Extracellular production of human parathyroid hormone as a thioredoxin fusion form in Escherichia coli by chemical permeabilization combined with heat treatment

A pET system encoding the fusion protein gene of thioredoxin (Trx) and human parathyroid hormone (hPTH) was introduced into Escherichia coli BL21 (DE3). Recombinant Trx-hPTH fusion protein was expressed in soluble form in the cytoplasm of the E. coli transformant. To recover Trx-hPTH from the E. coli culture efficiently, a novel tactic was developed by adding Triton X-100 into the fermentation culture at the exponential growth phase of E. coli and by heat treatment of the culture at the end of the fermentation. A concentration of 1% (v/v) Triton X-100 was added into the culture at the same time as IPTG addition after optimization. Under these conditions, addition of Triton X-100 had little effect on the cell growth, but more than 75% of the total recombinant Trx-hPTH was released into the fermentation broth. Also, a much higher volumetric yield of recombinant Trx-hPTH could be obtained with protein release compared to yield without protein release. Simultaneously, owing to the highly thermal stability of Trx-hPTH fusion protein, heat treatment of the fermentation broth at 80 degrees C for 15 min at the end of fermentation was employed for primary purification. Results demonstrated that heat treatment not only boosted further release of the recombinant Trx-hPTH fusion protein into the fermentation broth but also precipitated/denatured most of the nontarget proteins released in the broth. The tactics described herein integrated the fermentation process with subsequent recovery steps and thus provided a valuable and economical method for the production of Trx-hPTH and maybe some other Trx fusions in E. coli.     

Enhanced production of exocellular glucansucrase from Leuconostoc dextranicum NRRL B-1146 using response surface method

Statistically-based experimental designs were applied to optimize the fermentation for the production of glucosyltransferase by Leuconostoc dextranicum NRRL B-1146. Eleven medium components were examined for their significance on enzyme production using Plackett-Burman factorial design. Tween 80, sucrose and K2HPO4 significantly improved the enzyme production process. The combined effect of these nutrients on glucansucrase production were studied using a 2 2 full-factorial central composite design, a second-order polynomial was established to identify the relationship between the enzyme output and the three medium components. The optimal concentration of variables for maximum glucansucrase production were Tween 80 (0.55%, v/v); sucrose (5.6%, w/v) and K2HPO4 (1%, w/v). The maximum enzyme activity by predicted model was 6.53 U/ml that was in perfect agreement with the actual experimental value (6.40 U/ml).     

Selective permeabilization for the high-throughput measurement of compartmented enzyme activities in mammalian cells

Permeabilization was evaluated as a rapid method to prepare mammalian cells for subcellular enzyme activity measurement. It was observed that enzymes can be measured directly in cell suspensions permeabilized by Triton X-100 and digitonin with various concentrations. Total enzyme activities measured in permeabilized cells were identical to those measured in sonicated cells showing that permeabilization can replace the more complicated sonication method. Tuning of digitonin concentration allowed selective permeabilization of plasma and mitochondrial membranes. This was studied by analyzing the release of extramitochondrial and mitochondrial marker enzymes on treatment with different concentrations of the agent. Solely the plasma membrane was permeabilized by using 0.01–0.02% (w/v) digitonin. Access to all cellular enzymes was achieved by using 0.05% (v/v) Triton X-100. This selective permeabilization was further evaluated in a 96-well plate format by testing additional marker enzymes and additional cell lines, Hep G2 and CHO-K1, applying the developed protocol. The presented method is well suited for the high-throughput analysis of subcellular localization and activity of enzymes. The method is simple and enables one to distinguish between mitochondrial and extramitochondrial activities, which is usually achieved only by much more complicated and time-consuming cell preparation.     

Influence of phage population on the phage-mediated bioluminescent adenylate kinase (AK) assay for detection of bacteria

AIMS: The effect of phage concentration on the activity of adenylate kinase (AK) released from the cells lysed during infection was investigated in order to optimize a bioluminescent phage-mediated method for bacterial enumeration. METHODS AND RESULTS: The number of bacteria lysed by phages specific to Salmonella enteritidis and E. coli was determined using a bioluminescent method for the detection of AK released. In order to optimize the assay, the effect of phage concentration and time of infection on the amount of AK released was investigated. The release of AK was greatest at a multiplicity of infection (moi) of 10-100. CONCLUSION: The amount of AK released from Salmonella enteritidis and E. coli G2-2 cells by specific phages, SJ2 and AT20, respectively, depended on the type of bacteria, the stage of growth, the nature of phage, moi and time. SIGNIFICANCE AND IMPACT OF THE STUDY: An assay is described which allows detection of E. coli and Salmonella Enteritidis within 2 h at levels of 103 cfu ml-1.     

Factors effecting the solubilisation of stearoyl-coA desaturase of hen liver microsomes.

1. The lipid requirement for maximum desaturase activity was investigated using acetone/water mixtures. It was shown that for maximum stearoyl-CoA desaturase activity of hen liver microsomes neither the total neutral lipid fraction nor 44% of the phospholipid fraction were required. 2. The effect of sodium deoxycholate, Triton X-100, Nonidet P-40 and Bio-solv on the enzyme activity indicated that the neutral detergents had a milder effect than the ionic detergent but both classes could cause considerable irreversible loss of activity. 3. The treatment of the microsomes with 2.5% (v/v) water in acetone greatly improved the effective solubilising power of Triton X-100. The yield of desaturase in the 100 000 X g supernatant obtained by treating the microsomal fraction in this way was strongly dependent upon protein concentration. Maximum solubilisation was achieved with25 mg protein per ml 1% (w/v) Triton X-100 in 0.1 M potassium phosphate buffer pH 7.4. 4. A comparison of the properties of the solubilised and membrane-bound enzyme was made by an investigation of: (i) the temperature and pH optimum, (ii) activation energy and (iii) the effect of inhibitors on the enzyme activity.     

Lipid A modifications characteristic of Salmonella typhimurium are induced by NH4VO3 in Escherichia coli K12. Detection of 4-amino-4-deoxy-L-arabinose, phosphoethanolamine and palmitate.

Two-thirds of the lipid A in wild-type Escherichia coli K12 is a hexa-acylated disaccharide of glucosamine in which monophosphate groups are attached at positions 1 and 4'. The remaining lipid A contains a monophosphate substituent at position 4' and a pyrophosphate moiety at position 1. The biosynthesis of the 1-pyrophosphate unit is unknown. Its presence is associated with lipid A translocation to the outer membrane (Zhou, Z., White, K. A., Polissi, A., Georgopoulos, C., and Raetz, C. R. H. (1998) J. Biol. Chem. 273, 12466-12475). To determine if a phosphatase regulates the amount of the lipid A 1-pyrophosphate, we grew cells in broth containing nonspecific phosphatase inhibitors. Na2WO4 and sodium fluoride increased the relative amount of the 1-pyrophosphate slightly. Remarkably, NH4VO3-treated cells generated almost no 1-pyrophosphate, but made six major new lipid A derivatives (EV1 to EV6). Matrix-assisted laser desorption ionization/time of flight mass spectrometry of purified EV1 to EV6 indicated that these compounds were lipid A species substituted singly or in combination with palmitoyl, phosphoethanolamine, and/or aminodeoxypentose residues. The aminodeoxypentose residue was released by incubation in chloroform/methanol (4:1, v/v) at 25 degrees C, and was characterized by 1H NMR spectroscopy. The chemical shifts and vicinal coupling constants of the two anomers of the aminodeoxypentose released from EV3 closely resembled those of synthetic 4-amino-4-deoxy-L-arabinose. NH4VO3-induced lipid A modification did not require the PhoP/PhoQ two-component regulatory system, and also occurred in E. coli msbB or htrB mutants. The lipid A variants that accumulate in NH4VO3-treated E. coli K12 are the same as many of those normally found in untreated Salmonella typhimurium and Salmonella minnesota, demonstrating that E. coli K12 has latent enzyme systems for synthesizing these important derivatives.     

信号肽及化学通透剂对环糊精葡萄糖基转移酶胞外分泌的影响

【目的】研究不同的信号肽和化学通透剂对重组环糊精葡萄糖基转移酶(CGTase)胞外分泌的影响,提高CGTase的胞外分泌量。【方法】扩增地芽孢杆菌CHB1(Geobacillus sp.CHB1)的CGTase基因,构建带有地芽孢杆菌CHB1自身信号肽、Omp A、Pel B信号肽和不带信号肽的4种重组质粒;比较4种重组质粒对重组CGTase胞外分泌的影响,筛选最优的信号肽;考察甘氨酸、Triton X-100、SDS和Tween 80四种化学通透剂对重组CGTase胞外分泌的影响,确定最佳的化学通透剂及其浓度。【结果】Omp A信号肽介导的分泌效果最好,胞外酶活达到7.44 U/m L,分别是Pel B、CHB1信号肽的2.04倍和11.27倍,不带信号肽的重组质粒菌胞外检测不到酶活;携带Omp A信号肽的重组质粒菌发酵48 h,同时添加浓度为0.6%的甘氨酸和0.3%的Triton X-100,胞外酶活达最大到14.27 U/m L;SDS和Tween 80对该酶的胞外分泌具有明显的抑制作用。【结论】Omp A信号肽的介导效果最佳,同时添加浓度为0.6%和0.3%的甘氨酸和Triton X-100可以有效促进胞外分泌,为该重组酶的高效胞外分泌提供了一种有效的方法。     

A novel alkaline protease from wild edible mushroom Termitomyces albuminosus

A protease with a molecular mass of 30 kDa and the N-terminal sequence of GLQTNAPWGLARSS, was isolated from fresh fruiting bodies of the wild edible mushroom Termitomyces albuminosus. The purification protocol included ion exchange chromatography on DEAE-cellulose, Q-Sepharose, SP-Sepharose and FPLC-gel filtration on Superdex 75. The protein was unadsorbed on DEAE-cellulose and Q-Sepharose, but adsorbed on SP-Sepharose. The optimal pH and temperature of the purified enzyme were 10.6 and 60 °C, respectively. The enzyme was stable in the presence of 2 % (v/v) Tween 80 and 4 M urea. More than 80 % of the enzyme activity was retained in 2 % (v/v) Triton X 100, 54 % in 10 mM EDTA and 31 % in 2 % (w/v) SDS. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), but not inhibited by dithiothreitol (DTT), pepstatin or lima bean trypsin inhibitor suggesting that it was a serine protease but not a trypsin-like one. The protease was inhibited by Hg(2+), Cu(2+), and Fe(3+) ions. The K(m) and V(max) values of the purified enzyme for casein were 8.26 mg ? ml(-1) and 0.668 mg ? ml(-1) ? min(-1), respectively.     

Microencapsulation of alpha-tocopherol using sodium alginate and its controlled release properties

Response surface methodology (RSM) was used to optimize microencapsulation yield (MY) using three independent variables; the ratio of coating material to core material (w/w, X1), the emulsifier concentration (%, v/v, X2), and the CaCl2 concentration (%, w/v, X3). In the preparation of sodium alginate (SA) microcapsule, the regression model equation for the MY was predicted as follows; MY(%) = 56.02 + 3.64X2 + 3.18X1X2 - 3.74X2(2). The optimal conditions for the SA microcapsule were obtained at the [SA]/[alpha-TP] ratio of 6.6:3.4 (w/w), [emulsifier] of 1.35% (v/v), and [CaCl2] of 4.3% (w/v), and the predicted MY in this condition was of 57.2%. In vitro alpha-TP releasing test of the SA-based microcapsules was performed. The SA microcapsule released 28.8% of alpha-TP when exposed in the simulated gastric fluid (SGF, pH 1.2) for 24 h. In the simulated intestinal fluid (SIF, pH 7.4), the amount of released alpha-TP (81.5%) was significantly greater than that in the SGF. The duration time required for releasing 50 (T50%) and 70% (T70%) of alpha-TP from the SA-microcapsule were calculated to be 3.8 and 12.3 h, respectively. From these results, it was suggested that SA microcapsule would be structurally resistant against acidic environment, and it would rapidly release core material under mild alkali condition.     

Artificial intelligence based optimization of exocellular glucansucrase production from Leuconostoc dextranicum NRRL B-1146

Two different artificial intelligence techniques namely artificial neural network (ANN) and genetic algorithm (GA) were integrated for optimizing fermentation medium for the production of glucansucrase. The experimental data reported in a previous study were used to build the neural network. The ANN was trained using the back propagation algorithm. The ANN predicted values showed good agreement with the experimentally reported ones from a response surface based experiment. The concentrations of three medium components: viz Tween 80, sucrose and K(2)HPO(4) served as inputs to the neural network model and the enzyme activity as the output of the model. A model was generated with a coefficient of correlation (R(2)) of 1.0 for the training set and 0.90 for the test data. A genetic algorithm was used to optimize the input space of the neural network model to find the optimum settings for maximum enzyme activity. This artificial neural network supported genetic algorithm predicted a maximum glucansucrase activity of 6.92U/ml at medium composition of 0.54% (v/v) Tween 80, 5.98% (w/v) sucrose and 1.01% (w/v) K(2)HPO(4). ANN-GA predicted model gave a 6.0% increase of enzyme activity over the regression based prediction for optimized enzyme activity. The maximum enzyme activity experimentally obtained using the ANN-GA designed medium was 6.75+/-0.09U/ml which was in good agreement with the predicted value.     

Quantities of individual aminoacyl-tRNA families and their turnover in Escherichia coli.

The cellular content of all 20 aminoacyl-tRNA species was determined in small cultures of Escherichia coli by labeling cells with 3H-amino acids and extraction of 3H-amino acid-labeled nucleic acid by standard procedures. Of 3H-amino acid-labeled material, 25 to 90% was identified as 3H-aminoacyl-tRNA by the following criteria: sensitivity to base hydrolysis with expected kinetics; association of 3H counts released by base treatment of the 3H-amino acid-labeled nucleic acid with amino acid standards upon paper chromatography of the hydrolysate; and changes in the amount of 3H-amino acid-labeled nucleic acid recovered from cells as a function of time. Individual aminoacyl-tRNA content was determined with as few as 8 X 10(7) to 4 X 10(8) E. coli cells. Although the total number of aminoacyl-tRNA molecules per cell varied only by 10 to 20% among various strains of E. coli, some individual aminoacyl-tRNA families varied two- to threefold among strains. For a given amino acid, the number of aminoacyl-tRNA molecules per cell in E. coli strain K38 growing with a doubling time of 60 min varied from 730 (glutamyl-tRNA) to 7,910 (valyl-tRNA) with a mean value of 3,200. The total number of aminoacyl-tRNA molecules per cell (6.4 X 10(4)) in E. coli K38 was 5.5-fold higher than the number of ribosomes and was equal to 84% of the amount of elongation factor Tu molecules per cell. The ratio of aminoacyl-tRNA to synthetase for 10 amino acids varied from about 1 to 15 with a mean value of 4.7. The turnover of individual aminoacyl-tRNA families in E. coli cells was estimated to be in the range of 1.7 to 8.1 s-1 with a mean value of 3.7 s-1. An estimate of minimum in vivo molecular activity of aminoacyl-tRNA synthetases gives values of 2 to 48 s-1 for individual enzymes.     

Optimization of fermentation medium and conditions for mycelial growth and water-soluble exo-polysaccharides production by Isaria farinosa B05

The optimal fermentation medium and conditions for mycelial growth and water-soluble exo-polysaccharides production by Isaria farinosa B05 were investigated. The medium components and fermentation conditions were optimized according to the one at a time method, while the concentration of medium components was determined by the orthogonal matrix method. The results showed that the optimal fermentation medium was as follows: sucrose 3.5% (w/v), peptone 0.5%, yeast extract 0.2%, K(2)HPO(4) 0.1%, and MgSO(4) 0.05%. The suitable fermentation conditions were as follows: initial pH 7.0, temperature 25 degrees C, medium volume 75 mL/250 mL, inoculum volume 5% (v/v), time 5d. In such optimal nutrition and environmental conditions, the maximal mycelial yield was 2.124 g/100 mL after 4 day's fermentation, while maximal water-soluble exo-polysaccharides production reached 2.144 g/L after 5 day's fermentation.     

Human neutrophil N-acetyl-beta-D-glucosaminidase: heparin inhibition

To determine N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) in human neutrophil granules separated by a method requiring heparin, the inhibition of this enzyme by heparin was studied. Neutrophils were purified from blood of five donors by modifications of the Hypaque-Ficoll and dextran separation methods resulting in a suspension which was 96% neutrophils. Neutrophil lysates were assayed for N-acetyl-beta-D-glucosaminidase by measuring the amount of p-nitrophenol released from p-nitrophenyl-N-acetyl-beta-D-glucosaminide. The reaction showed first-order kinetics with regard to enzyme concentration. Triton X-100, 0.1% v/v, enhanced enzyme activity. Heparin was shown to reduce neutrophil lysate N-acetyl-beta-D-glucosaminidase to a specific activity of 46% at a heparin concentration of 2 units per assay and to 43% (maximal inhibition) at 17 and 50 units of heparin per assay. Substantially higher heparin concentrations partially restored the inhibited activity, the maximal restoration being a return to 80% of the original activity at 1700 units of heparin per assay. Protamine sulfate was assessed for its ability to restore N-acetyl-beta-D-glucosaminidase activity in the presence of heparin. At 1.0 mg/10 units of heparin, protamine restores enzyme activity to its heparin-free activity. These studies of human neutrophil N-acetyl-beta-D-glucosaminidase demonstrate: (1) specific enzyme activity is 28.8 +/- 7.0 nmole p-nitrophenol released per minute per milligram of protein or 1.7 +/- 0.5 nmole p-nitrophenol released per minute per 10(6) neutrophils; (2) heparin rapidly but finitely inhibits enzyme activity at very low concentrations and paradoxically restores it toward normal at high concentrations; and (3) protamine sulfate restores enzyme activity inhibited by heparin.     

Refolding and purification of Zymomonas mobilis levansucrase produced as inclusion bodies in fed-batch culture of recombinant Escherichia coli

Zymomonas mobilis levansucrase was overproduced by the fed-batch culture of recombinant Escherichia coli harboring a novel expression system that is constitutively expressed by the promoter from the Rahnella aquatilis levansucrase gene. Most of the levansucrase was produced as inclusion bodies in the bacterial cytoplasm, accounting for approximately 20% of the total cellular protein. Refolding after complete denaturation by high concentrations of urea or guanidine hydrochloride was not successful, resulting in large amounts of insoluble aggregates. During the development of the refolding method, it was found that direct solubilization of the inclusion bodies with Triton X-100 reactivated the enzyme, with a considerable refolding efficiency. About 65% of inclusion body levansucrase was refolded into active levansucrase in the renaturation buffer containing 4% (v/v) Triton X-100. The in vitro refolded enzyme was purified to 95% purity by single-step DEAE-Sepharose ion exchange chromatography. Triton X-100 was removed by this ion exchange chromatography.     

Control of autolysis of Bacillus subtilis for selective production of the intracellular enzyme glucose-6-phosphate dehydrogenase in aqueous two-phase systems

A method for the release of intracellular enzyme by autolysis of Bacillus subtilis cells is presented. Both the growth and lysis processes were further applied to aqueous two-phase systems (ATPS). Lysis induced by the addition of Triton X-100 and by low-temperature treatment facilitated the release of cytoplasmic enzyme glucose-6-phosphate dehydrogenase (G6PDH) in ATPS. The release selectivity increased when lysis was regulated by the addition of 50 μM or 100 μM Triton X-100. Cardiolipin efficiently inhibited the autolytic process. Control of the autolytic system promoted the selective release of G6PDH. B. subtilis cells could be grown and lysed in aqueous two-phase systems in a similar fashion to the conventional single-phase medium solutions. The released enzymes were partitioned according to their surface properties. G6PDH were extracted to the top phase in a PEG1540/Dex100K-200K sytem. Cells were partitioned to the bottom phase or the interface, and could be recycled into the fermentor. The selectivity of enzyme production was also increased in two-phase systems by the addition of cardiolipin.     

Low molecular weight chitosans--preparation with the aid of pronase, characterization and their bactericidal activity towards Bacillus cereus and Escherichia coli

The homogeneous low molecular weight chitosans (LMWC) of molecular weight 9.5-8.5 kDa, obtained by pronase catalyzed non-specific depolymerization (at pH 3.5, 37 degrees C) of chitosan showed lyses of Bacillus cereus and Escherichia coli more efficiently (100%) than native chitosan (<50%). IR and (1)H-NMR data showed decrease in the degree of acetylation (14-19%) in LMWC compared to native chitosan ( approximately 26%). Minimum inhibitory concentration of LMWC towards 10(6) CFU ml(-1) of B. cereus was 0.01% (w/v) compared to 0.03% for 10(4) CFU ml(-1) of E. coli. SEM revealed pore formation as well as permeabilization of the bacterial cells, as also evidenced by increased carbohydrate and protein contents as well as the cytoplasmic enzymes in the cell-free supernatants. N-terminal sequence analyses of the released proteins revealed them to be cytoplasmic/membrane proteins. Upon GLC, the supernatant showed characteristic fatty acid profiles in E. coli, thus subscribing to detachment of lipopolysaccharides into the medium, whereas that of B. cereus indicated release of surface lipids. The mechanism for the observed bactericidal activity of LMWC towards both Gram-positive and Gram-negative bacteria has been discussed.     

Orthophosphate anion enhances the stability and activity of endoxylanase from Bacillus sp

Endoxylanase, for which the optimum temperature is 60 degrees C (optimum pH 7), is labile to heat. Because the isoelectric point (pI) value of this xylanase is 10.6, the net charge of this enzyme is positive at pH 7. Thus, ions are likely to influence its enzyme structure and the thermal stability of endoxylanase may improve. Among the various ions tested, orthophosphate anion (HPO(4)(2-)) was found to significantly improve not only the stability but the activity of xylanase. When K(2)HPO(4) concentration was increased from 50 mM to 1.2 M, the T(m )value of xylanase was increased from 60.0 degrees C to 74.5 degrees C. The affinity of xylanase on xylan also increased along with K(2)HPO(4) concentration. Thus, the xylanase activity at 0.6 M K(2)HPO(4) was 2.3-fold higher than that at 50 mM K(2)HPO(4), and 120.2-fold higher than that in 40 mM MOPS buffer. This enhanced activity in the presence of K(2)HPO(4 )probably takes place because the orthophosphate anion affects the binding and catalytic residues of endoxylanase.     

Purification and characterization of the phosphate/hydroxyl ion antiport protein from rat liver mitochondria.

The phosphate transport protein was purified from rat liver mitochondria by extraction in an 8% (v/v) Triton X-100 buffer followed by adsorption chromatography on hydroxyapatite and Celite. SDS/polyacrylamide-gel electrophoresis (10%, w/v) demonstrated that the purified polypeptide was apparently homogeneous when stained with Coomassie Blue and had a subunit Mr of 34,000. However, lectin overlay analysis of this gel with 125I-labelled concanavalin A demonstrated the presence of several low- and high-Mr glycoprotein contaminants. To overcome this problem, mitochondria were pre-extracted with a 0.5% (v/v) Triton X-100 buffer as an additional step in the purification of phosphate transport protein. SDS/polyacrylamide gradient gel electrophoresis (14-20%, w/v) of the hydroxyapatite and Celite eluates revealed one major band of Mr 34,000 when stained with Coomassie Blue. The known thiol group sensitivity of the phosphate transporter was employed to characterize the isolated polypeptide further. Labelling studies with N-[2-3H]ethylmaleimide showed that only the 34,000-Mr band was labelled in both the hydroxyapatite and Celite fractions, when purified from rat liver mitochondria. Further confirmation of its identity has been provided with an antiserum directed against the 34,000-Mr protein. Specific partial inhibition of phosphate uptake, as measured by iso-osmotic swelling in the presence of (NH4)2HPO4, was achieved when mitoplasts (mitochondria minus outer membrane) were incubated with this antiserum. Finally, amino acid analysis of the rat liver mitochondrial phosphate/hydroxyl ion antiport protein indicates that it is similar in composition to the equivalent protein isolated from ox heart.     

Statistically designed optimisation of enzyme catalysed starch removal from potato pulp

Potato pulp is a high-volume, low-value byproduct stream resulting from the industrial manufacture of potato starch. The pulp is a rich source of biologically functional dietary fibers, but the targeted valorisation of the fibers requires removal of the residual starch from the pulp. The objective of this study was to release the residual starch, making up 21–22% by weight of the dry matter, from the potato pulp in a rational way employing as few steps, as few enzyme activities, as low enzyme dosages, as low energy input (temperature and time), and as high pulp dry matter as possible. Starch removal to obtain dietary fibers is usually accomplished via a three step, sequential enzymatic treatment procedure using a heat stable α-amylase, protease, and amyloglucosidase. Statistically designed experiments were performed to investigate the influence of enzyme dose, amount of dry matter, incubation time and temperature on the amount of starch released from the potato pulp. The data demonstrated that all the starch could be released from potato pulp in one step when 8% (w/w) dry potato pulp was treated with 0.2% (v/w) (enzyme/substrate (E/S)) of a thermostable Bacillus licheniformis α-amylase (Termamyl^® SC) at 70 °C for at least 65 min. The study also indicated that the amount of other carbohydrates released from the pulp during the release of starch was less than using the AOAC Official Method 985.29 and another recently published starch release method employed as a pretreatment for enzymatic upgrading of a pectinaceous potato pulp fiber.     

Construction of deoxyriboaldolase-overexpressing Escherichia coli and its application to 2-deoxyribose 5-phosphate synthesis from glucose and acetaldehyde for 2'-deoxyribonucleoside production

The gene encoding a deoxyriboaldolase (DERA) was cloned from the chromosomal DNA of Klebsiella pneumoniae B-4-4. This gene contains an open reading frame consisting of 780 nucleotides encoding 259 amino acid residues. The predicted amino acid sequence exhibited 94.6% homology with the sequence of DERA from Escherichia coli. The DERA of K. pneumoniae was expressed in recombinant E. coli cells, and the specific activity of the enzyme in the cell extract was as high as 2.5 U/mg, which was threefold higher than the specific activity in the K. pneumoniae cell extract. One of the E. coli transformants, 10B5/pTS8, which had a defect in alkaline phosphatase activity, was a good catalyst for 2-deoxyribose 5-phosphate (DR5P) synthesis from glyceraldehyde 3-phosphate and acetaldehyde. The E. coli cells produced DR5P from glucose and acetaldehyde in the presence of ATP. Under the optimal conditions, 100 mM DR5P was produced from 900 mM glucose, 200 mM acetaldehyde, and 100 mM ATP by the E. coli cells. The DR5P produced was further transformed to 2'-deoxyribonucleoside through coupling the enzymatic reactions of phosphopentomutase and nucleoside phosphorylase. These results indicated that production of 2'-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase is possible with the addition of a suitable energy source, such as ATP.