Production of glutamine synthetase in <Emphasis Type="Italic">Escherichia coli</Emphasis> using SUMO fusion partner and application to <Emphasis Type="SmallCaps">l</Emphasis>-glutamine synthesis

l-glutamine (Gln) is an important conditionally necessary amino acid in human body and potential demand in food or medicine industry is expected. High efficiency of l-Gln production by coupling genetic engineered bacterial glutamine synthetase (GS) with yeast alcoholic fermentation system has been developed. We report here first the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of recombinant Bacillus subtilis GS. In order to obtain GS with high Gln-forming activity, safety and low cost for food and pharmaceutics industry, 0.1% (w/v) lactose was selected as inducer. The fusion protein was expressed in totally soluble form in E. coli, and expression was verified by SDS–PAGE and western blot analysis. The fusion protein was purified to 90% purity by nickel nitrilo-triacetic acid (Ni–NTA) resin chromatography with a yield of 625 mg per liter fermentation culture. After the SUMO/GS fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni–NTA column. Finally, about 121 mg recombinant GS was obtained from 1 l fermentation culture with no less than 96% purity. The recombinant purified GS showed great transferase activity (23 U/mg), with 25 U recombinant GS in a 50 ml reaction system, a biosynthesis yield of 27.5 g/l l-Gln was detected by high pressure liquid chromatography (HPLC) or thin-layer chromatography. Thus, the application of SUMO technology to the expression and purification of GS potentially could be employed for the industrial production of l-Gln.     

Production of Bioactive γ-Glutamyl Transpeptidase in <Emphasis Type="Italic">Escherichia coli</Emphasis> Using SUMO Fusion Partner and Application of the Recombinant Enzyme to <Emphasis Type="SmallCaps">l</Emphasis>-Theanine Synthesis

The amino acid l-theanine (γ-glutamylethylamide) has potential important applications in the food and pharmaceutical industries and increased demand for this compound is expected. It is the major “umami” (good taste) component of tea and its favorable physiological effects on mammals have been reported. An enzymatic method for the synthesis of l-theanine involving recombinant Escherichia coli γ-glutamyltranspeptidase (GGT) has been developed. We report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of recombinant Escherichia coli γ-GGT. In order to obtain γ-GGT with high theanine-forming activity, safety, and low cost for food and pharmaceutics industry, M9 (consisting of glycerol and inorganic salts) and 0.1% (w/v) lactose were selected as culture medium and inducer, respectively. The fusion protein was expressed in soluble form in E. coli, and expression was verified by SDS-PAGE and western blot analysis. The fusion protein was purified to 90% purity by nickel–nitrilotriacetic acid (Ni–NTA) resin chromatography with a yield of 115 mg per liter fermentation culture. After the SUMO/γ-GGT fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni–NTA column. Finally, about 62 mg recombinant γ-GGT was obtained from 1 l fermentation culture with no less than 95% purity. The recombinant γ-GGT showed great transpeptidase activity, with 1500 U of purified recombinant γ-GGT in a 1-l reaction system, a biosynthesis yield of 41 g of l-theanine was detected by paper chromatography or high pressure liquid chromatography (HPLC). Thus, the application of SUMO technology to the expression and purification of γ-GGT potentially could be employed for the industrial production of l-theanine.     

Enzymatic production of α-<Emphasis Type="SmallCaps">d</Emphasis>-galactose 1-phosphate by lactose phosphorolysis

α-d-Galactose 1-phosphate (αGal1P) is an important building block for the synthesis of nucleotide sugars that are substrates for glycosyltransferases. We have previously reported the creation of novel lactose phosphorylase enzymes that are useful for the synthesis of αGal1P from the cheap and abundant lactose. Here we describe the application of such a lactose phosphorylase in a production system using permeabilized Escherichia coli cells. After purification of the product from the reaction mixture by anion-exchange chromatography and ethanol precipitation, 9.5 grams of highly pure αGal1P were obtained from a 1 l reaction volume.     

Heterologous expression and characterization of <Emphasis Type="Italic">Bacillus coagulans</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase

Bacillus coagulans has been of great commercial interest over the past decade owing to its strong ability of producing optical pure l-lactic acid from both hexose and pentose sugars including l-arabinose with high yield, titer and productivity under thermophilic conditions. The l-arabinose isomerase (L-AI) from Bacillus coagulans was heterologously over-expressed in Escherichia coli. The open reading frame of the L-AI has 1,422 nucleotides encoding a protein with 474 amino acid residues. The recombinant L-AI was purified to homogeneity by one-step His-tag affinity chromatography. The molecular mass of the enzyme was estimated to be 56 kDa by SDS-PAGE. The enzyme was most active at 70°C and pH 7.0. The metal ion Mn²⁺ was shown to be the best activator for enzymatic activity and thermostability. The enzyme showed higher activity at acidic pH than at alkaline pH. The kinetic studies showed that the K _m, V _max and k _cat/K _m for the conversion of l-arabinose were 106 mM, 84 U/mg and 34.5 mM⁻¹min⁻¹, respectively. The equilibrium ratio of l-arabinose to l-ribulose was 78:22 under optimal conditions. l-ribulose (97 g/L) was obtained from 500 g/l of l-arabinose catalyzed by the enzyme (8.3 U/mL) under the optimal conditions within 1.5 h, giving at a substrate conversion of 19.4% and a production rate of 65 g L⁻¹ h⁻¹.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> using semicontinuous fermentation in bioreactor

Semicontinuous fermentation using pellets of Rhizopus oryzae has been recognized as a promising technology for l-lactic acid production. In this work, semicontinuous fermentation of R. oryzae AS 3.819 for l-lactic acid production has been developed with high l-lactic acid yield and volumetric productivity. The effects of factors such as inoculations, CaCO₃ addition time, and temperature on l-lactic acid yield and R. oryzae morphology were researched in detail. The results showed that optimal fermentation conditions for the first cycle were: inoculation with 4% spore suspension, CaCO₃ added to the culture medium at the beginning of culture, and culture temperature of 32–34°C. In orthogonal experiments, high l-lactic acid yield was achieved when the feeding medium was (g/l): glucose, 100; (NH₄)₂SO₄, 2; KH₂PO₄, 0.1; ZnSO₄·7H₂O, 0.33; MgSO₄·7H₂O, 0.15; CaCO₃, 50. Twenty cycles of semicontinuous fermentation were carried out in flask culture. l-lactic acid yield was 78.75% for the first cycle and 80–90% for the repeated cycles; the activities of lactate dehydrogenases (LDH) were 7.2–9.2 U/mg; fermentation was completed in 24 h for each repeated cycle. In a 7-l magnetically stirred fermentor, semicontinuous fermentation lasted for 25 cycles using pellets of R. oryzae AS 3.819 under the optimal conditions determined from flask cultures. The final l-lactic acid concentration (LLAC) reached 103.7 g/l, and the volumetric productivity was 2.16 g/(l·h) for the first cycle; in the following 19 repeated cycles, the final LLAC reached 81–95 g/l, and the volumetric productivities were 3.40–3.85 g/(l·h).     

Isolation and characterization of Glyceraldehyde-3-phosphate dehydrogenase from the common octopus (<Emphasis Type="Italic">Octopus vulgaris </Emphasis>Cuvier, 1797)

The NAD⁺ dependent cytosolic Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) from arms of Octopus vulgaris, Cuvier, 1787, (Octopoda, Cephalopoda) was purified to homogeneity and its kinetic properties investigated. The purification method consisted of ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulting in a 26-fold increase in specific activity and a final yield of approximately 16%. The apparent molecular weight of the purified native enzyme was 153 kDa. The protein is an homotetramer, composed of identical subunits with an apparent molecular weight of approximately 36 kDa. The Michaelis constants K_m for both NAD⁺ and d-G3P were 66 μM and 320 μM, respectively. The maximal velocity V_max of the purified enzyme was estimated to be 21.8 U/mg. Only one GAPDH isoform (pI 6.6) was obtained by isoelectrofocusing in polyacrylamide slab gels holding ampholyte generated pH gradients. Under the conditions of assay, the optimum activity occurs at pH 7.0 and at temperature of 35°C. Polyclonal antibodies raised in rabbits against the purified GAPDH immunostained a single 36 kDa GAPDH band on crude extract protein preparations blotted onto nitrocellulose.     

Continuous <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid production by a novelthermotolerant <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> QU 41

We isolated and characterized a d-lactic acid-producing lactic acid bacterium (d-LAB), identified as Lactobacillus delbrueckii subsp. lactis QU 41. When compared to Lactobacillus coryniformis subsp. torquens JCM 1166 ^T and L. delbrueckii subsp. lactis JCM 1248 ^T, which are also known as d-LAB, the QU 41 strain exhibited a high thermotolerance and produced d-lactic acid at temperatures of 50 °C and higher. In order to optimize the culture conditions of the QU 41 strain, we examined the effects of pH control, temperature, neutralizing reagent, and initial glucose concentration on d-lactic acid production in batch cultures. It was found that the optimal production of 20.1 g/l d-lactic acid was acquired with high optical purity (>99.9% of d-lactic acid) in a pH 6.0-controlled batch culture, by adding ammonium hydroxide as a neutralizing reagent, at 43 °C in MRS medium containing 20 g/l glucose. As a result of product inhibition and low cell density, continuous cultures were investigated using a microfiltration membrane module to recycle flow-through cells in order to improve d-lactic acid productivity. At a dilution rate of 0.87 h⁻¹, the high cell density continuous culture exhibited the highest d-lactic acid productivity of 18.0 g/l/h with a high yield (ca. 1.0 g/g consumed glucose) and a low residual glucose (<0.1 g/l) in comparison with systems published to date.     

Enhanced <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine production by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> BR-42 (pAP-B03) resistant to bacteriophage BP-1 via a two-stage feeding approach

The l-phenylalanine (l-Phe) production by Escherichia coli WSH-Z06 (pAP-B03) was frequently prevented by bacteriophage BP-1 infestation. To cope with the bacteriophage BP-1 problem for an improved l-Phe production, one bacteriophage BP-1-resistant mutant, E. coli BR-42, was obtained from 416 mutant colonies of E. coli WSH-Z06 after N-methyl-N’-nitro-N-nitrosoguanidine (NTG) mutagenesis by selection for resistance to bacteriophage BP-1. The recombinant E. coli BR-42-carrying plasmid pAP-B03 had a high capacity in l-Phe production and a remarkable tolerance to 1 × 10¹⁰ pfu (plaque-forming unit)/ml bacteriophage stock. For an enhanced l-Phe production by E. coli BR-42 (pAP-B03), the effects of different feeding strategies including pH–stat, constant rate feeding, linear decreasing rate feeding, and exponential feeding on l-Phe production were investigated; and a two-stage feeding strategy, namely exponential feeding at μ _set = 0.18 h⁻¹ in the first 20 h and a following linear varying rate feeding with F = (−0.55 × t + 18.6) ml/h, was developed to improve l-Phe production. With this two-stage feeding approach, a maximum l-Phe titer of 57.63 g/l with a high l-Phe productivity (1.15 g/l/h) was achieved, which was 15% higher than the highest level (50 g/l) reported so far according to our knowledge. The recombinant E. coli BR-42 (pAP-B03) is a potential l-Phe over-producer in substantial prevention of bacteriophage BP-1 infestation compared to its parent strain WSH-Z06 (pAP-B03).     

Application of immobilized thrombin for production of S-thanatin expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

S-thanatin, a small antimicrobial peptide with 21 amino acid residues, was expressed as a fusion protein containing thrombin cleavage site in Escherichia coli BL21 (DE3). To reduce the production cost, immobilization of thrombin in polyacrylamide gel for cleavage was studied in this work. The immobilized thrombin exhibited excellent activity within wider ranges of pH value and temperature for reaction than free enzyme, and the residual activity could remain above 75% after ten times of usage. Tricine–SDS–PAGE result showed that the immobilized thrombin could cleave the S-thanatin fusion protein effectively. After cleavage, recombinant S-thanatin was purified by preparative reversed-phase high-performance liquid chromatography and mass spectrum showed that the molecular weight (2,448.86) was close to the theoretical value (2,448.98). After purification, about 7 mg of S-thanatin was obtained from 1 l of culture and the recombinant exhibited excellent bioactivity to E. coli ATCC 25922, with the minimum inhibitory concentration of 12 μg/ml. The purification method could be applied to prepare other peptides with similar properties at low cost.     

Strain improvement of <Emphasis Type="Italic">Lactobacillus lactis</Emphasis> for <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid production

Three mutants, isolated by repeated UV mutagenesis of Lactobacillus lactis NCIM 2368, produced increased d-lactic acid concentrations. These mutants were compared with the wild type using 100 g hydrolyzed cane sugar/l in the fermentation medium. One mutant, RM2-24, produced 81 g lactic acid/l which was over three times that of the wild type. The highest d-lactic acid (110 g/l) in batch fermentation was obtained with 150 g cane sugar/l with a 73% lactic acid yield. The mutant utilizes cellobiose efficiently, converting it into d-lactic acid suggesting the presence of cellobiase. Thus, this strain could be used to obtain d-lactic acid from cellulosic materials that are pre-hydrolyzed with cellulase.     

Metabolic engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for improving <Emphasis Type="SmallCaps">l</Emphasis>-3,4-dihydroxyphenylalanine (<Emphasis Type="SmallCaps">l</Emphasis>-DOPA) synthesis from glucose

l-3,4-dihydroxyphenylalanine (l-DOPA) is an aromatic compound employed for the treatment of Parkinson's disease. Metabolic engineering was applied to generate Escherichia coli strains for the production of l-DOPA from glucose by modifying the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and aromatic biosynthetic pathways. Carbon flow was directed to the biosynthesis of l-tyrosine (l-Tyr), an l-DOPA precursor, by transforming strains with compatible plasmids carrying genes encoding a feedback-inhibition resistant version of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase, transketolase, the chorismate mutase domain from chorismate mutase-prephenate dehydratase from E. coli and cyclohexadienyl dehydrogenase from Zymomonas mobilis. The effects on l-Tyr production of PTS inactivation (PTS⁻ gluc⁺ phenotype), as well as inactivation of the regulatory protein TyrR, were evaluated. PTS inactivation caused a threefold increase in the specific rate of l-Tyr production (q _l-Tyr), whereas inactivation of TyrR caused 1.7- and 1.9-fold increases in q _l-Tyr in the PTS⁺ and the PTS⁻ gluc⁺ strains, respectively. An 8.6-fold increase in l-Tyr yield from glucose was observed in the PTS⁻ gluc⁺ tyrR ⁻ strain. Expression of hpaBC genes encoding the enzyme 4-hydroxyphenylacetate 3-hydroxylase from E. coli W in the strains modified for l-Tyr production caused the synthesis of l-DOPA. One of such strains, having the PTS⁻ gluc⁺ tyrR ⁻ phenotype, displayed the best production parameters in minimal medium, with a specific rate of l-DOPA production of 13.6 mg/g/h, l-DOPA yield from glucose of 51.7 mg/g and a final l-DOPA titer of 320 mg/l. In a batch fermentor culture in rich medium this strain produced 1.51 g/l of l-DOPA in 50 h.     

<Emphasis Type="SmallCaps">l</Emphasis>(+)-Lactic acid production by co-fermentation of glucose and xylose with <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> obtained by low-energy ion beam irradiation

Low-energy ion beam irradiation (10–200 keV) has been proved to have a wide range of biological effects in recent years. When Rhizopus oryzae PW352 was irradiated with a 15-keV low-energy ion beam an l(+)-lactic acid high-yield mutant, RQ4015, was obtained. When 150 g/l glucose was used as the sole carbon source, l(+)-lactic acid of RQ4015 reached 121 g/l after 36 h shake-flask cultivation. However, the highest lactic acid concentration 74 g/l was obtained when 100 g/l xylose was present in the medium as the sole carbon source. When mixed xylose (25 g/l) and glucose (75 g/l) were present in a bubble column, l(+)-lactic acid production of RQ4015 reached 83 g. A high mutation rate and a wide mutation spectrum of low-energy ion implantation were observed in the experiment, suggesting that ion implantation can be a highly efficient mutagenic means for microorganism breeding in many commercial applications.     

Production of recombinant mink growth hormone in <Emphasis Type="Italic">E. coli</Emphasis>

Escherichia coli cells expressing mink (Mustela vison) growth hormone were grown in a batch fermentation process. The expression level was estimated to be 27% of the total cellular protein after 3 h of induction with 1 mM isopropyl β-d-thiogalactoside (IPTG). If the expression of mink growth hormone (mGH) was induced with 0.2 mM IPTG, the concentration of target protein was slightly lower and was found to be 23% at the same time after induction. mGH expressed as inclusion bodies was solubilized in 8 M urea and renatured by dilution protocol at a protein concentration of 1.4–2.1 mg/ml in the presence of glutathione pair in a final concentration of 11.3 mM. [GSH]/[GSSG] ratio equal to 2/1 was used. Two-step purification process comprising of ion-exchange chromatography on Q-Sepharose and hydrophobic chromatography on Phenyl-Sepharose was developed. Some 25–30 mg of highly purified and biologically active mGH was obtained from 4 g of biomass. The method presented in this study allows producing large quantities of mGH and considering initiation of scientific investigation on mGH effect on mink in vivo and availability in fur industry.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-Lysine from starch by <Emphasis Type="BoldItalic">Corynebacterium glutamicum</Emphasis> displaying α-amylase on its cell surface

We engineered a Corynebacterium glutamicum strain displaying α-amylase from Streptococcus bovis 148 (AmyA) on its cell surface to produce amino acids directly from starch. We used PgsA from Bacillus subtilis as an anchor protein, and the N-terminus of α-amylase was fused to the PgsA. The genes of the fusion protein were integrated into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. l-Lysine fermentation was carried out using C. glutamicum displaying AmyA in the growth medium containing 50 g/l soluble starch as the sole carbon source. We performed l-lysine fermentation at various temperatures (30–40°C) and pHs (6.0–7.0), as the optimal temperatures and pHs of AmyA and C. glutamicum differ significantly. The highest l-lysine yield was recorded at 30°C and pH 7.0. The amount of soluble starch was reduced to 18.29 g/l, and 6.04 g/l l-lysine was produced in 24 h. The l-lysine yield obtained using soluble starch as the sole carbon source was higher than that using glucose as the sole carbon source after 24 h when the same amount of substrates was added. The results shown in the current study demonstrate that C. glutamicum displaying α-amylase has a potential to directly convert soluble starch to amino acids.     

Biotransformation of glycerol to <Emphasis Type="SmallCaps">d</Emphasis>-glyceric acid by <Emphasis Type="Italic">Acetobacter tropicalis</Emphasis>

Bacterial strains capable of converting glycerol to glyceric acid (GA) were screened among the genera Acetobacter and Gluconacetobacter. Most of the tested Acetobacter and Gluconacetobacter strains could produce 1.8 to 9.3 g/l GA from 10% (v/v) glycerol when intact cells were used as the enzyme source. Acetobacter tropicalis NBRC16470 was the best GA producer and was therefore further investigated. Based on the results of high-performance liquid chromatography analysis and specific rotation, the enantiomeric composition of the produced GA was d-glyceric acid (d-GA). The productivity of d-GA was enhanced with the addition of both 15% (v/v) glycerol and 20 g/l yeast extract. Under these optimized conditions, A. tropicalis NBRC16470 produced 22.7 g/l d-GA from 200 g/l glycerol during 4 days of incubation in a jar fermentor.     

Mannose production from fructose by free and immobilized <Emphasis Type="SmallCaps">d</Emphasis>-lyxose isomerases from <Emphasis Type="Italic">Providencia stuartii</Emphasis>

A recombinant d-lyxose isomerase from Providencia stuartii was immobilized on Duolite A568 beads which gave the highest conversion of d-fructose to d-mannose among the various immobilization beads evaluated. Maximum activities of both the free and immobilized enzymes for fructose isomerization were at pH 7.5 and 45°C in the presence of 1 mM Mn²⁺. Enzyme half-lives were 14 and 30 h at 35°C and 3.4 and 5.1 h at 45°C, respectively. The immobilized enzyme in 300 g fructose/l (replaced hourly), produced 75 g mannose/l at 35°C = 25% (w/w) yield with a productivity of 75 g mannose l⁻¹ h⁻¹ after 23 cycles.     

Optimisation of the purification process of a tumour-targeting antibody produced in <Emphasis Type="Italic">N. benthamiana</Emphasis> using vacuum-agroinfiltration

It was previously demonstrated that the tumour-targeting antibody mAb H10 can be transiently expressed and purified at high levels in Nicotiana benthamiana by using a vacuum-agroinfiltration system boosted by the use of a virus silencing suppressor protein. Scope of this work was to analyse different steps of protein extraction from agroinfiltrated leaves to optimise the purification process of the secretory mAb H10 providing new insights in the field of large-scale plant production. Two different extraction procedures (mechanical shearing/homogenisation and recovery of intercellular fluids -IFs-) were evaluated and compared in terms of purified antibody yields, antibody degradation and total phenolic compounds content. Mechanical grinding from fresh leaf tissues gave the highest purification yield (75 mg/kg Fresh Weight -75% intact tetrameric IgG-) and total phenolics concentration in the range of 420 μg/g FW. The second extraction procedure, based on the recovery of IFs, gave purification yields of 15–20 mg/kg FW (corresponding to 27% of total soluble protein) in which about 40% of purified protein is constituted by fully assembled IgG with a total phenolic compounds content reduced by one order of magnitude (21 μg/g FW). Despite a higher antibody degradation, purification from intercellular fluids demonstrated to be very promising since extraction procedures resulted extremely fast and amenable to scaling-up. Overall data highlight that different extraction procedures can dramatically affect the proteolytic degradation and quality of antibody purified from agroinfiltrated N. benthamiana leaves. Based on these results, we optimised a pilot-scale purification protocol using a two-step purification procedure from batches of fresh agroinfiltrated leaves (250 g) allowing purification of milligram quantities (average yield 40 mg/kg FW) of fully assembled and functional IgG with a 99.4% purity, free of phenolic and alkaloid compounds with low endotoxin levels (<1 EU/ml).     

Purification,characterization, and substrate specificity of a glucoamylase with steroidal saponin-rhamnosidase activity from <Emphasis Type="Italic">Curvularia lunata</Emphasis>

It has been previously reported that a glucoamylase from Curvularia lunata is able to hydrolyze the terminal 1,2-linked rhamnosyl residues of sugar chains at C-3 position of steroidal saponins. In this work, the enzyme was isolated and identified after isolation and purification by column chromatography including gel filtration and ion-exchange chromatography. Analysis of protein fragments by MALDI-TOF/TOF™ proteomics Analyzer indicated the enzyme to be 1,4-alpha-D-glucan glucohydrolase EC 3.2.1.3, GA and had considerable homology with the glucoamylase from Aspergillus oryzae. We first found that the glucoamylase was produced from C. lunata and was able to hydrolyze the terminal rhamnosyl of steroidal saponins. The enzyme had the general character of glucoamylase, which hydrolyze starch. It had a molecular mass of 66 kDa and was optimally active at 50°C, pH 4, and specific activity of 12.34 U mg of total protein⁻¹ under the conditions, using diosgenin-3-O-α-L-rhamnopyranosyl(1→4)-[α-L-rhamnopyranosyl (1→2)]-β-D-glucopyranoside (compound II) as the substrate. Furthermore, four kinds of commercial glucoamylases from Aspergillus niger were investigated in this work, and they had the similar activity in hydrolyzing terminal rhamnosyl residues of steroidal saponin. This project was supported by the National Natural Science Foundation of China (NSFC; 30572333).     

Immobilization of <Emphasis Type="Italic">Escherichia coli</Emphasis> cells with <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase activity for <Emphasis Type="SmallCaps">d</Emphasis>(−)-tartaric acid production

Immobilization of cis-epoxysuccinate hydrolase-containing E. coli for d(−)-tartaric acid production was screened by various methods. The highest recovery of activity was obtained by entrapment in κ-carrageenan gel. 23.6 g biomass/l and 43.4 g κ-carrageenan/l were the best immobilization conditions optimized by response surface methodology with 83% yield (114 U/g). Cell autolysis was observed after immobilization. Immobilized cells showed high pH (5–10) stability, thermal (up to 65°C) stability, conversion rate (>99.5%), enantioselectivity (ee > 99.6%), and were less affected by metal ions and surfactants compared with free cells. Conversion rate for immobilized cells preserved 93% after 10 repeated batches (5% for free cells).     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine from glycerol by a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

The production of l-phenylalanine is conventionally carried out by fermentations that use glucose or sucrose as the carbon source. This work reports on the use of glycerol as an inexpensive and abundant sole carbon source for producing l-phenylalanine using the genetically modified bacterium Escherichia coli BL21(DE3). Fermentations were carried out at 37°C, pH 7.4, using a defined medium in a stirred tank bioreactor at various intensities of impeller agitation speeds (300–500 rpm corresponding to 0.97–1.62 m s⁻¹ impeller tip speed) and aeration rates (2–8 L min⁻¹, or 1–4 vvm). This highly aerobic fermentation required a good supply of oxygen, but intense agitation (impeller tip speed ~1.62 m s⁻¹) reduced the biomass and l-phenylalanine productivity, possibly because of shear sensitivity of the recombinant bacterium. Production of l-phenylalanine was apparently strongly associated with growth. Under the best operating conditions (1.30 m s⁻¹ impeller tip speed, 4 vvm aeration rate), the yield of l-phenylalanine on glycerol was 0.58 g g⁻¹, or more than twice the best yield attainable on sucrose (0.25 g g⁻¹). In the best case, the peak concentration of l-phenylalanine was 5.6 g L⁻¹, or comparable to values attained in batch fermentations that use glucose or sucrose. The use of glycerol for the commercial production of l-phenylalanine with E. coli BL21(DE3) has the potential to substantially reduce the cost of production compared to sucrose- and glucose-based fermentations.