactor (CSTR) was observed to be about 150 h.     

Isomaltulose production from sucrose by Protaminobacter rubrum immobilized in calcium alginate

Different culture conditions for Protaminobacter rubrum and enzymatic reaction parameters were evaluated with the goal of improving isomaltulose production. P. rubrum was grown in a medium with 1% (w/v) cane molasses and 0.5% yeast extract and achieved a maximum cell yield Y_x/s of 0.295 g of cells/g sucrose and a specific growth rate (μ) of 0.192 h⁻¹. The immobilization of P. rubrum cells was carried out with calcium alginate, glutaraldehyde and polyethyleneimine. Stabile immobilized cell pellets were obtained and used 24 times in batch processes. Enzymatic conversion was carried out at different sucrose concentrations and in pH 6 medium with 70% (w/v) sucrose at 30 °C an isomaltulose yield of 89–94% (w/v) was obtained. The specific activity of the P. rubrum immobilized pellets in calcium alginate at 30 °C ranged from 1.6 to 4.0 g isomaltulose g⁻¹ pellet h⁻¹, respectively with 70% and 65% sucrose solution, while in lower sucrose concentration had higher specific activities presumably due to substrate inhibition of the isomaltulose synthase in higher sucrose concentrations.     

Potato tubers as bioreactors for palatinose production

Palatinose (isomaltulose, 6-O-alpha-D-glucopyranosyl-D-fructose) is a structural isomer of sucrose with very similar physico-chemical properties. Due to its non-cariogenicity and low calorific value it is an ideal sugar substitute for use in food production. Palatinose is produced on an industrial scale from sucrose by an enzymatic rearrangement using immobilized bacterial cells. To explore the potential of transgenic plants as alternative production facilities for palatinose, a chimeric sucrose isomerase gene from Erwinia rhapontici under control of a tuber-specific promoter was introduced into potato plants. The enzyme catalyses the conversion of sucrose into palatinose. Expression of the palI gene within the apoplast of transgenic tubers led to a nearly quantitative conversion of sucrose into palatinose. Despite the soluble carbohydrates having been altered within the tubers, growth of palI expressing transgenic potato plants was indistinguishable from wild type plants. Therefore, expression of a bacterial sucrose isomerase provides a valid tool for high level palatinose production in storage tissues of transgenic crop plants.     

Synthesis of l-tyrosine by immobilized Escherichia intermedia cells

Summary Cells of Escherichia intermedia were immobilized by entrapment in a polyacrylamide gel and used for the enzymatic production of l-tyrosine from phenol, pyruvate, and ammonia. A preparation containing 50 mg of cells/g of gel retained 60% of its original activity. The effect of temperature, pH and substrate concentration on the activity of free cells was almost identical with the effect on immobilized cells. Phenol showed inhibition and inactivation of the catalyst at high concentration. Synthesis of l-tyrosine (up to 10 g/l) was demonstrated in batch reactors with high conversion yields (95–100%) and a maximal productivity of 2 g/l/h. In continuous reactor the catalyst showed a very high operational stability (more than 54 days without losses).     

Direct immobilization of tyrosinase enzyme from natural mushrooms (Agaricus bisporus) on D-sorbitol cinnamic ester

Mushroom tyrosinase was immobilized from an extract onto the totally cinnamoylated derivative of D-sorbitol by direct adsorption as a result of the intense hydrophobic interactions that took place. The immobilization pH value and mass of lyophilized mushrooms were important parameters that affected the immobilization efficiency, while the immobilization time and immobilization support concentration were not important in this respect. The extracted/immobilized enzyme could best be measured above pH 3.5 and the optimum measuring temperature was 55 degrees C. The apparent Michaelis constant using 4-tert-butylcatechol as substrate was 0.38+/-0.02 mM, which was lower than for the soluble enzyme from Sigma (1.41+/-0.20 mM). Immobilization stabilized the extracted enzyme against thermal inactivation and made it less susceptible to activity loss during storage. The operational stability was higher than in the case of the tyrosinase supplied by Sigma and immobilized on the same support. The results show that the use of p-nitrophenol as enzyme-inhibiting substrate during enzyme extraction and immobilization made the use of ascorbic acid unnecessary and is a suitable method for extracting and immobilizing the tyrosinase enzyme, providing good enzymatic activity and stability.     

Characterization of a denitrifying bacterium immobilized in κ-carrageenan

Summary Cells of a pure strain of a denitrifying bacterium have been immobilized in -carrageenan. The influence of pH and temperature on the immobilized cells was determined, as well as the operational stability in a continuous gas-lift loop reactor. Several steady states at different nitrate loadings were reached with respect to activity and dry cell weight. The results show that the immobilized cells form a stable system which rapidly reacts to changes in substrate (nitrate) supply. The maximum conversion rate of the immobilized cells is higher than the rate usually observed with immobilized denitrifying cells but lower than in fluidized-bed systems with attached biomass. It is shown that with increasing immobilized concentration the K _m value apparently increases to such an extent that for growing cells no zero-order kinetics may be assumed. Offprint requests to: R. H. Wijffels     

Characterization of Pantoea dispersa UQ68J: producer of a highly efficient sucrose isomerase for isomaltulose biosynthesis

AIMS: Isolation, identification and characterization of a highly efficient isomaltulose producer. METHODS AND RESULTS: After an enrichment procedure for bacteria likely to metabolize isomaltulose in sucrose-rich environments, 578 isolates were screened for efficient isomaltulose biosynthesis using an aniline/diphenylamine assay and capillary electrophoresis. An isolate designated UQ68J was exceptionally efficient in sucrose isomerase activity. Conversion of sucrose into isomaltulose by UQ68J (enzyme activity of 90-100 U mg(-1) DW) was much faster than the current industrial strain Protaminobacter rubrum CBS574.77 (41-66 U mg(-1) DW) or a reference strain of Erwinia rhapontici (0.3-0.9 U mg(-1) DW). Maximum yield of isomaltulose at 78-80% of supplied sucrose was achieved in less than half the reaction time needed by CBS574.77, and the amount of contaminating trehalulose (4%) was the lowest recorded from an isomaltulose-producing microbe. UQ68J is a Gram negative, facultatively anaerobic, motile, noncapsulate, straight rod-shaped bacterium producing acid but no gas from glucose. Based on 16S rDNA analysis UQ68J is closest to Klebsiella oxytoca, but it differs from Klebsiella in defining characteristics and most closely resembles Pantoea dispersa in phenotype. SIGNIFICANCE AND IMPACT OF STUDY: This organism is likely to have substantial advantage over previously characterized sucrose isomerase producers for the industrial production of isomaltulose.     

Enzymatic synthesis of γ-glutamylmethylamide from L-glutamylhydrazine and methylamine catalysed by immobilized recombinant γ-glutamyltranspeptidase

In many organisms, γ-glutamylmethylamide is a significant amino acid constituent. In this research, a novel method of γ-glutamylmethylamide synthesis is presented. The synthesis of γ-glutamylmethylamide was catalysed by immobilized recombinant γ-glutamyltranspeptidase and used L-glutamylhydrazine as an economical substrate. The optimal enzymes and γ-glutamyltranspeptidase reaction conditions for the production of γ-glutamylmethylamide were 200?mM L-glutamylhydrazine, 1?M methylamine, and 0.1?g/ml immobilized γ-glutamyltranspeptidase cells at pH 10 and 37?°C for 10?h. The immobilized γ-glutamyltranspeptidase cells were used for 10 reactions, and the average conversion ratio from L-glutamylhydrazine to γ-glutamylmethylamide reached 93.2%. The activity of immobilized recombinant γ-glutamyltransferase was not inhibited by 200?mM L-glutamylhydrazine. The immobilized γ-glutamyltranspeptidase cells exhibited favourable operational stability.     

Stability of immobilized maltotetraose-forming amylase from Pseudomonas stutzeri

The stability of immobilized maltotetraose (G(4))-forming amylase (1,4-alpha-D-glucan maltoteraohydrolase, EC 3.2.1.60) from Pseudomonas stutzeri was investigated in both batch and continous processes. The inactivation process of the immobilized enzyme seemed to obey first-order kinetics, and the immobilized enzyme became more stable when coexisting with 20-30 wt % substrate and calcium ions. From intensive studies on the operational stability in the continuous process, the apparent half-life of G(4) productivity in a constant-flow system was mainly affected by the reaction temperature, substrate concentration, and initial immobilized enzyme activity. A new factor, immobilized enzyme stability factor f(s), was proposed to evaluate the half-life of the immobilized enzyme system.     

Enzymatic synthesis of theanine from glutamic acid γ-methyl ester and ethylamine by immobilized Escherichia coli cells with γ-glutamyltranspeptidase activity

Theanine (γ-glutamylethylamide) is the main amino acid component in green tea. The demand for theanine in the food and pharmaceutical industries continues to increase because of its special flavour and multiple physiological effects. In this research, an improved method for enzymatic theanine synthesis is reported. An economical substrate, glutamic acid γ-methyl ester, was used in the synthesis catalyzed by immobilized Escherichia coli cells with γ-glutamyltranspeptidase (GGT) activity. The results show that GGT activity with glutamic acid γ-methyl ester as substrate was about 1.2-folds higher than that with glutamine as substrate. Reaction conditions were optimized by using 300 mmol/l glutamic acid γ-methyl ester, 3,000 mmol/l ethylamine, and 0.1 g/ml of immobilized GGT cells at pH 10 and 50°C. Under these conditions, the immobilized cells were continuously used ten times, yielding an average glutamic acid γ-methyl ester to theanine conversion rate of 69.3%. Bead activity did not change significantly the first six times they were used, and the average conversion rate during the first six instances was 87.2%. The immobilized cells exhibited favourable operational stability.     

Enhancing the biocatalytic potential of carbonyl reductase of Candida viswanathii using aqueous-organic solvent system

In the present work, the toxic effect of various solvents with different Log P values was studied on the whole cells of Candida viswanathii. Experiments showed that the lower concentrations of some solvent increased both the activity retention and enzyme activity as compared to the control while this was not the case with higher concentrations of the same solvents. The model compound taken in the present study was 1-acetophenone. The percentage conversion improved from 76 to 94%. Addition of 2-propanol increased the substrate tolerance, giving the conversion of 90% compared to 9% in control at a substrate concentration of 70 mM in 1h. The operational stability increased at higher temperatures with the addition of 2-propanol in the reaction mixture with good conversion (90%) and enantiomeric excess (>99%) at 45 degrees C and 50 degrees C. The effect was also found to be prominent in other tested substrates. In order to further stabilize the cells for long term use in higher concentration of organic solvents, the cells were further immobilized, and were found to have higher activity retention than that of free cells.     

Carbon nanotubes tuned foam structures as novel nanostructured biocarriers for lignocellulose hydrolysis

The use of immobilized enzymes during saccharification of lignocelluloses enables the continuous process of enzymatic hydrolysis and repeatable use of enzyme, resulting in reduced operational cost. Novel nano-biocarriers were developed by layer-by-layer deposition of carbon nanotube (CNT) on the foam structures, and their efficiency for enzyme immobilization was demonstrated with cellulase and β-glucosidase. A three-fold enhancement was achieved in the activity of cellulase immobilized on CNT coated polyurethane foam. In addition, both cellulase and β-glucosidase immobilized on the CNT-foam showed much better storage stability and operational stability than the ones immobilized on the commercial biocarrier (Celite), which is critical for a continuous operation. CNT coated monolith was also developed as a biocarrier, offering high surface area and geometric stability. These nano-biocarriers are promising candidates for the efficient saccharification of biomass and to reduce carbon footprint and cost of the equipment.     

Immobilization of α-amylase and amyloglucosidase onto ion-exchange resin beads and hydrolysis of natural starch at high concentration

α-Amylase was immobilized on Dowex MAC-3 with 88 % yield and amyloglucosidase on Amberlite IRA-400 ion-exchange resin beads with 54 % yield by adsorption process. Immobilized enzymes were characterized to measure the kinetic parameters and optimal operational parameters. Optimum substrate concentration and temperature were higher for immobilized enzymes. The thermal stability of the enzymes enhanced after the immobilization. Immobilized enzymes were used in the hydrolysis of the natural starch at high concentration (35 % w/v). The time required for liquefaction of starch to 10 dextrose equivalent (DE) and saccharification of liquefied starch to 96 DE increased. Immobilized enzymes showed the potential for use in starch hydrolysis as done in industry.     

Enhancing isomaltulose production by recombinant Escherichia coli producing sucrose isomerase: culture medium optimization containing agricultural wastes and cell immobilization

Isomaltulose is a structural isomer of sucrose commercially used in food industries. In this work, recombinant Escherichia coli producing sucrose isomerase (SIase) was used to convert sucrose into isomaltulose. To develop an economical industrial medium, untreated cane molasses (10.63 g l^?1), yeast extract (25.93 g l^?1), and corn steep liquor (10.45 g l^?1) were used as main culture compositions for SIase production. The relatively high SIase activity (14.50 ± 0.11 U mg DCW^?1) was obtained by the recombinant cells. To the best of our knowledge, this is the first investigation on SIase production by engineered E. coli using untreated cane molasses. The recombinant E. coli cells expressing the SIase gene were immobilized in calcium alginate gel in order to improve the efficiency of recycling. The immobilization was most effective with 2 % (w/v) sodium alginate and 3 % (w/v) calcium chloride. The optimal initial biomass for immobilization was 20 % (w/v, wet wt.), with a hardening time of 8 h for cell immobilization. The immobilized E. coli cells exhibited good stability for 30 batches with the productivity of 0.45 g isomaltulose g pellet^?1 h^?1. A continuous isomaltulose formation process using a column reactor remained stable for 40 days with 83 ± 2 % isomaltulose yield, which would be beneficial for economical production of isomaltulose.     

Substrate induction of isomaltulose synthase in a newly isolated Klebsiella sp. LX3

AIMS: Production of isomaltulose by newly isolated Klebsiella sp. LX3. METHODS AND RESULTS: The bacterial isolate LX3, which transforms sucrose to isomaltulose and trehalulose, has been isolated from a soil sample in Singapore. Morphological and biochemical analysis, as well as 16s rRNA sequence demonstrated that the isolate could represent a new member of genus Klebsiella. The strain has several interesting features. The immobilized cells of Klebsiella sp. LX3 convert more than 99% of sucrose to products that consist of more than 87% of isomaltulose, 11.6% of trehalulose, and <1% of glucose. CONCLUSIONS: The production of isomaltulose synthase in isolate LX3 is inducible by its substrate sucrose and the sugars containing a fructofuranosyl group. SIGNIFICANCE AND IMPACT OF STUDY: It would be useful for future biotechnological applications to understand the structural features or motifs of the isomaltulose synthases that determine the sucrose conversion efficiency and the ratio of the conversion products.