Molecular basis of the amylose-like polymer formation catalyzed by Neisseria polysaccharea amylosucrase

Amylosucrase from Neisseria polysaccharea is a remarkable transglucosidase from family 13 of the glycoside-hydrolases that synthesizes an insoluble amylose-like polymer from sucrose in the absence of any primer. Amylosucrase shares strong structural similarities with alpha-amylases. Exactly how this enzyme catalyzes the formation of alpha-1,4-glucan and which structural features are involved in this unique functionality existing in family 13 are important questions still not fully answered. Here, we provide evidence that amylosucrase initializes polymer formation by releasing, through sucrose hydrolysis, a glucose molecule that is subsequently used as the first acceptor molecule. Maltooligosaccharides of increasing size were produced and successively elongated at their nonreducing ends until they reached a critical size and concentration, causing precipitation. The ability of amylosucrase to bind and to elongate maltooligosaccharides is notably due to the presence of key residues at the OB1 acceptor binding site that contribute strongly to the guidance (Arg415, subsite +4) and the correct positioning (Asp394 and Arg446, subsite +1) of acceptor molecules. On the other hand, Arg226 (subsites +2/+3) limits the binding of maltooligosaccharides, resulting in the accumulation of small products (G to G3) in the medium. A remarkable mutant (R226A), activated by the products it forms, was generated. It yields twice as much insoluble glucan as the wild-type enzyme and leads to the production of lower quantities of by-products.     

Facilitated diffusion of p-nitrophenyl-alpha-D-maltohexaoside through the outer membrane of Escherichia coli. Characterization of LamB as a specific and saturable channel for maltooligosaccharides

LamB, an outer membrane protein of Escherichia coli, is a component of the maltose-maltooligosaccharide transport system. We used p-nitrophenyl-alpha-D-maltohexaoside, a chromogenic analog of maltohexaose, and a periplasmic amylase that hydrolyzes this compound to study the LamB-mediated diffusion of p-nitrophenyl-alpha-D-maltohexaoside into the periplasm. Using this approach, we were able to characterize LamB in vivo as a saturable channel for maltooligosaccharides. Permeation through LamB follows Michaelis-Menten kinetics, with a Km of 0.13 mM and a Vmax of 3.3 nmol/min/10(9) cells. Previous studies suggested that maltose-binding protein increases the rate of maltooligosaccharide diffusion through LamB. We show here that, at least in strains that are unable to transport maltooligosaccharides into the cytoplasm, maltose-binding protein does not influence the rate of substrate diffusion. The periplasmic amylase had been previously described as being of the alpha-type. We have now purified this protein and analyzed its mode of action using chromogenic maltooligosaccharides of varying length. Analysis of the hydrolytic products revealed that the enzyme recognizes its substrate from the nonreducing that the enzyme recognizes its substrate from the nonreducing end and preferentially liberates maltohexaose, in contrast to the behavior of classical alpha-amylases that are endohydrolases. Using p-nitrophenyl-alpha-D-maltohexaoside as a substrate, we determined a Km of 3 microM and a Vmax of 0.14 mumol/min/mg of protein.     

Membrane-associated nucleotide sugar reactions: influence of mutations affecting lipopolysaccharide on the first enzyme of O-antigen synthesis.

Both the synthesis of lipopolysaccharide O-antigen and the synthesis of peptidoglycan in Salmonella typhimurium proceed via membrane-bound glycosylated lipid intermediates. The first enzyme of each pathway transfers a sugar phosphate from a nucleotide sugar to the glycosyl carrier lipid (P-GCL). Each enzyme catalyzes an exchange reaction between the reaction product urine monophosphate, and the nucleotide sugar substrate. Several strains of S. typhimurium defective in lipopolysaccharide synthesis accumulate glycosylated lipid intermediates under appropriate conditions. In addition, strains lysogenic for phage P22 synthesize a glucose derivative of the carrier lipid. These strains were used to demonstrate the P/GCL requirement of the exchange reaction catalyzed by galactose-diphosphoglycosyl carrier lipid (GCL-PP-Gal) synthetase, the first enzyme of O-antigen synthesis. Enzyme activity is greatly reduced when glycosylated P-GCL accumulates on the cytoplasmic membrane. The exchange reaction catalyzed by the first enzyme of peptidoglycan synthesis is unaffected by the accumulation of O-antigen fragments on the carrier lipid and may interact with a different pool of P-GCL within the membrane. GCL-PP-Gal synthetase activity cannot be detected in the membranes of two rfa mutants that synthesize incomplete lipopolysaccharide core. Either the synthesis of GCL-PP-Gal synthetase or the stable integration of the enzyme into the membrane structure may be disrupted in the rfa mutants. Peptidoglycan synthesis is unaffected by the mutations affecting the core glycosyltransferases.     

黑曲霉突变株葡萄糖淀粉酶的底物特异性

黑曲霉(Aspergillus niger)突变株T-21葡萄糖淀粉酶(GAI)仅能水解多种淀粉及麦芽低聚糖生成唯一产物β-葡萄糖,其水解麦芽糖及麦芽三糖的速度分别为200和570mg葡萄糖·h~(-1)·mg~(-1).GAI水解α-1,4键的速度比水解α-1.6键快100多倍.除了马铃薯淀粉外,对其它淀粉及麦芽低聚糖几乎都能100%地水解,但不能水解环状糊精,其水解各麦芽低聚糖的最先产物都比原底物少一个葡萄糖单位,说明GAI为一外切型淀粉酶.GAI对麦芽糖、麦芽三糖、可溶性淀粉、糯米淀粉、糊精及糖原的Km值分别1.92mmol/L、0.38mmol/L、0.053%、0.045%、0.059%、及0.076%,V_(max)分别为590、1370、1270、1520、1120和1220mg葡萄糖·h~(-1)·mg~(-1).D-葡萄糖酸-δ-内酯及麦芽糖醇对此酶分别具有反竞争性抑制和混合性抑制.     

Subsite structure and catalytic mechanism of a new glycosyltrehalose-producing enzyme isolated from the hyperthermophilic archaeum, Sulfolobus solfataricus KM1

A glycosyltrehalose-producing enzyme from Sulfolobus solfataricus KM1 catalyzes a conversion of maltooligosaccharides to glycosyltrehaloses and also hydrolyzes maltooligosaccharides to liberate glucose, as a side reaction. From the sum of the conversion and hydrolysis reaction rates, the rate parameters involved in the "splitting" of the alpha-1,4 glucosidic linkage were calculated. From the data obtained, the subsite structure for maltooligosaccharides was identified. From the analysis of the hydrolysate of maltotriose in [18O labeled H2O, the hypothesis of the C1-O bond splitting and the formation of a glycosyl (maltosyl)-enzyme intermediate was strongly supported. From the analysis of the reaction product in the presence of [3H] labeled glucose, the occurrence of intermolecular transglycosylation was confirmed. These data strongly support the suggestion that the catalytic mechanism of this enzyme is a transglycosylation.     

4,6-α-Glucanotransferase activity occurs more widespread in Lactobacillus strains and constitutes a separate GH70 subfamily

Family 70 glycoside hydrolase glucansucrase enzymes exclusively occur in lactic acid bacteria and synthesize a wide range of α-d-glucan (abbreviated as α-glucan) oligo- and polysaccharides. Of the 47 characterized GH70 enzymes, 46 use sucrose as glucose donor. A single GH70 enzyme was recently found to be inactive with sucrose and to utilize maltooligosaccharides [(1→4)-α-d-glucooligosaccharides] as glucose donor substrates for α-glucan synthesis, acting as a 4,6-α-glucanotransferase (4,6-αGT) enzyme. Here, we report the characterization of two further GH70 4,6-αGT enzymes, i.e., from Lactobacillus reuteri strains DSM 20016 and ML1, which use maltooligosaccharides as glucose donor. Both enzymes cleave α1→4 glycosidic linkages and add the released glucose moieties one by one to the non-reducing end of growing linear α-glucan chains via α1→6 glycosidic linkages (α1→4 to α1→6 transfer activity). In this way, they convert pure maltooligosaccharide substrates into linear α-glucan product mixtures with about 50% α1→6 glycosidic bonds (isomalto/maltooligosaccharides). These new α-glucan products may provide an exciting type of carbohydrate for the food industry. The results show that 4,6-αGTs occur more widespread in family GH70 and can be considered as a GH70 subfamily. Sequence analysis allowed identification of amino acid residues in acceptor substrate binding subsites +1 and +2, differing between GH70 GTF and 4,6-αGT enzymes.     

Combinatorial engineering to enhance thermostability of amylosucrase

Amylosucrase is a transglucosidase that catalyzes amylose-like polymer synthesis from sucrose substrate. About 60,000 amylosucrase variants from two libraries generated by the MutaGen random mutagenesis method were submitted to an in vivo selection procedure leading to the isolation of more than 7000 active variants. These clones were then screened for increased thermostability using an automated screening process. This experiment yielded three improved variants (two double mutants and one single mutant) showing 3.5- to 10-fold increased half-lives at 50 degrees C compared to the wild-type enzyme. Structural analysis revealed that the main differences between wild-type amylosucrase and the most improved variant (R20C/A451T) might reside in the reorganization of salt bridges involving the surface residue R20 and the introduction of a hydrogen-bonding interaction between T451 of the B' domain and D488 of flexible loop 8. This double mutant is the most thermostable amylosucrase known to date and the only one usable at 50 degrees C. At this temperature, amylose synthesis by this variant using high sucrose concentration (600 mM) led to the production of amylose chains twice as long as those obtained by the wild-type enzyme at 30 degrees C.     

Evaluation of the rate constants of sugar transport through maltoporin (LamB) of Escherichia coli from the sugar-induced current noise

LamB (maltoporin) of Escherichia coli outer membrane was reconstituted into artificial lipid bilayer membranes. The channel contains a binding site for sugars and is blocked for ions when the site is occupied by a sugar. The on and off reactions of sugar binding cause an increase of the noise of the current through the channel. The sugar-induced current noise of maltoporin was used for the evaluation of the sugar-binding kinetics for different sugars of the maltooligosaccharide series and for sucrose. The on rate constant for sugar binding was between 10(6) and 10(7) M-1.s-1 for the maltooligosaccharides and corresponds to the movement of the sugars from the aqueous phase to the central binding site. The off rate (corresponding to the release of the sugars from the channel) decreased with increasing number of glucose residues in the maltooligosaccharides from approximately 2,000 s-1 for maltotriose to 180 s-1 for maltoheptaose. The kinetics for sucrose movement was considerably slower. The activation energies of the stability constant and of the rate constants for sugar binding were evaluated from noise experiments at different temperatures. The role of LamB in the transport of maltooligosaccharides across the outer membrane is discussed.     

Relationship between membrane physical properties and secretory phospholipase A2 hydrolysis kinetics in S49 cells during ionophore-induced apoptosis

During apoptosis, changes occur in lymphocyte membranes that render them susceptible to hydrolysis by secretory phospholipase A(2) (sPLA(2)). To study the relevant mechanisms, a simplified model of apoptosis using a calcium ionophore was applied. Kinetic and flow cytometry experiments provided key observations regarding ionophore treatment: the initial rate of hydrolysis was elevated at all enzyme concentrations, the total amount of reaction product was increased fourfold, and adsorption of the enzyme to the membrane surface was unaltered. Analysis of these results suggested that susceptibility during calcium-induced apoptosis is limited by availability of substrate rather than adsorption of enzyme. Fluorescence experiments identified three membrane alterations during apoptosis that might affect substrate access to the sPLA(2) active site. First, intercalation of merocyanine 540 into the membrane was improved, suggesting an increase in lipid spacing. Second, laurdan detected increased solvation of the lower headgroup region of the membrane. Third, the rate at which fluorescent lipids could be removed from the membrane by albumin was enhanced, implying greater vertical mobility of phospholipids. Thus, it is proposed that the membranes of apoptotic cells become susceptible to sPLA(2) through a reduction in lipid-neighbor interactions that facilitates migration of phospholipids into the enzyme active site.     

Immobilization of biocatalysts with bombyx mori silk fibroin by several kinds of physical treatment and its application to glucose sensors

Biocatalyst-immobilized Bombyx mori silk fibroin membrane was prepared. The insolubilization of the water-soluble membranes was performed by physical treatments only, i.e. stretching, compressing and standing under high humidity and methanol-immersion treatment, without any use ofcovalently binding reagent. All physical treatments performed were effective for the purpose of the immobilization of the enzymes in the membranes. The structural characterization of the glucose oxidase (GOD) immobilized membrane was performed in detail. The permeability of the substrate depends on the crystalline structure, i.e. the fraction of Silk I and Silk II of the membrane. The activity yield of the immobilized GOD was more than 80% of the value of free enzyme when 0–002% of the enzyme was entrapped in the membrane, but it decreased with increasing the concentration of the GOD in the membrane. This seems to result from diffusion limitation of the substrate. The pH and thermal stabilities of the immobilized enzyme were much improved, and were essentially independent of the methods of the immobilization. Development of the GOD or microorganism, Pseudomonas fluorescens immobilized silk fibroin membranes as glucose sensors are described.     

Study of an enzyme membrane reactor with immobilized fumarase for production of L-malic acid

The conversion of fumaric acid into L-malic acid by fumarase immobilized in a membrane reactor was analyzed experimentally. The enzyme was entrapped in asymmetric capillary membranes made of polysulfone. The performance of the reactor was evaluated in terms of conversion degree, reaction rate, and stability. The influence of operating conditions, such as amount of immobilized enzyme, substrate concentration, residence time, and axial flow rate, were investigated. The kinetic parameters K(m), V(max), and k(+2) were also measured. The stability of the immobilized enzyme was very good, showing no activity decay during more than 2 weeks of continuous operation.     

A continuous membrane bioreactor for ester synthesis in organic media: I. Operational characterization and stability

The feasibility of continuous ester synthesis in a membrane bioreactor (MBR) by a recombinant cutinase from Fusarium solani pisi was investigated, using the optimal conditions previously attained by medium engineering. The objective was to analyze the MBR behavior as a differential or an integral reactor. The main component of the reactor was an anisotropic ceramic membrane with 15,000 NMWCO. The operating variables included the influence of substrates ratio and flow rate on the conversion degree and on the productivity. The highest conversion degree was obtained using 1M of hexanol and 0.1M of butyl acetate as acyl donor. The use of these substrate concentrations led to a conversion degree of 79.3% and a specific productivity of 41 g hexyl acetate/(d x g cutinase), when the permeate flow rate was 0.025 mL/min. The increase of flow rate to 0.4 mL/min decreased the conversion to 35.6%, although the productivity was enhanced to 294 g product/day x g enzyme. The MBR characterization involved the calculations of mass balance, recirculation rate, conversion per pass, number of cycles, and hydraulic residence time. The operational stability was also evaluated in a longterm experiment over 900 hours and the enzyme half-life was estimated to be approximately 2 years.     

Substrate and energy costs of the production of exocellular enzymes by Bacillus licheniformis

Substrate and energy costs of the production of exocellular enzymes from glucose and citrate by B. Iicheniformis S1684 as well as molar growth yields corrected for these costs of product formation were calculated using data from chemostat experiments. The calculations showed that 1.46-1.73 mol glucose and 2.31-2.77 mol citrate are needed for formation and excretion of 1 mol protein. Consequently, the values of the maximal product yield from substrate (Y(psm') g/mol) are 80 < Y(psm) < 95 when product is formed from glucose and 50 < Y(psm) < 60 when product is formed from citrate. The higher substrate costs for product formation from citrate are due to a higher level of CO(2) production during protein formation and a higher substrate requirement for the energy supply of product formation and excretion than when product is formed from glucose. The theoretical ATP requirement for protein synthesis could be determined reasonably well, but the energy costs of protein excretion could not be determined exactly. The energy costs of protein formation are higher than those of biomass formation or protein excretion. Molar growth yields corrected for the substrate costs of product formation were high, indicating a high efficiency of growth.Growth and production parameters were determined as well from experimental data of recycling fermentor experiments using a parameter optimization procedure based on a mathematical model describing biomass growth as a linear function of the substrate consumption rate and the rate of product formation as a linear function of biomass growth rate. The fitting procedure yielded two growth and production domains during glucose limitation. In the first domain the values for the maximal growth yield and maintenance coefficient were in agreement with those found in chemostat experiments at corresponding values of Y(spm). Domain 2 could be described best with linear growth and product formation. In domain 2 the rate of product formation decreased and more substrate became available for biomass formation. As a consequence the specific growth rate increased in the shift from domain 1 to 2. Domain 2 behavior most probably is caused by the rel-status of B. Iicheniformis S1684.     

Combinatorial engineering to enhance amylosucrase performance: construction, selection, and screening of variant libraries for increased activity

Amylosucrase is a glucosyltransferase belonging to family 13 of glycoside hydrolases and catalyses the formation of an amylose-type polymer from sucrose. Its potential use as an industrial tool for the synthesis or the modification of polysaccharides, however, is limited by its low catalytic efficiency on sucrose alone, its low stability, and its side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling, and selective screening (directed evolution) was started, in order to generate more efficient variants of the enzyme. A convenient zero background expression cloning strategy was developed. Mutant gene libraries were generated by error-prone polymerase chain reaction (PCR), using Taq polymerase with unbalanced dNTPs or Mutazyme™, followed by recombination of the PCR products by DNA shuffling. A selection method was developed to allow only the growth of amylosucrase active clones on solid mineral medium containing sucrose as the sole carbon source. Automated protocols were designed to screen amylosucrase activity from mini-cultures using dinitrosalicylic acid staining of reducing sugars and iodine staining of amylose-like polymer. A pilot experiment using the described mutagenesis, selection, and screening methods yielded two variants with significantly increased activity (five-fold under the screening conditions). Sequence analysis of these variants revealed mutations in amino acid residues which would not be considered for rational design of improved amylosucrase variants. A method for the characterisation of amylosucrase action on sucrose, consisting of accurate measurement of glucose and fructose concentrations, was introduced. This allows discrimination between hydrolysis and transglucosylation, enabling a more detailed comparison between wild-type and mutant enzymes.     

Enzymatic activity under tangential flow conditions of photochemically grafted membranes containing immobilized catalase

Catalase has been immobilized within sandwich membranes prepared by the photoinduced grafting of an epoxy-diacrylate prepolymer onto commercial asymmetric cellulose membranes. The enzymatic activity of the membrane composite of hydrogen peroxide decomposition has been studied in a recirculation apparatus under tangential flow conditions without ultrafiltration. The enzymatic membranes were exposed to very low mechanical stresses and showed a very good catalytic performance and durability. Initial reaction rates, measured at 25 degrees C as a function of both substrate concentration and enzyme amount immobilized per unit membrane surface, indicate that the mechanism of action of catalase is not altered after immobilization, although substrate diffusion through the original thin layer of membranes may become rate controlling. (c) 1993 John Wiley & Sons, Inc.     

The kinetic behavior of an artificial bienzyme membrane.

Artificial membranes bearing immobilized enzymes can be used to study some effects of membrane structure on enzyme kinetic behavior. The bienzyme system described is a mixture of beta-glucosidase and glucose oxidase. Gluconolactone, the product of thesecond enzyme, is an inhibitor of the first one. The resulting feedback effect has been compared using a mixed two-enzyme membrane, two separated one-enzyme membranes, and astirred bienzyme solution. The feedback effect is quicker and more efficient in the two-enzyme membrane than in solution; it is slower and less efficient in the case of the separated one-enzyme membranes. Effects of enzyme proximity in the structure are discussed. Conclusions are drawn concerning the efficiency of feedback mechanisms when enzymes are embedded within a single structure.     

Determination of the distribution of catalyst activity across a permeable membrane containing an immobilized enzyme. Indeterminacy of a functional approach to a structural problem.

Porous membranes were fabricated from collodion and impregnated with papain, inhomogeneously through the thickness of the membrane. These membranes were placed between reservoirs containing N-alpha-benzoyl arginineamide, a substrate for the enzyme papain. The progress of the reaction was monitored by sampling the reservoirs on each side for ammonia, a reaction product. From these data the diffusion coefficient, enzyme activity, and distribution of enzyme activity of the membrane were estimated. The limitations of this approach are discussed in the context of the analysis of biological transport systems.     

Distribution of immobilized enzymes on porous membranes

In this article, the results from a theoretical and experimental investigation of enzyme immobilization in porous membranes are reported. A theoretical model of the immobilization process, which accounts for restricted diffusion of enzyme in the pores of the membrane, has been developed. The model predicts the effect of immobilization kinetics and time of immobilization on the enzyme distribution in the pores of the membrane. The immobilization of glucose oxidase and glucose oxidase-biotin conjugate on porous alumina membranes was experimentally investigated. Enzyme uptake data was correlated to the theory to determine the rate constant of imobilization and the distribution of the enzyme in the pore. Immobilization studies were carried out for enzyme adsorption and for enzyme attachment by covalent coupling. The distribution of enzyme was experimentally studied by assembling five membranes in the diffusion cell. Following immobilization, the membranes were separated and each was assayed for activity. The amount of active enzyme present in each membrane yielded a discrete distribution that compared well with that predicted by theory. (c) 1992 John Wiley & Sons, Inc.     

Outer membrane mutation effects on UDP-glucose permeability and whole-cell catalysis rate

In whole-cell biocatalysis, cell envelopes represent a formidable barrier for substrates to permeate. The present research addresses this critical issue by investigating the effects of outer membrane mutation on uridine diphosphate (UDP)-glucose-utilizing enzymes in whole-cell systems. Owing to the severe limitation in substrate permeability, the wild-type Escherichia coli cells only exhibited as low as 4% of available enzyme activities. The reduction of the barriers of the outer membrane permeability (by mutations in its structure) led to a striking acceleration (up to 14-fold) of the reaction rate in cells expressing UDP-glucose dehydrogenase. Mutations in the lipopolysaccharide synthesis pathway or Braun’s lipoprotein are both effective. The acceleration was dependent upon the substrate concentrations as well as the enzyme expression level. In addition, the mutation has been demonstrated to be much more effective than the freeze–thaw permeabilizing method. An application of outer membrane mutants was illustrated with the synthesis of a disaccharide (N-acetyllactosamine) from UDP-glucose. Both reaction rate and product yield were enhanced significantly (more than twofold) in the lipoprotein mutant, demonstrating the importance of the outer membrane permeability barrier and the advantages of using outer membrane mutants in synthesis. This research and the results outlined in this paper point to a valid strategy in addressing permeability issues in whole-cell biocatalysis. It also highlights a need for an assessment of substrate permeability in biocatalysis research and development.     

Purification and characterization of a cyclodextrin-degrading enzyme from Flavobacterium sp.

A cell-bound cyclodextrin-degrading enzyme with a relative molecular mass (M_r) of around 62 000 and an isoelectric point (pI) near 8.0 was isolated and purified to 94% homogeneity from Flavobacterium sp. The enzyme hydrolysed maltooligosaccharides and cyclodextrins to glucose, maltose, and maltotriose. Less glucose, but larger amounts of the line of maltooligosaccharides from maltose to (in case of cyclodextrins) the linearized substrates were found in short-term digests. Digestion of maltotriose yielded glucose, maltose, and some maltotetraose to maltohexaose, i.e. the enzyme catalysed both hydrolysis and transglycosylation. Starch was a poorer substrate, and was hydrolysed to mainly glucose and maltose, presumably by a kind of exo-attack. Pullulan was slightly digested, the products being glucose, panose/isopanose, and larger saccharides containing -1,6-glucosidic bonds. Since maltohexaose to maltooctaose were hydrolysed at higher rates than the cyclodextrins of corresponding lengths, the enzyme of Flavobacterium sp. was proposed to be classified as a decycling maltodextrinase. Correspondence to: H. Bender