Co-immobilization of dextransucrase and dextranase for the facilitated synthesis of isomalto-oligosaccharides: Preparation, characterization and modeling

Co-Immobilization of dextransucrase (DS) and dextranase (DN) into calcium alginate includes the co-entrapment of soluble DS and adsorbed DN. DS converts sucrose into dextran, which is the substrate for DN, so that isomalto-oligosaccharides (IMOs) are follow-up products of dextran hydrolysis. The boundary conditions for the successful preparation are investigated with respect to choice of DN adsorbate, surface modifications using blotting agents and optimal enzyme activity ratios. Further, repetitive batch experiments suggest the selection of medium activity ratios for continuous use (0.3 U(DN)U(-1) (DS), e.g.). Product formation at various cosubstrate:substrate concentrations as well as at different DN:DS ratios are discussed. Moreover, the complexity of the bi-enzymatic system can be reduced considering the molar ratios of cosubstrate:substrate (glucose:sucrose). Based on these factors, a mechanistic kinetic model is developed, which distinguishes the corresponding contributions of the two enzymes upon overall product formation. In general, at low glucose:sucrose ratios isomaltose synthesis is featured primarily by DN action. Yet with increasing amounts of glucose both the quantity and quality of DN substrate changes, so that its contribution to product formation decreases in an exponential manner; still the overall product yield continuously increases due to enhanced DS contribution.     

Co-immobilization of dextransucrase and dextranase in alginate

Dextransucrase from Leuconostoc mesenteroides and dextranase from Penicillium lilacinum were co-immobilized and used to produce isomaltooligosaccharides from sucrose. The enzymes were co-immobilized by encapsulating soluble dextransucrase and dextranase covalently attached to Eupergit C in alginate (beads, fibers, and capsules). The alginate capsule co-immobilization was done in the presence of soluble starch and resulted in a high immobilization yield (71%), and the enzymes retained their activities during 20 repeated batch reactions and for a month in storage at 4 °C. The presence of starch was essential for the stability of dextransucrase in alginate capsules. Furthermore, it is important that the dextranase be pre-immobilized prior to alginate capsule co-immobilization to prevent dextranase leakage and inactivation of dextransucrase. The co-immobilized enzymes formed oligosaccharides from sucrose, which can be used as prebiotics. In addition, the oligosaccharides that were produced after the addition of sucrose reacted with the alginate fiber-encapsulted dextransucrase, thus increasing the amount of prebiotics. Co-immobilization in alginate fiber and beads also resulted in high yields (70 and 64%), but enzymatic activities decreased by 74 and 99%, respectively, after a month in storage at 4 °C. The newly developed alginate capsule method for co-immobilization of dextransucrase and dextranase is simple yet effective and has the potential for industrial-scale production of isomaltooligosaccharides.     

NahK/GlmU fusion enzyme: characterization and one-step enzymatic synthesis of UDP-N-acetylglucosamine

The availability of uridine 5'-diphosphate N-acetylglucosamine (UDP-GlcNAc) is a prerequisite for the GlcNAc-transferase-catalyzed glycosylation reaction. UDP-GlcNAc has already been synthesized using an N-acetylhexosamine 1-kinase (NahK) and a GlcNAc-1-P uridyltransferase (truncated GlmU) and here, a fusion enzyme was constructed with truncated GlmU and NahK. After determination of the optimum catalytic condition (pH 8.0 at 40 °C), the fusion enzyme was used to synthesize UDP-GlcNAc in a single step with a yield of 88 % from GlcNAc, ATP and UTP. Furthermore, a simplified purification method was demonstrated using separation by gel filtration after by-product digestion with alkaline phosphatase. An overall yield of 77 % and a purity of over 90 % were achieved.     

Volume changes during enzyme reactions. The influence of pressure on the action of invertase, dextranase and dextransucrase.

The pressure dependence of the maximum velocities and the Michaelis constants for the enzymes invertase and dextranase was measured up to 1400 bar. The corresponding activation volumes deltaV not equal to c and deltaV not equal to Km proved to be independent of pressure. Together with data from other sources the meaning of deltaV not equal to c and deltaV not equal to Km is established and the volume profiles of the reactions are constructed. These profiles are similar in contour to the volume profile of the dextran formation catalyzed by the enzyme dextransucrase, but the amount of the volume changes is very much larger for dextransucrase. The evaluation of salt effects shows, that for all three enzymes solvent interactions are not important in explaining the results. The reaction mechanisms seem to be governed by conformation changes of the enzymes. The larger effects in dextransucrase are explained by the produced dextran chain remaining tightly bound to the enzyme and being transported relative to the enzymes position in each reaction cycle.     

The use of an oscillating-tube densitometer as a tool in enzyme kinetics. Determination of the influence of sodium ascorbate on invertase, dextransucrase and dextranase

The use of a commercial oscillating-tube densitometer with an accuracy of 4 . 10(-7) g/cm3 for the determination of enzyme-kinetics constants is tested. This method is applied to the investigation of the influence of vitamin C (sodium ascorbate) on the glycolytic enzymes invertase, dextransucrase and dextranase. Invertase is inhibited uncompetitively, dextransucrase non-competitively. There is no significant effect of the vitamin on dextranase. The comparison of the mechanisms of the three enzymes suggests that only those reaction steps are inhibited by vitamin C in which fructose is released from the enzyme.     

Synthesis of isomaltooligosaccharides and oligodextrans in a recycle membrane bioreactor by the combined use of dextransucrase and dextranase

A recycle ultrafiltration membrane reactor was used to develop a continuous synthesis process for the production of isomaltooligosaccharides (IMO) from sucrose, using the enzymes dextransucrase and dextranase. A variety of membranes were tested and the parameters affecting reactor stability, productivity, and product molecular weight distribution were investigated. Enzyme inactivation in the reactor was reduced with the use of a non-ionic surfactant but its use had severe adverse effects on the membrane pore size and porosity. During continuous isomaltooligosaccharide synthesis, dextransucrase inactivation was shown to occur as a result of the dextranase activity and it was dependent mainly on the substrate availability in the reactor and the hydrolytic activity of dextranase. Substrate and dextranase concentrations (50-200 mg/mL(-1) and 10-30 U/mL(-1), respectively) affected permeate fluxes, reactor productivity, and product average molecular weight. The oligodextrans and isomaltooligosaccharides formed had molecular weights lower than in batch synthesis reactions but they largely consisted of oligosaccharides with a degree of polymerization (DP) greater than 5, depending on the synthesis conditions. No significant rejection of the sugars formed was shown by the membranes and permeate flux was dependent on tangential flow velocity.     

Construction and evaluation of a novel bifunctional N-carbamylase-D-hydantoinase fusion enzyme

A fully enzymatic process employing two sequential enzymes, D-hydantoinase and N-carbamylase, is a typical case requiring combined enzyme activity for the production of D-amino acids. To test the possibility of generating a bifunctional fusion enzyme, we constructed a fusion protein via end-to-end fusion of a whole gene that encodes an intact protein at the N terminus of the D-hydantoinase. Firstly, maltose-binding protein (MBP) gene of E. coli was fused with D-hydantoinase gene from Bacillus stearothermophilus SD1, and the properties of the resulting fusion protein (MBP-HYD) were compared with those of native D-hydantoinase. Gel filtration and kinetic analyses clearly demonstrated that the typical characteristics of D-hydantoinase are maintained even in a fusion state. Based on this result, we constructed an artificial fusion enzyme composed of the whole length of N-carbamylase (304 amino acids [aa]) from Agrobacterim radiobacter NRRL B11291 and D-hydantoinase (471 aa). The fusion enzyme (CAB-HYD) was functionally expressed with an expected molecular mass of 86 kDa and efficiently converted exogenous hydantoin derivatives to the D-amino acids. A related D-hydantoinase (HYD1) gene from Bacillus thermocatenulatus GH2 was also fused with the N-carbamylase gene at its N terminus. The resulting enzyme (CAB-HYD1) was bifunctional as expected and showed better performance than the CAB-HYD fusion enzyme. The conversion of hydantoin derivatives to corresponding amino acids by the fusion enzymes was much higher than that by the separately expressed enzymes, and comparable to that by the coexpressed enzymes. Thus, the fusion enzyme might be useful as a potential biocatalyst for the production of nonnatural amino acids.     

Construction of a fusion enzyme exhibiting superoxide dismutase and peroxidase activity

A chimeric gene construct encoding human peroxiredoxin 6 and Mn-superoxide dismutase from Escherichia coli was developed. Conditions for expression of the fusion protein in E. coli cell were optimized. Fusing of the enzymes into a single polypeptide chain with peroxiredoxin 6 at the N-terminus (PSH) did not affect their activities. On the contrary, the chimeric protein with reverse order of enzymes (SPH) was not obtained in a water-soluble active form. The active chimeric protein (PSH) exhibiting both peroxidase and superoxide dismutase activities was prepared and its physicochemical properties were characterized.     

Directed evolution of a dextransucrase for increased constitutive activity and the synthesis of a highly branched dextran

An Escherichia coli transformant (pDSRB742CK) was obtained from the DSRB742 clone by using ultrasoft X-rays for the expression of a dextransucrase. The enzyme differed in several aspects from DSRB742 dextransucrase: it (1) was constitutive; (2) was extracellular; (3) had 2.6 times greater activity (0.035 IU/ml and 0.23 IU/mg); and (4) synthesized a highly (15.6%) -(1→3) branched dextran. Seven nucleotides of the parent gene (dsrB742) were changed in the nucleotide sequence; four nucleotides were changed in the open reading frame (ORF) that resulted in a 30 amino acid deletion in the N-terminus.     

N-(2-chlorobenzyloxycarbonyloxy)-succinimide as a terminating agent for solid-phase peptide synthesis: Application to a one-step purification procedure

Summary The solid-phase synthesis of peptides is limited by the ability to separate the target sequence from chromatographically similar deletion and truncated impurities. Earlier we have reported the development of a one-step purification procedure for Boc- and Fmoc-synthesised peptides, which involves the incorporation of a base-labile probe with enhanced chromatographic properties at the N-terminal residue of the peptidyl-resin. To prevent the coderivatisation of deletion peptides, an efficient capping procedure is required at each step of chain assembly to terminate unreacted amino groups. N-(2-Chlorobenzyloxycarbonyloxy)-succinimide (Z(2-Cl)-OSu) was found to be a highly effective capping agent for automated SPPS, because it is (i) a solid which can be dissolved when required to limit possible degradation; (ii) stable to the reagents commonly used for Boc/Fmoc chemistries; and (iii) sufficiently reactive so as not to significantly extend cycle times. We demonstrate the effectiveness of a 5 min capping cycle, using Z(2-Cl)-OSu, by synthesising several peptides ranging from 12 to 101 residues in length, by both the Fmoc and Boc chemical strategies.     

Simplified gene synthesis: a one-step approach to PCR-based gene construction

PCR-based gene synthesis conventionally requires two steps: first, all overlapping oligonucleotides are assembled by self-priming; then an additional pair of primers is used to amplify the full-length gene product. Here we propose a simplified method of gene synthesis which combines these two steps into one. We have found that the efficiency of this one-step method, which we term "Simplified Gene Synthesis", is affected by multiple parameters of the PCR reactions. In particular, the choice of polymerase is critical for successful one-step assembly. Other important factors include the concentration of assembly oligonucleotides and amplification primers. Moreover, we offer a general method to estimate, given a known mutation rate, how many clones should be sequenced in order to be confident of obtaining at least one correct gene product. Having determined the accuracy of gene products synthesized under optimal conditions with Simplified Gene Synthesis, we show that our estimation works well. Overall, the simplified gene synthesis provides an easier and more efficient approach to gene synthesis, providing a further step towards the future goal of generalized automation for this process.     

On the kinetics of dextransucrase and dextran synthesis in batch reactors

Summary Basic dextransucrase kinetics are described by the substrate inhibition model and the parameters are evaluated; the model describes fairly well the behaviour in a batch stirred reactor. Optimum conditions for maximum productivity are experimentally and theoretically determined.     

Acetohydroxyacid synthase: a new enzyme for chiral synthesis of R-phenylacetylcarbinol

We have found that acetohydroxyacid synthase (AHAS) is an efficient catalyst for the enantiospecific (> or =98% enantiomeric excess) synthesis of (R)-phenylacetylcarbinol (R-PAC) from pyruvate and benzaldehyde, despite the fact that its normal physiological role is synthesis of (S)-acetohydroxyacids from pyruvate and a second ketoacid. (R)-phenylacetylcarbinol is the precursor of important drugs having alpha and beta adrenergic properties, such as L-ephedrine, pseudoephedrine, and norephedrin. It is currently produced by whole-cell fermentations, but the use of the isolated enzyme pyruvate decarboxylase (PDC) for this purpose is the subject of active research and development efforts. Some of the AHAS isozymes of Escherichia coli have important advantages compared to PDC, including negligible acetaldehyde formation and high conversion of substrates (both pyruvate and benzaldehyde) to PAC. Acetohydroxyacid synthase isozyme I is particularly efficient. The reaction is not limited to condensation of pyruvate with benzaldehyde and other aromatic aldehydes may be used.     

A self-cleavable sortase fusion for one-step purification of free recombinant proteins

A new protein fusion system has been developed to generate free recombinant protein in a single affinity chromatographic step. The key component in the fusion is the catalytic core of sortase A from Staphylococcus aureus (SrtAc), which recognizes and cleaves the Thr-Gly bond at an LPXTG sequence with moderate activity. The fusion here consists of an N-terminal His6 tag, SrtAc, and an LPETG linker followed by protein of interest at the C-terminus. The fusion protein is expressed in Escherichia coli and purified by immobilized metal-ion affinity chromatography (IMAC). The immobilized fusion then undergoes on-column SrtAc-mediated cleavage at the LPETG site in the presence of Ca2+ and/or triglycine. The target protein with an extra N-terminal glycine is released from the fusion while the N-terminal portion remains bound to the column. Because the cleavage enzyme SrtAc is co-expressed as a fusion with the target protein, the purification system eliminates exogenous proteolysis. This purification approach is simple, robust, inexpensive, time saving, and allows purification of free recombinant protein via one-step chromatography.     

Construction of a fusion enzyme system by gene splicing as a new molecular recognition element for a sequence biosensor.

A bifunctional fusion enzyme system constructed by gene splicing is proposed as a new model to develop sequence biosensors, taking maltose biosensor as an example. The cDNA fragment of Aspergillus niger glucoamylase (E.C 3.2.1.3, GA) was fused to the 3' end of Aspergillus niger glucose oxidase (E.C 1.1.3.4, GOD) gene with the insertion of a flexible linker peptide [-(Ser-Gly)5-] coding sequence. The fusion gene was cloned into the vector pPIC9 and expressed in Pichia pastoris GS115 under the control of the AOX1 promoter. It was found that a bifunctional hybrid protein with a molecular weight of 430 kDa was secreted after induction with methanol. The fusion enzyme GOD-(Ser-Gly)5-GA (GLG) was purified using Q Sepharose Fast Flow ion-exchange chromatography. Kinetic analysis demonstrated that GLG retained the typical kinetic properties of both GA and GOD. After being immobilized on an aminosilanized glass slide through covalent bonding by glutaraldehyde, GLG showed much higher sequential catalytic efficiency than the mixture of separately expressed GA and GOD (GA/GOD). Maltose biosensors were fabricated with GLG and GA/GOD, respectively. The performance characteristics of the maltose biosensor with respect to reproducibility, signal level, and linearity were effectively improved by using the fusion enzyme. Our findings offer a basis for the development of other sequence biosensors.     

Removal of ampholines by hollow fiber dialysis: Application to dextransucrase

Ampholines must be removed from proteins that have been purified using isoelectric focusing chromatography before hydrolysis since ninhydrin positive fragments result. Hollow fiber dialysis was shown to be a fast and efficient method for the removal of ampholines from dextransucrase after isoelectric focusing. The purified protein was then subjected to amino acid analysis.     

The mechanism of dextransucrase action: Activation of dextransucrase from Streptococcus mutans OMZ 176 by dextran and modified dextran and the nonexistence of the primer requirement for the synthesis of dextran

Streptococcus mutans OMZ 176 was grown in a sucrose-free medium containing fructose as a carbohydrate source. Dextransucrase was precipitated from the culture supernatant with 40% saturated ammonium sulfate. The activity of dextransucrase was shown to be stimulated by exogenous dextran. Maximum activity was reached when the concentration of exogenous dextran was 2 mg/ml. Dextrans modified at the nonreducing ends by reaction with tripsyl chloride and/or by hydrolysis with an exodextranase also activated dextransucrase four to six times over that of a control. The exodextranasemodified dextrans have nonreducing chains that are very short in comparison with unmodified dextran and the tripsyl-modified dextrans have chains that are blocked at the nonreducing ends with a triisopropylbenzenesulfonyl group on the C₆ hydroxyl group. Because the nonreducing ends of the modified dextrans are not available for reaction, the activation of dextransucrase by these modified dextrans cannot be due to primer reactions with the nonreducing ends. The activation of dextransucrase, thus, must be by an alternate mechanism. Two alternative mechanisms discussed are an allosteric effect and nucleophilic displacement reactions by the added dextran. It was also found that the addition of increasing amounts of dextran shifted the synthesis from an insoluble dextran to a soluble dextran.     

Construction and characterization of a bifunctional fusion enzyme of Bacillus-sourced beta-glucanase and xylanase expressed in Escherichia coli

A chimeric gene, Glu-Xyl, encoding Bacillus amyloliquefaciens glucanase (Glu, 24.4 kDa) and Bacillus subtilis xylanase (Xyl, 21.2 kDa), was constructed via end-to-end fusion and expressed successfully in Escherichia coli. The purified fusion protein (46.1 kDa) exhibited both glucanase and xylanase activities. Compared with parental enzymes, the Glu moiety was characterized by kinetic parameters of decreased K(m) (0.66-fold) and increased K(cat) (2.75-fold), whereas the Xyl moiety had an increased K(m) (1.37-fold) and decreased K(cat) (0.79-fold). These indicate a 3.15-fold net increase and a 31% decrease in catalytic efficiency (K(cat)/K(m)) of the Glu and Xyl moieties. Activities and stabilities of both moieties at 40-90 degrees C or pH 3.0-10.0 were compared with those of the parental enzymes. Despite some variations, common optima were 40 degrees C and pH 9.0 for the Glu moiety and parent, and 50-60 degrees C and pH 9.0 for the Xyl counterparts. Thus, the fusion enzyme Glu-Xyl was bifunctional, with greatly enhanced glucanase activity associated with a decrease in xylanase activity.     

Expression and characterization of a DNase I-Fc fusion enzyme

Recombinant human deoxyribonuclease I (DNase I) is an important clinical agent that is inhaled into the airways where it degrades DNA to lower molecular weight fragments, thus reducing the viscoelasticity of sputum and improving the lung function of cystic fibrosis patients. To investigate DNases with potentially improved properties, we constructed a molecular fusion of human DNase I with the hinge and Fc region of human IgG1 heavy chain, creating a DNase I-Fc fusion protein. Infection of Sf9 insect cells with recombinant baculovirus resulted in the expression and secretion of the DNase I-Fc fusion protein. The fusion protein was purified from the culture medium using protein A affinity chromatography followed by desalting by gel filtration and was characterized by amino-terminal sequence, amino acid composition, and a variety of enzyme-linked immunosorbent assays (ELISA) and activity assays. The purified fusion contains DNase I, as determined by a DNase I ELISA and an actin-binding ELISA, and an intact antibody Fc region, which was quantified by an Fc ELISA, in a 2:1 stoichiometric ratio, respectively. The dimeric DNase I-Fc fusion was functionally active in enzymatic DNA digestion assays, albeit about 10-fold less than monomeric DNase I. Cleavage of the DNase I-Fc fusion by papain resulted in a specific activity comparable to the monomeric enzyme. Salt was inhibitory for wild type monomeric DNase I but actually enhanced the activity of the dimeric DNase I-Fc fusion. The DNase I-Fc fusion protein was also less Ca2+-dependent than DNase I itself. These results are consistent with a higher affinity of the dimeric fusion protein to DNA than monomeric DNase I. The engineered DNase I-Fc fusion protein described herein has properties that may have clinical benefits.