•S. sp. carrying of vgb was constructed to improve welan gum yield.•An increase of 34.6 g/L welan gum was attained with VHb expression.•The rheological behavior of welan gum solutions had no change after vgb insertion.•VHb expression enhanced oxygen uptake rate and ATP level for welan gum production.Welan gum is a microbial polysaccharide produced by Sphingomonas sp. Its production is limited by the dissolved oxygen levels in the highly viscous fermentation. A strategy of heterologous expression of the Vitreoscilla hemoglobin gene in Sphingomonas sp. HT-1 was investigated to alleviate oxygen limitation and improve the yield of welan gum. Ultimately, the welan gum production increased from 25.3 g/L to 34.6 g/L, whereas the rheological behavior of welan gum solutions remained virtually unchanged. The transcriptional levels of the key genes in the electron transfer chain, TCA cycle and welan gum synthesis pathway, as well as ATP level revealed that the VHb expression in Sphingomonas sp. HT-1 enhanced welan gum biosynthesis by improving respiration and ATP supply. This study would pave the genetic manipulation way for enhancing welan gum yield, and it’s also of great importance for the industrial applications of welan gum under harsh conditions.

Rhamsan gum is a type of water-soluble exopolysaccharide produced by species of Sphingomonas bacteria. The optimal fermentation medium for rhamsan gum production by Sphingomonas sp. CGMCC 6833 was explored definition. Single-factor experiments indicate that glucose, soybean meal, K2HPO4 and MnSO4 compose the optimal medium along with and initial pH 7.5. To discover ideal cultural conditions for rhamsan gum production in a shake flask culture, response surface methodology was employed, from which the following optimal ratio was derived: 5.38 g/L soybean meal, 5.71 g/L K2HPO4 and 0.32 g/L MnSO4. Under ideal fermentation rhamsan gum yield reached 19.58 g/L ± 1.23 g/L, 42.09% higher than that of the initial medium (13.78 g/L ± 1.38 g/L). Optimizing the fermentation medium results in enhanced rhamsan gum production.

l-Arabinose isomerase (l-AI) catalyzes the isomerization of l-arabinose to l-ribulose and d-galactose to d-tagatose. Most reported l-AIs exhibit neutral or alkaline optimum pH, which is less beneficial than acidophilic ones in industrial d-tagatose production. Lactobacillus fermentuml-AI (LFAI) is a thermostable enzyme that can achieve a high conversion rate for d-galactose isomerization. However, its biocatalytic activity at acidic conditions can still be further improved. In this study, we report the single- and multiple-site mutagenesis on LFAI targeting three aspartic acid residues (D268, D269, and D299). Some of the lysine mutants, especially D268K/D269K/D299K, exhibited significant optimum pH shifts (from 6.5 to 5.0) and enhancement of pH stability (half-life time increased from 30 to 62 h at pH 6.0), which are more favorable for industrial applications. With the addition of borate, d-galactose was isomerized into d-tagatose by D268K/D269K/D299K at pH 5.0, resulting in a high conversion rate of 62 %. Based on the obtained 3.2-Å crystal structure of LFAI, the three aspartic acid residues were found to be distant from the active site and possibly did not participate in substrate catalysis. However, they were proven to possess similar optimum pH control ability in other l-AI, such as that derived from Escherichia coli. This study sheds light on the essential residues of l-AIs that can be modified for desired optimum pH and better pH stability, which are useful in d-tagatose bioproduction.

L-Arabinose isomerase (AI), a key enzyme in the microbial pentose phosphate pathway, has been regarded as an important biological catalyst in rare sugar production. This enzyme could isomerize L-arabinose into L-ribulose, as well as D-galactose into D-tagatose. Both the two monosaccharides show excellent commercial values in food and pharmaceutical industries. With the identification of novel AI family members, some of them have exhibited remarkable potential in industrial applications. The biological production processes for D-tagatose and L-ribose (or L-ribulose) using AI have been developed and improved in recent years. Meanwhile, protein engineering techniques involving rational design has effectively enhanced the catalytic properties of various AIs. Moreover, the crystal structure of AI has been disclosed, which sheds light on the understanding of AI structure and catalytic mechanism at molecular levels. This article reports recent developments in (i) novel AI screening, (ii) AI-mediated rare sugar production processes, (iii) molecular modification of AI, and (iv) structural biology study of AI. Based on previous reports, an analysis of the future development has also been initiated.

In this paper, the production of rhamsan gum from Sphingomonas sp. CGMCC 6833 at different pH values was investigated. Based on kinetic analysis, a two-stage strategy for pH control was proposed. During the first 10 h, pH was controlled at 7.5 to maintain high specific cell growth rate and specific glucose consumption rate. After 10 h, pH decreased naturally to 7.0; this value was retained to maintain high specific rhamsan gum formation rate. Using this method, the maximum concentration and productivity of rhamsan gum reached 18.56 ± 1.68 g/L and 0.290 ± 0.026 g/L/h, which are 12.83 and 12.84 % higher than the optimum results obtained at natural pH, respectively.

Streptomyces albulus PD-1 can co-produce antimicrobial homo-polymers poly(ε-lysine) (ε-PL) and poly(l-diaminopropionic acid) (PDAP). In this study, a novel feeding strategy of citric acid coupled with glucose-(NH4)2SO4 feeding was employed to S. albulus PD-1. When the pH of the culture broth dropped to 4.0, the feeding solution was added continuously to maintain the concentrations of glucose and citric acid at 10 and 4 g L−1, respectively. As a result, the final concentration of ε-PL increased from 21.7 to 29.7 g L−1 and the final concentration of PDAP decreased from 4.8 to 3.2 g L−1. Assays on intracellular nucleotide levels and key enzyme activities were performed to elucidate the underlying regulation mechanism. The addition of citric acid increased NADH/NAD+ ratio and decreased intracellular ATP level; meanwhile, the activities of pyruvate kinase, citrate synthase and isocitrate dehydrogenase decreased while aspartate aminotransferase activity increased. Therefore, we deduced that citric acid feeding resulted in metabolic flux redistribution at the node of phosphoenolpyruvate; the metabolic pathway from phosphoenolpyruvate directed into tricarboxylic acid cycle was weakened and thus PDAP production was inhibited. On the other hand, the metabolic pathway from phosphoenolpyruvate directed into oxaloacetate and l-aspartate was enhanced, thereby improving ε-PL production. This fermentation strategy may be potentially useful in ε-PL production because it can effectively inhibit the formation of by-products, such as PDAP.

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

The effects of oxidoreduction potential (ORP) regulation on the process of propionic acid production by Propionibacterium freudenreichii CCTCC M207015 have been investigated. Potassium ferricyanide and sodium borohydride were determined as ORP control agents through serum bottle experiment. In batch fermentation, cell growth, propionic acid and by-products distribution were changed with ORP levels in the range of 0–160 mV. Based on these analysis results, an ORP-shift control strategy was proposed: at first 156 h, ORP was controlled at 120 mV to obtain higher cell growth rate and propionic acid formation rate, and then it was shifted to 80 mV after 156 h to maintain the higher propionic acid formation rate. By applying this strategy, the optimal parameters were obtained as follows: the propionic acid concentration 45.99 g L−1, productivity 0.192 g L−1 h−1, the proportion of propionic acid to total organic acids 92.26 % (w/w) and glycerol conversion efficiency 76.65 %. The mechanism of ORP regulation was discussed by the ratio of NADH/NAD+, ATP levels, and metabolic flux analysis. The results suggest that it is possible to redistribute energy and metabolic fluxes by the ORP-shift control strategy, and the strategy could provide a simple and efficient tool to realize high purity propionic acid production with glycerol as carbon source.

Poly(ε-l-lysine) (ε-PL) producer strain Streptomyces albulus PD-1 secreted a novel polymeric substance into its culture broth along with ε-PL. The polymeric substance was purified to homogeneity and identified. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and nuclear magnetic resonance spectroscopy as well as other analytical techniques revealed that the substance was poly(l-diaminopropionic acid) (PDAP). PDAP is an l-α,β-diaminopropionic acid oligomer linking between amino and carboxylic acid functional groups. The molecular weight of PDAP ranged from 500 to 1500 Da, and no co-polymers composed of l-diaminopropionic acid and l-lysine were present in the culture broth. Compared with ε-PL, PDAP exhibited stronger inhibitory activities against yeasts but weaker activities against bacteria. ε-PL and PDAP co-production was also investigated. Both ε-PL and PDAP were synthesized during the stationary phase of growth, and the final ε-PL and PDAP concentration reached 21.7 and 4.8 g L-1, respectively, in fed-batch fermentation. Citric acid feeding resulted in a maximum ε-PL concentration of 26.1 g L-1 and a decrease in the final concentration of PDAP to 3.8 g L-1. No studies on ε-PL and PDAP co-production in Streptomyces albulus have been reported previously, and inhibition of by-products such as PDAP is potentially useful in ε-PL production.

The effect of additives on welan gum production produced by fermentation with Alcaligenes sp. CGMCC2428 was studied. Tween-40 was the best additive for improving welan gum production and welan gum displayed better rheological properties than that obtained by control fermentation without additives. Response surface methodology was employed to optimize the culture conditions for welan gum production in the shake flask culture, including Tween-40 concentration, pH and culture temperature. The optimal conditions were determined as follows: Tween-40 concentration 0.94 g/l, pH 6.9 and temperature 29.6 °C. The corresponding experimental concentration of welan gum was 23.62 ± 0.60 g/l, which was agreed closely with the predicted value (23.48 g/l). Validation experiments were also carried out to prove the adequacy and the accuracy of the model obtained. The welan gum fermentation in a 7.5 l bioreactor reached 24.90 ± 0.68 g/l.Highlights► The effect of additives on exopolysaccharide welan gum was studied. ► Tween-40 was proved to be the best additive. ► Response surface methodology was employed to optimize the culture conditions. ► The maximum welan gum concentration (24.90 ± 0.68 g/l) was obtained.

In order to obtain genetically stable, high-yield, laccase-producing strains, Ceriporiopsis subvermispora was induced by N+ ion implantation and subcultured. The results revealed that, with energy of 30 keV and a dose of 80×1014 ions/cm2, a relatively high increase in mutations and positive mutations were achieved. Three screened high-yield strains (NL3, NL4, and NL6) were obtained and subcultured. The results of the comparison showed that, NL4 had stable genetic traits and the highest laccase activity (323 U/L). In the course of fermentation, NL4 entered a vigorous growth period 24 h ahead of the original strain, and produced a large amount of laccase during the stationary phase. Up until the sixth day of fermentation achieved the highest laccase activity of 377 U/L, and a corresponding biomass dry weight of 4.2 mg/mL. The relative laccase activity of the per gram dry cells was 89.76 U, which was 4.79 times that of the original strain. The results indicated that N+ ion implantation was an ideal technique for microbial breeding, which could be applied for the improvement of Ceriporiopsis subvermispora.

d-tagatose is a ketohexose that can be used as a novel functional sweetener in foods, beverages, and dietary supplements. This study was aimed at developing a high-yielding d-tagatose production process using alginate immobilized Lactobacillus fermentum CGMCC2921 cells. For the isomerization from d-galactose into d-tagatose, the immobilized cells showed optimum temperature and pH at 65 °C and 6.5, respectively. The alginate beads exhibited a good stability after glutaraldehyde treatment and retained 90% of the enzyme activity after eight cycles (192 h at 65 °C) of batch conversion. The addition of borate with a molar ratio of 1.0 to d-galactose led to a significant enhancement in the d-tagatose yield. Using commercial β-galactosidase and immobilized L. fermentum cells, d-tagatose was successfully obtained from lactose after a two-step biotransformation. The relatively high conversion rate and productivity from d-galactose to d-tagatose of 60% and 11.1 g l−1 h−1 were achieved in a packed-bed bioreactor. Moreover, lactobacilli have been approved as generally recognized as safe organisms, which makes this L. fermentum strain an attracting substitute for recombinant Escherichia coli cells among d-tagatose production progresses.

Bacillus subtilis CGMCC 0833 is a poly(γ-glutamic acid) (γ-PGA)-producing strain. It has the capacity to tolerate high concentration of extracellular glutamate and to utilize glutamate actively. Such a high uptake capacity was owing to an active transport system for glutamate. Therefore, a specific transport system for l-glutamate has been observed in this strain. It was a novel transport process in which glutamate was symported with at least two protons, and an inward-directed sodium gradient had no stimulatory effect on it. Km and Vm for glutamate transport were estimated to be 67 μM and 152 nmol−1 min−1 mg−1 of protein, respectively. The transport system showed structural specificity and stereospecificity and was strongly dependent on extracellular pH. Moreover, it could be stimulated by Mg2+, NH4+, and Ca2+. In addition, the glutamate transporter in this strain was studied at the molecular level. As there was no important mutation of the transporter protein, it appeared that the differences of glutamate transporter properties between this strain and other B. subtilis strains were not due to the differences of the amino acid sequence and the structure of transporter protein. This is the first extensive report on the properties of glutamate transport system in γ-PGA-producing strain.

Batch fermentative production of welan gum by Alcaligenes sp. CGMCC2428 was investigated under various oxygen supply conditions using regulating agitation speed. Based on a three kinetic parameters analysis that includes specific cell growth rate (μ), specific glucose consumption rate (qs), and specific welan formation rate (qp), a two-stage agitation speed control strategy was proposed to achieve high concentration, high yield, and high viscosity of welan. During the first 22 h, the agitation speed in 7.5 L fermenter was controlled at 800 rpm to maintain high μ for cell growth. The agitation was then reduced step-wise to 600 rpm to maintain a changing profile with stable dissolved oxygen levels and obtain high qp for high welan accumulation. Finally, the maximum concentration of welan was reached at 26.3 ± 0.89 g L−1 with a yield of 0.53 ± 0.003 g g−1 and the welan gum viscosity of 3.05 ± 0.10 Pa s, which increased by an average of 15.4, 15.2, and 20.1% over the best results controlled by constant agitation speeds.

In this study, the production of poly(γ-glutamic acid) by Bacillus subtilis NX-2 (PGA) at different agitation speeds was investigated. Based on the analysis of specific cell growth rate (μ) and specific PGA formation rate (qp), a two-stage strategy for agitation speed control was proposed. During the first 24 h, an agitation speed of 600 rpm was used to maintain a high μ for better cell growth, which then reduced to 400 rpm after 24 h to maintain a high qp to enhance PGA production. Using this method, the maximum concentration of PGA reached 40.5 ± 0.91 g/L and the PGA productivity was 0.56 ± 0.012 g/L/h, which was 17.7 and 9.8% higher, respectively, than the best results obtained when a constant agitation speed was used. The flux distributions and the related enzymes of 2-oxoglutarate could be affected by this two-stage strategy for agitation speed. The activity of isocitrate dehydrogenase and glutamate dehydrogenase at the key node of 2-oxoglutarate increased, and more flux distribution was directed to glutamate. The flux distribution from extracellular to intracellular glutamate also increased and improved PGA production as the glutamate uptake rates increased using the agitation-shift control method.

The sucrose isomerase (SIase) gene from an efficient strain of Erwinia rhapontici NX-5 for isomaltulose hyperproduction was cloned and overexpressed in Escherichia coli. Protein sequence alignment revealed that SIase was a member of the glycoside hydrolase 13 family. The molecular mass of the purified recombinant protein was estimated at 66 kDa by SDS-PAGE. The SIase had an optimal pH and temperature of 5.0 and 30 °C, respectively, with a Km of 257 mmol/l and Vmax of 48.09 μmol/l/s for sucrose. To the best of our knowledge, the recombinant SIase has the most acidic optimum pH for isomaltulose synthesis. When the recombinant E. coli (pET22b- palI) cells were used for isomaltulose synthesis, almost complete conversion of sucrose (550 g/l solution) to isomaltulose was achieved in 1.5 h with high isomaltulose yields (87%). The immobilized E. coli cells remained stable for more than 30 days in a “batch”-type enzyme reactor. This indicated that the recombinant SIase could continuously and efficiently produce isomaltulose.

Linoleic acid isomerase from Lactobacillus delbrueckii subsp. bulgaricus 1.1480 was purified by DEAE ion-exchange chromatography and gel filtration chromatography. An overall 5.1% yield and purification of 93-fold were obtained. The molecular weight of the purified protein was ~41 kDa which was analyzed by SDS-PAGE. The purified enzyme was immobilized on palygorskite modified with 3-aminopropyltriethoxysilane. The immobilized enzyme showed an activity of 82 U/g. The optimal temperature and pH for the activity of the free enzyme were 30 °C and pH 6.5, respectively; whereas those for the immobilized enzyme were 35 °C and pH 7.0, respectively. The immobilized enzyme was more stable than the free enzyme at 30–60 °C, and the operational stability result showed that more than 85% of its initial activity was retained after incubation for 3 h. The Km and Vmax values of the immobilized enzyme were found to be 0.0619 mmol l−1 and 0.147 mmol h−1 mg−1, respectively. The immobilized enzyme had high operational stability and retained high enzymatic activity after seven cycles of reuse at 37 °C.

A new yeast, isolated from natural osmophilic sources, produces d-arabitol as the main metabolic product from glucose. According to 18S rRNA analysis, the NH-9 strain belongs to the genus Kodamaea. The optimal culture conditions for inducing production of d-arabitol were 37 °C, neutral pH, 220 rpm shaking, and 5% inoculum. The yeast produced 81.2 ± 0.67 g L−1d-arabitol from 200 g L−1d-glucose in 72 h with a yield of 0.406 g g−1 glucose and volumetric productivity \( Q_{\text{P}} \) of 1.128 g L−1 h−1. Semi-continuous repeated-batch fermentation was performed in shaker-flasks to enhance the process of d-arabitol production by Kodamaea ohmeri NH-9 from d-glucose. Under repeated-batch culture conditions, the highest volumetric productivity was 1.380 g L−1 h−1.

Propionic acid was produced in a multi-point fibrous-bed (MFB) bioreactor by Propionibacterium freudenreichii CCTCC M207015. The MFB bioreactor, comprising spiral cotton fiber packed in a modified 7.5-l bioreactor, was effective for cell-immobilized propionic acid production compared with conventional free cell fermentation. Batch fermentations at various glucose concentrations were investigated in the MFB bioreactor. Based on analysis of the time course of production, a fed-batch strategy was applied for propionic acid production. The maximum propionic acid concentration was 67.05 g l−1 after 496 h of fermentation, and the proportion of propionic acid to total organic acids was approximately 78.28% (w/w). The MFB bioreactor exhibited excellent production stability during batch fermentation and the propionic acid productivity remained high after 78 days of fermentation.

Glycerol would stimulate the production of poly(γ-glutamic acid) (γ-PGA) and decrease its molecular weight in Bacillus subtilis NX-2. When 20 g/l glycerol was added in the medium, the yield of γ-PGA increased from 26.7 ± 1.0 to 31.7 ± 1.3 g/l, and molecular weight of γ-PGA decreased from 2.43 ± 0.07 × 106 to 1.86 ± 0.06 × 106 Da. In addition, it was found that the decrease of γ-PGA chain length by glycerol would lead to the decrease of broth viscosity during the fermentation and enhanced the uptake of substrates, which could not only improve cell growth but also stimulate γ-PGA production. Moreover, it was also found that glycerol could effectively regulate molecular weight between 2.43 ± 0.07 × 106 and 1.42 ± 0.05 × 106 Da with the concentration ranging from 0 to 60 g/l. This was the first time to discover such contribution of glycerol on γ-PGA production in Bacillus genus. And the effects of glycerol on molecular weight of γ-PGA would be developed to be an approach for the regulation of microbial γ-PGA chain length, which is of practical importance for future commercial development of this polymer.

Low-energy nitrogen ion beam implantation technique was used for the strain improvement of Alcaligenes sp. NX-3 for the production of exopolysaccharide welan gum. A high welan gum producing mutant, Alcaligenes sp. NX-3-1, was obtained through 20 keV N+ ion beam irradiation. Starting at a concentration of 50 g/L of glucose, mutant NX-3-1 produced 25.0 g/L of welan gum after 66 h of cultivation in a 7.5 L bioreactor, which was 34.4% higher than that produced by the wild-type strain. The results of metabolic flux analysis showed that the glucose-6-phosphate and acetyl coenzyme A nodes were the principle and flexible nodes, respectively. At the glucose-6-phosphate node, the fraction of carbon measured from glucose-6-phosphate to glucose-1-phosphate was enhanced after mutagenesis, which indicated that more flux was used to synthesize welan gum in the mutant. By analyzing the activities of related enzymes in the biosynthetic pathway of sugar nucleotides essential for welan gum production, we found that the specific activities of phosphoglucomutase, UDP-glucose pyrophosphorylase, UDP-glucose dehydrogenase, and dTDP-glucose pyrophosphorylase in the mutant strain were higher than those in the wild-type strain. These improvements in enzyme activities could be due to the affected of ion beam implantation.

The production of propionic acid by Propionibacterium freudenreichii CCTCC M207015 was investigated in a 7.5-l stirred-tank fermentor. Batch fermentations by P. freudenreichii CCTCC M207015 at various pH values ranging from 5.5 to 7.0 were studied. Based on the analysis of the time course of specific cell growth rate (μx) and specific propionic acid formation rate (μp), a two-stage pH-shift control strategy was proposed. At first 48 h, pH was controlled at 6.5 to obtain the maximal μx, subsequently pH 6.0 was used to maintain high μp to enhance the production of propionic acid. By applying this pH-shift control strategy in propionic acid fermentation, the maximal propionic acid and glucose conversion efficiency had a significant improvement and reached 19.21 g/l and 48.03%, respectively, compared with those of constant pH operation (14.58 g/l and 36.45%). Fed-batch fermentation with pH-shift control strategy was also applied to produce propionic acid; the maximal propionic acid yield and glucose conversion efficiency reached 25.23 g/l and 47.76%, respectively.

Bacillus subtilis NX-2 produces γ-polyglutamic acid (γ-PGA) when using glucose and l-glutamate as carbon sources. The conversion of carbon sources into γ-PGA was analyzed with the 13C-NMR method after enriching the media with 13C-labeled glucose. The results showed that the percentage of γ-PGA monomers derived from glucose was relatively low, approximately 6% and 9%, respectively, with an initial glucose concentration of 30 and 40 g L−1. It was concluded that glucose was utilized mainly as the growth-limiting substrate for cell growth and supplied the required energy during γ-PGA biosynthesis, while l-glutamate was preferred as the main substrate for γ-PGA formation. To achieve an efficient conversion of l-glutamate and enhance the γ-PGA production, a fed-batch culture was proposed by feeding of glucose. By this method, supplied l-glutamate (40 g L−1) was completely depleted, and γ-PGA yield was attained 42 g L−1.

Tween-80, dimethyl sulfoxide (DMSO), and glycerol could be used as novel materials to regulate the central carbon metabolic pathway and improve γ-PGA biosynthesis by Bacillus subtilis CGMCC 0833. With glycerol in the medium, the activity of 2-oxoglutarate dehydrogenase complex at the key node of 2-oxoglutarate was depressed, more carbon flux distribution was directed to synthesize glutamate, the substrate of γ-PGA, which led to overproducing of γ-PGA, reached 31.7 g/l, compared to the original value of 26.7 g/l. When Tween-80 or DMSO was in the medium, the activity of isocitrate dehydrogenase was stimulated, the branch flux from 2-oxoglutarate to glutamate was also enhanced due to the increasing of total flux from iso-citrate to 2-oxoglutarate, then a large amount of glutamate was produced, and formation of γ-PGA was also improved, which was a different process compared with that of glycerol. Moreover, with the addition of Tween-80 or DMSO, cell membrane permeability was increased, which facilitated the uptake of extracellular substrates and the secretion of γ-PGA by this strain; therefore, γ-PGA production was further stimulated, and 34.4 and 32.7 g/l γ-PGA were obtained, respectively. This work firstly employed additives to improve the biosynthesis of γ-PGA and would be helpful in understanding the biosynthesis mechanism of γ-PGA by Bacillus species deeply.

In this study, poly(γ-glutamic acid)-coated Fe3O4 magnetic nanoparticles (γ-PGA/Fe3O4 MNPs) were successfully fabricated using the co-precipitation method. Fe3O4 MNPs were also prepared for comparison. The average size and specific surface area results reveal that γ-PGA/Fe3O4 MNPs (52.4 nm, 88.41 m2·g−1) have smaller particle size and larger specific surface area than Fe3O4 MNPs (62.0 nm, 76.83 m2·g−1). The γ-PGA/Fe3O4 MNPs can remove over 99% of Cr3+, Cu2+ and Pb2+, and over 77% of Ni2+ in deionized water, much higher than γ-PGA and Fe3O4 MNPs, attributed to the larger specific surface area of γ-PGA/Fe3O4 MNPs. With the solution pH higher than 6.0, γ-PGA/Fe3O4 MNPs demonstrate better removal activity. The adsorption isotherm of γ-PGA/Fe3O4 MNPs for Cr3+ fits the Freundlich model well, with the adsorption capacity of 24.60 mg·g−1. γ-PGA/Fe3O4 MNPs are strongly attracted by permanent magnet, so it is easy to separate them completely from water. With their high efficiency for heavy metal removal and easier separation, γ-PGA/Fe3O4 MNPs have great potential applications in water treatment.

A plant fibrous-bed bioreactor (PFB) was constructed for propionic acid production. Sugar cane bagasse was applied to the PFB as immobilizing material. Starting at a concentration of 80 g/L of glucose, Propionibacterium freudenreichii CCTCC M207015 produced 41.20 ± 2.03 g/L of propionic acid at 108 h in the PFB. The value was 21.07% higher than that produced by free cell fermentation. Intermittent and constant fed-batch fermentations were performed in the PFB to optimize the fermentation results. The highest propionic acid concentration obtained from constant fed-batch fermentation was 136.23 ± 6.77 g/L, which is 1.40 times higher than the highest concentration (97.00 g/L) previously reported. Scanning electron microscopy analysis showed that cells exhibited striking changes in morphology after PFB domestication. Compared with free cell fermentation, the fluxes of propionic acid synthesis and the pentose phosphate pathway in PFB fermentation increased by 84.65% and 227.62%, respectively. On the other hand, a decrease in succinic and acetic acid fluxes was also observed. The metabolic flux distributions of the two PFB fed-batch fermentation strategies also demonstrated that constant fed-batch fermentation is a more beneficial method for the immobilized production of propionic acid. The relevant key enzyme activities and metabolic flux variations of the batch cultures showed good consistency. These results suggest that the PFB was effective in high-concentration propionic acid production.Highlights► A plant fibrous-bed bioreactor was designed for propionic acid production. ► The highest propionic acid concentration of 136.23 g/L was obtained. ► Physiological changes, metabolic fluxes, and related enzyme activities were analyzed to elucidate the mechanism of PFB fermentation. ► PFB fermentation is very effective in high concentration propionic acid production.

The araA gene encoding l-arabinose isomerase (l-AI) from the acidophilus bacterium Lactobacillus fermentum CGMCC2921 was cloned and over-expressed in Escherichia coli. The open reading frame of the l-AI consisted of 1425 nucleotides encoding 474 amino acid residues. The molecular mass of the enzyme was estimated to be approximately 53 kDa on SDS–PAGE. The purified recombinant enzyme showed maximum activity at 65 °C and pH 6.5, which were extremely suitable for industrial applications. It required divalent metal ions, either Mn2+ or Co2+, for enzymatic activity and thermostability improvement at higher temperatures. The enzyme was active and stable at acidic pH, it exhibited 83% of its maximal activity at pH 6.0 and retained 88% of the original activity after incubation at pH 6.0 for 24 h. Kinetic parameter study showed that the catalytic efficiency was relatively high, with a kcat/Km of 9.02 mM−1 min−1 for d-galactose. The purified L. fermentum CGMCC2921 l-AI converted d-galactose into d-tagatose with a high conversion rate of 55% with 1 mM Mn2+ after 12 h at 65 °C, suggesting its excellent potential in d-tagatose production.Graphical abstractDownload full-size imageResearch highlights► LFAI showed maximum activity at 65 °C and pH 6.5. ► LFAI behaved preferable stability at acidic pH and higher temperatures. ► The catalytic efficiency for d-galactose (kcat/Km = 9.02 mM−1 min−1) was relatively high. ► LFAI required Mn2+ and Co2+ for enzymatic activity. ► The thermostability of LFAI was proved to be Mn2+ dependent.

The preparation of γ-polyglutamic acid (γ-PGA) from Bacillus subtilis NX-2 has been previously investigated, and its depolymerization during the batch culture was studied in this paper. The results suggested that the γ-PGA depolymerase was present and active extracellularly in the culture. The ywtD gene from B. subtilis NX-2, encoding the γ-PGA depolymerase was cloned and expressed in Escherichia coli. The YwtD protein was purified by metal-chelating affinity chromatography. YwtD was proved to be an endo-hydrolase enzyme and exhibited a remarkable activity in γ-PGA degradation at a wide range of temperature (30–40 °C) and pH (5.0–8.0). On an optimal condition of 30 °C and pH 5.0, an efficient γ-PGA enzymatic degradation was achieved. The molecular weight of γ-PGA could be reduced within the range of 1000–20 kDa and the polydispersity also decreased as a function of depolymerization time. Therefore, a controllable degradation of γ-PGA could be available by enzymatic depolymerization.