Effect of sucrose on dynamic mechanical characteristics of maize and potato starch films

The dynamic mechanical properties of prepared maize and potato starch films were evaluated for mixtures containing 0%, 10% and 15% (w/w) of sucrose at temperatures ranging from 40.0 to 140.0 °C. The spectra of storage modulus (G′), loss modulus (G″), and loss factor (tan δ) of starch films were acquired. Remarkable reduction in the glass transition temperature of maize and potato starch films was observed with the increasing sucrose content. The spectra of storage modulus (G′), loss modulus (G″), and loss factor (tan δ) were measured for the second and third time after two and seven days, respectively. The peaks of loss factor (tan δ) appeared at 59.81 ± 1.86 °C and 95.96 ± 1.67 °C after two-day-storage, but only one peak appeared at 85.46 ± 5.50 °C after seven days. A shifting trend from higher to lower temperature for loss factor was observed after seven days.     

Amylolytically-resistant tapioca starch modified by combined treatment of branching enzyme and maltogenic amylase

Tapioca starch was modified using branching enzyme (BE) isolated from Bacillus subtilis 168 and Bacillus stearothermophilus maltogenic amylase (BSMA), and their molecular fine structure and susceptibility to amylolytic enzymes were investigated. By BE treatment, the molecular weight decreased from 3.1 × 10⁸ to 1.7 × 10⁶, the number of shorter branch chains (DP 6–12) increased, the number of longer branch chains (DP >25) decreased, and amylose content decreased from 18.9% to 0.75%. This indicated that α–1,4 linkages of amylose and amylopectin were cleaved, and moiety of glycosyl residues were transferred to another amylose and amylopectin to produce branched glucan and BE-treated tapioca starch by forming α–1,6 branch linkages. The product was further modified with BSMA to produce highly-branched tapioca starch with 9.7% of extra branch points. When subject to digestion with human pancreatic α-amylase (HPA), porcine pancreatic α-amylase (PPA) and glucoamylase, highly-branched tapioca starch gave significantly lowered α-amylase susceptibility (7.5 times, 14.4 times and 3.9 times, respectively), compared to native tapioca starch.     

Purification,kinetic and thermodynamic characterization of soluble acid invertase from sugarcane (Saccharum officinarum L.)

We report for the first time kinetic and thermodynamic properties of soluble acid invertase (SAI) of sugarcane (Saccharum officinarum L.) salt sensitive local cultivar CP 77-400 (CP-77). The SAI was purified to apparent homogeneity on FPLC system. The crude enzyme was about 13 fold purified and recovery of SAI was 35%. The invertase was monomeric in nature and its native molecular mass on gel filtration and subunit mass on SDS-PAGE was 28 kDa. SAI was highly acidic having an optimum pH lower than 2. The acidic limb was missing. Proton transfer (donation and receiving) during catalysis was controlled by the basic limb having a pKa of 2.4. Carboxyl groups were involved in proton transfer during catalysis. The kinetic constants for sucrose hydrolysis by SAI were determined to be: k_m = 55 mg ml^?1, k_cat = 21 s^?1, k_cat/k_m = 0.38, while the thermodynamic parameters were: ΔH* = 52.6 kJ mol^?1, ΔG* = 71.2 kJ mol^?1, ΔS* = ?57 J mol^?1 K^?1, ΔG*_E–S = 10.8 kJ mol^?1 and ΔG*_E–T = 2.6 kJ mol^?1. The kinetics and thermodynamics of irreversible thermal denaturation at various temperatures 53–63 °C were also determined. The half -life of SAI at 53 and 63 °C was 112 and 10 min, respectively. At 55 °C, surprisingly the half -life increased to twice that at 53 °C. ΔG*, ΔH* and ΔS* of irreversible thermal stability of SAI at 55 °C were 107.7 kJ mol^?1, 276.04 kJ mol^?1 and 513 J mol^?1K^?1, respectively.     

Sugar gustatory thresholds and sugar selection in two species of Neotropical nectar-eating bats

Nectar-feeding bats play an important role in natural communities acting as pollinators; however, the characteristics that affect their food selection are unclear. Here we explore the role that sugar gustatory thresholds and sugar concentration play on sugar selection of Glossophaga soricina and Leptonycteris yerbabuenae. We offered bats paired feeders containing sugar solutions of sucrose (S), glucose (G) or fructose (F) vs. pure water, and sucrose vs. 1:1 equicaloric solutions of glucose–fructose at 5, 15 and 35% (wt./vol.). To see the effect of sweetness on sugar selection, we habituated the bats with a diet containing either sucrose or hexoses and subsequently evaluated sugar preferences. Sugar thresholds were S < G,F for G. soricina and G < S < F for L. yerbabuenae. These thresholds did not match with sugar preferences when the bats fed on dilute nectars. L. yerbabuenae changed its sugar preferences with concentration while G. soricina did not. Finally, the bats consistently preferred the sugar they were habituated to. Our results suggest that bats become accustomed to the sugar found in the most abundant plants they use, and thus prefer the most common sugars included in their diet. This could confer an advantage by allowing them shifting sugar preferences on the most common food present in their environment.     

Purification and biochemical characterization of a novel SDS and surfactant stable,raw starch digesting,and halophilic α-amylase from a moderately halophilic bacterium,Nesterenkonia sp. strain F

An extracellular halophilic α-amylase from Nesterenkonia sp. strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography, with a 10.8-fold increase in specific activity. The molecular mass of the amylase was estimated to be 100 kDa and 106 kDa by SDS–PAGE and gel filtration chromatography, respectively. The enzyme showed maximal activity at pH 7.5 and 45 °C. The amylase was active in a wide range of salt concentrations (0–4 M) with its maximum activity at 0.5 M NaCl or 1 M KCl and was stable at the salts concentrations between 1 M and 4 M. Fe³⁺, Cu²⁺, Zn²⁺ and Al³⁺ strongly inhibited the enzyme, whereas Ca²⁺ stimulated the amylase activity. The α-amylase was inhibited by EDTA, but was not inhibited by PMSF and β-mercaptoethanol. The enzyme showed remarkable stability towards 0.5% SDS and sarcosyl, and 2% each of Triton X-100, Tween 80 and Tween 20. K_m value of the amylase for soluble starch was 4.5 mg/ml. The amylase hydrolyzed 38% of raw wheat starch and 20% of corn starch in a period of 48 h. The major products of soluble starch hydrolysis were maltose, maltotriose and maltotetraose, indicating an α-amylase activity.     

Inhibition of biofilm formation by conformationally constrained indole-based analogues of the marine alkaloid oroidin

Herein, we describe indole-based analogues of oroidin as a novel class of 2-aminoimidazole-based inhibitors of methicillin-resistant Staphylococcus aureus biofilm formation and, to the best of our knowledge, the first reported 2-aminoimidazole-based inhibitors of Streptococcus mutans biofilm formation. This study highlighted the indole moiety as a dibromopyrrole mimetic for obtaining inhibitors of S. aureus and S. mutans biofilm formation. The most potent compound in the series, 5-(trifluoromethoxy)indole-based analogue 4b (MBIC₅₀ = 20 μM), emerged as a promising hit for further optimisation of novel inhibitors of S. aureus and S. mutans biofilms.     

The stability and thermodynamic parameters of a very thermostable and calcium-independent α-amylase from a newly isolated bacterium,Anoxybacillus beppuensis TSSC-1

Anoxybacillus beppuensis TSSC-1 (GenBank Number, EU710556), a thermophilic bacterium isolated from a hot spring reservoir, was found to optimally secrete a monomeric α-amylase at 55 °C and pH 7. The enzyme was purified to homogeneity by a single-step purification on phenyl sepharose 6FF, achieving a 58% yield, 10,000 U/mg specific activity and 19.5 fold purification. The molecular weight, K_m and V_max were 43 kD, 0.5 mg ml^?1 and 3571.42 μmol ml^?1 m^?1, respectively. The enzymatic catalysis of soluble starch was optimum at 80 °C and pH 7. The thermodynamic parameters, K_d, t_1/2, ΔH*, ΔS*, E and ΔG*, were consistent. The very compact structure of the enzyme and the transitional enzyme–substrate complex resisted denaturation at extreme temperatures and alkaline pH. The K_d and t_1/2 measurements were consistent with the high thermostability and pH tolerance observed. The structural stability of the enzyme was also reflected by the values of ΔH*, ΔS*, E and ΔG*. While the enzyme did not exhibit metal ion dependency, it was resistant to chemical denaturation. The broad thermo- and pH-tolerance of this enzyme suggests potential commercial opportunities.     

Synthesis of cellobiose-containing oligosaccharides by intermolecular transglucosylation of cyclodextrin glycosyltransferase from Paenibacillus sp. A11

Intermolecular transglucosylation of cyclodextrin glycosyltransferase (CGTase) was investigated for its use in oligosaccharide synthesis. From the kinetic parameters of the CGTase-catalyzed transglucosylation reaction, using β-cyclodextrin (β-CD) as the glucosyl donor and various saccharides or derivatives as acceptors, the efficient acceptors of the Paenibacillus sp. A11 enzyme were glucose, sorbose, lactose and cellobiose. Amongst these acceptors, cellobiose showed the highest k_cat/K_m value. The transglucosylation yields of the reactions for cellobiose, sorbose and glucose acceptors were 78, 57 and 54%, respectively, making cellobiose the most efficient acceptor of the tested saccharides in coupling with β-CD. The optimal condition for the coupling reaction was determined as: 2% (w/v) β-CD and 0.5% (w/v) cellobiose, incubated with 64 U/mL of CGTase at 30 °C for 2 h. Two main transfer products detected by HPLC, PC1 and PC2, with retention times of 3.81 and 4.42 min, respectively, and a product ratio of 3:1, had a molecular mass of 504 and 666 Da, respectively, as analyzed by mass spectrometry. The structures suggested by NMR were a trisaccharide and a novel tetrasaccharide-containing cellobiose of the structures glc (α1 → 4) glc (β1 → 4) glc and glc (α1 → 4) glc (α1 → 4) glc (β1 → 4) glc, respectively. The products were found to be resistant to hydrolysis by α-amylase.     

Glass transition temperatures and water sorption isotherms of cassava starch

The effect of water content on the glass transition temperatures of cassava starch was determined by differential scanning calorimetry (DSC) and dynamic mechanical thermal analysis (DMTA). Samples were transformed to the amorphous state by compression molding at high temperature (as demonstrated by wide angle X-ray diffraction, WAXS), and then the samples were moisture conditioned. Both DSC and DMTA showed that water anti-plasticized cassava starch at lower moisture contents, and plasticized it at higher water contents. Samples with higher moisture contents stored at room temperature, 45 °C and 80 °C underwent retrogradation as indicated by WAXS. Sorption isotherms of cassava starch showed that for a_w values lower than around 0.85, the sorption capacity decreased with increasing temperature; while the opposite behavior was observed at a_w > 0.85. This inversion point (a_w = 0.85) was attributed to the fact that more active sites were exposed to the adsorption processes, due to the enhanced molecular mobility promoted in the amorphous regions by starch crystallization.     

The Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 employs a new glycoside hydrolase family 70 4,6-α-glucanotransferase enzyme (GtfD) to synthesize a reuteran like polymer from maltodextrins and starch

BackgroundOriginally the glycoside hydrolase (GH) family 70 only comprised glucansucrases of lactic acid bacteria which synthesize α-glucan polymers from sucrose. Recently we have identified 2 novel subfamilies of GH70 enzymes represented by the Lactobacillus reuteri 121 GtfB and the Exiguobacterium sibiricum 255-15 GtfC enzymes. Both enzymes catalyze the cleavage of (α1 → 4) linkages in maltodextrin/starch and the synthesis of consecutive (α1 → 6) linkages. Here we describe a novel GH70 enzyme from the nitrogen-fixing Gram-negative bacterium Azotobacter chroococcum, designated as GtfD.MethodsThe purified recombinant GtfD enzyme was biochemically characterized using the amylose-staining assay and its products were identified using profiling chromatographic techniques (TLC and HPAEC-PAD). Glucans produced by the GtfD enzyme were analyzed by HPSEC-MALLS-RI, methylation analysis, 1D/2D [1]H/[13]C NMR spectroscopy and enzymatic degradation studies.ResultsThe A. chroococcum GtfD is closely related to GtfC enzymes, sharing the same non-permuted domain organization also found in GH13 enzymes and displaying 4,6-α-glucanotransferase activity. However, the GtfD enzyme is unable to synthesize consecutive (α1 → 6) glucosidic bonds. Instead, it forms a high molecular mass and branched α-glucan with alternating (α1 → 4) and (α1 → 6) linkages from amylose/starch, highly similar to the reuteran polymer synthesized by the L. reuteri GtfA glucansucrase from sucrose.ConclusionsIn view of its origin and specificity, the GtfD enzyme represents a unique evolutionary intermediate between family GH13 (α-amylase) and GH70 (glucansucrase) enzymes.General significanceThis study expands the natural repertoire of starch-converting enzymes providing the first characterization of an enzyme that converts starch into a reuteran-like α-glucan polymer, regarded as a health promoting food ingredient.     

Substitution of the catalytic acid–base Glu237 by Gln suppresses hydrolysis during glucosylation of phenolic acceptors catalyzed by Leuconostoc mesenteroides sucrose phosphorylase

Stereoselective glycosylation of a phenolic hydroxyl is a key transformation in the (bio)synthesis of natural products. Biocatalytic transglycosylation usually provides the desired glycosidic product in exquisite anomeric purity. However, loss of substrate and product to hydrolysis often limits application of the method. Kinetic studies and in situ proton NMR analysis of reaction time courses were used here to characterize glucosylation of substituted phenol acceptors by Leuconostoc mesenteroides sucrose phosphorylase in the presence of α-d-glucose 1-phosphate (αG1P) as donor substrate. In the wild-type enzyme, hydrolysis of the sugar 1-phosphate strongly prevailed (about 10-fold, ∼1.6 U/mg) over glucosyl transfer to the 2,6-difluorophenol acceptor (∼0.17 U/mg) used. A mutated phosphorylase in which the catalytic acid–base Glu²³⁷ had been replaced by Gln (E237Q) did not display hydrolase activity under transglucosylation conditions and therefore provided substantial (∼7-fold) enhancement of transfer yield. Utilization of the donor substrate was however slowed down (about 400-fold, ∼0.004 U/mg) in E237Q as compared to wild-type enzyme (∼1.6 U/mg). In a series of mono- and disubstituted phenols differing in hydroxyl pK_a between 7.02 and 8.71, the transferase activity of E237Q was found to be dependent on steric rather than electronic properties of the acceptor used. Both wild-type and mutated enzyme employed 4-nitrophenyl-α-d-glucopyranoside (4-NPG) as a slow artificial substrate for phosphorolysis and hydrolysis (native: ∼0.12 U/mg; E237Q: ∼0.02 U/mg).     

Grazing of cover crops in integrated crop-livestock systems

Conventional agriculture is specializing rapidly into the management of few monoculture crops, threatening crop diversity and questioning the sustainability of extensive cropping systems. The grazing of cover crops in integrated crop-livestock systems could be a feasible biologically based technology to restore crop diversity and mitigate ecological issues in cropping systems. However, there is limited evidence on plausible synergies or trade-offs for the practice, and about how grazing plans could affect the herbage production and services from cover crops. This work assessed the effects of cattle grazing on the primary and secondary production of annual ryegrass (Lolium multiflorum) in an integrated ryegrass–soybean rotation system. Specifically, the prediction for synergistic effects of cattle grazing on the ryegrass herbage production, residual crop cover and animal performance were tested in a 2-year (2014 and 2015) study comprising a randomized complete block design of four grazing intensity treatments, replicated three times. A no-cattle grazing treatment (NG), used as control, or continuous grazing with Holstein heifers (~ 220 kg live weight) at targeted sward heights of 5, 10, 15 and 20 cm (hereafter referred as G₅, G₁₀, G₁₅ and G₂₀, respectively) was applied to ryegrass plots. The herbage production and residual herbage cover of ryegrass, and the average daily gain (ADG, kg/day) and live weight gain per hectare (LWG, kg/ha) of heifers were analyzed by ANOVA (P < 0.05) and compared by the Tukey_HSD test (P < 0.05). Regression models were used to estimate relationships between herbage production, animal performance and sward height. The herbage production was 60% higher (P < 0.01) for the grazing treatments compared to NG. The residual herbage for G₁₅ and G₂₀ was not different than that for NG and increased linearly as sward heights increased, reaching highest values for G₁₅ and G_20. Maximum ADG was 1.10 kg/day for ryegrass grazed at a 20.6 cm height, whereas maximum LWG was 427 kg/ha for ryegrass grazed to a 16.1 cm height. The results support the hypothesis for synergistic effects of using annual ryegrass as a dual forage and service cover crop. Moderate grazing intensity to sward height of 12–18 cm with continuous stocking led to optimized forage production and utilization by dairy heifers.     

Kinetic and thermodynamic analysis of the inhibitory effects of maltose,glucose, and related carbohydrates on wheat β-amylase

Inhibition of wheat β-amylase (WBA) by glucose and maltose was studied by kinetics and thermodynamics. The inhibitory effects of fructose, difructose, sucrose, trehalose, cellobiose, acarbose, and 1-deoxynojirimycin on WBA were also evaluated. The half maximal inhibitory concentrations (IC₅₀) of acarbose, maltose and glucose were 0.06 ± 0.01 M, 0.22 ± 0.09 M, and 1.41 ± 0.17 M, respectively. The inhibitor constant (K_i) and the thermodynamic parameters such as changes in Gibbs energy (ΔG), enthalpy (ΔH), and entropy (ΔS) of the dissociation reactions of the WBA-glucose and WBA-maltose complexes were temperature and pH-dependent. The dissociation reactions were endothermic and enthalpy-driven. Both glucose and maltose behaved as competitive inhibitors at pH 3.0 and 5.4 at a temperature of 25 °C with respective K_i values of 0.33 ± 0.02 M and 0.12 ± 0.03 M. In contrast, both sugars exhibited uncompetitive inhibition at pH 9 at a temperature of 25 °C with K_i values of 0.21 ± 0.03 M for glucose and 0.11 ± 0.04 M for maltose. The pH-dependence of the inhibition type and K_i values indicate that the ionizing groups of WBA influence drastically the interaction with these carbohydrates. This evidence enables us to consider temperature and pH in the WBA-catalyzed hydrolysis to manipulate the inhibition by end-product, maltose, and even by glucose.     

Unmarked insertional inactivation in the gfo gene improves growth and ethanol production by Zymomonas mobilis ZM4 in sucrose without formation of sorbitol as a by-product,but yields opposite effects in high glucose

Sorbitol, one of the main by-products of growth on high sucrose concentrations, is catalyzed by glucose-fructose oxidoreductase (GFOR, EC 1.1.99.28) in Zymomonas mobilis, which decreases the ethanol yield. In this study, an unmarked gfo mutant from Z. mobilis ZM4 was constructed using a site-specific FLP recombinase, and growth and ethanol production were evaluated with or without the addition of sorbitol to the media. The inactivation of gfo had contrasting effects in different substrates, especially at high concentrations. The maximum specific growth rate (μ_m) and theoretical ethanol yield value (Y_m) increased from 0.065 h⁻¹ and 60.56% to 0.094 h⁻¹ and 83.87% in 342 g/L sucrose, respectively. Conversely, in 200 g/L glucose, gfo inactivation decreased μ_m and Y_m from 0.15 h⁻¹ and 89.85% to 0.10 h⁻¹ and 67.59%, respectively, and prolonged the lag period from 16 h to 40 h. The addition of sorbitol slightly accelerated growth and sucrose hydrolysis by the gfo mutant in 342 g/L sucrose; however, addition of sorbitol restored the μ_m and Y_m of the gfo mutant in 200 g/L glucose to 0.14 h⁻¹ and 82.50%, respectively. Inactivation of gfo had a small effect on fructose utilization, and a positive one on mixture of glucose and fructose similar to that on sucrose. These results provide further understanding of the osmoregulation mechanisms in Z. mobilis and may help to exploit the biotechnological applications of this industrially important bacterium.     

Efficient production of α-arbutin by whole-cell biocatalysis using immobilized hydroquinone as a glucosyl acceptor

The aim of the present study is to develop an efficient and cost-effective method for α-arbutin production by using whole-cell of Xanthomonas maltophilia BT-112 as a biocatalyst. Hydroquinone (HQ), substrate for the bioconversion as glucosyl acceptor, was immobilized on H107 macroporous resin to reduce its toxic effect on the cells, and the optimal reaction conditions for α-arbutin synthesis were investigated. When 350 g/L H107 resin (254.5 mM HQ) and 20 g/L (4.2 U/g) of cells were shaken in 10 mL Na₂HPO₄–KH₂PO₄ buffer (50 mM, pH 6.5) containing 509 mM sucrose at 35 °C with 150 rpm for 48 h, the final yield of α-arbutin reached 65.9 g/L with a conversion yield of 95.2% based on the amount of HQ supplied. The α-arbutin production was 202% higher than that of the control (free HQ) and the cells maintained its full activity for almost six consecutive batch reactions, indicating a potential for reducing production costs. Additionally, the product was one-step isolated and identified as α-arbutin by ¹³C NMR and ¹H NMR analysis. In conclusion, the combination of whole cells and immobilized hydroquinone (IMHQ) is a promising approach for economical and industrial-scale production of α-arbutin.     

Secretory expression of functional barley limit dextrinase by Pichia pastoris using high cell-density fermentation

Heterologous production of large multidomain proteins from higher plants is often cumbersome. Barley limit dextrinase (LD), a 98 kDa multidomain starch and α-limit dextrin debranching enzyme, plays a major role in starch mobilization during seed germination and is possibly involved in starch biosynthesis by trimming of intermediate branched α-glucan structures. Highly active barley LD is obtained by secretory expression during high cell-density fermentation of Pichia pastoris. The LD encoding gene fragment without signal peptide was subcloned in-frame with the Saccharomyces cerevisiae α-factor secretion signal of the P. pastoris vector pPIC9K under control of the alcohol oxidase 1 promoter. Optimization of a fed-batch fermentation procedure enabled efficient production of LD in a 5-L bioreactor, which combined with affinity chromatography on β-cyclodextrin–Sepharose followed by Hiload Superdex 200 gel filtration yielded 34 mg homogenous LD (84% recovery). The identity of the recombinant LD was verified by N-terminal sequencing and by mass spectrometric peptide mapping. A molecular mass of 98 kDa was estimated by SDS–PAGE in excellent agreement with the theoretical value of 97419 Da. Kinetic constants of LD catalyzed pullulan hydrolysis were found to K_m,app = 0.16 ± 0.02 mg/mL and k_cat,app = 79 ± 10 s^?1 by fitting the uncompetitive substrate inhibition Michaelis–Menten equation, which reflects significant substrate inhibition and/or transglycosylation. The resulting catalytic coefficient, k_cat,app/K_m,app = 488 ± 23 mL/(mg s) is 3.5-fold higher than for barley malt LD. Surface plasmon resonance analysis showed α-, β-, and γ-cyclodextrin binding to LD with K_d of 27.2, 0.70, and 34.7 μM, respectively.     

Response surface methodology: Synthesis of short chain fructooligosaccharides with a fructosyltransferase from Aspergillus aculeatus

A transferase was isolated, purified and characterised from Aspergillus aculeatus. The enzyme exhibited a pH and temperature optima of 6.0 and 60 °C, respectively and under such conditions remained stable with no decrease in activity after 5 h. The enzyme was purified 7.1 fold with a yield of 22.3% and specific activity of 486.1 U mg^?1 after dialysis, concentration with polyethyleneglycol (30%) and DEAE-Sephacel chromatography. It was monomeric with a molecular mass of 85 kDa and K_m and V_max values of 272.3 mM and 166.7 μmol min^?1 ml^?1. The influence of pH, temperature, reaction time, and enzyme and sucrose concentration on the formation of short-chain fructooligosaccharides (FOS) was examined by statistical response surface methodology (RSM). The enzyme showed both transfructosylation and hydrolytic activity with the transfructosylation ratio increasing to 88% at a sucrose concentration of 600 mg ml^?1. Sucrose concentration (400 mg ml^?1) temperature (60 °C), and pH (5.6) favoured the synthesis of high levels of GF₃ and GF₄. Incubation time had a critical effect on the yield of FOS as the major products were GF₂ after 4 h and GF₄ after 8 h. A prolonged incubation of 16 h resulted in the conversion of GF₄ into GF₂ as a result of self hydrolase activity.     

Osmoregulation in Dunaliella,Part II: Photosynthesis and starch contribute carbon for glycerol synthesis during a salt stress in Dunaliella tertiolecta

In response to an osmotic stress, Dunaliella tertiolecta osmoregulates by metabolizing intracellular glycerol as compatible solute. Upon the application of a salt stress to 0.17 M or 0.7 M NaCl grown D. tertiolecta cells, rates of total glycerol synthesis were substantially higher than that arising from photosynthetic ¹⁴CO₂ fixation into glycerol. The source of this extra carbon is the reserve starch pool. The contribution of carbon from the starch breakdown to glycerol synthesis was estimated from the difference between the total glycerol synthesized and that arising from ¹⁴CO₂ fixation. The maximum observed flux of carbon from ¹⁴CO₂ to glycerol from photosynthesis was of the order of 15–20 μmol ¹⁴C-glycerol mg⁻¹ Chl h⁻¹, whereas the total glycerol synthesis reached about 70 μmol glycerol mg⁻¹ Chl h⁻¹. The contribution of products of starch breakdown to glycerol synthesis increased progressively with increasing salt stress. In light, contrary to prevailing assumptions, both the photosynthesis and the starch breakdown contribute carbon to glycerol biosynthesis. The relative contributions of these two processes in the light, while cells were actively photosynthesizing, depended on the magnitude of the salt stress. On application of dilution stress, the flux of carbon from newly photosynthetically fixed ¹⁴CO₂ into glycerol was reduced progressively with increasing dilution stress that was also accompanied by a decline in total glycerol contents of the cell. The maximum observed rate of glycerol dissimilation was about 135 μmol glycerol mg⁻¹ Chl h⁻¹.     

A multi-tolerant low molecular weight mannanase from Bacillus sp. CSB39 and its compatibility as an industrial biocatalyst

Bacillus sp. CSB39, isolated from popular traditional Korean food (Kimchi), produced a low molecular weight, thermostable mannanase (MnCSB39); 571.14 U/mL using locust bean gum galactomannan as a major substrate. It was purified to homogeneity using a simple and effective two-step purification strategy, Sepharose CL-6B and DEAE Sepharose Fast Flow, which resulted in 25.47% yield and 19.32-fold purity. The surfactant-, NaCl-, urea-, and protease-tolerant monomeric protein had a mass of ∼30 kDa as analyzed by SDS-PAGE and galactomannan zymography. MnCSB39 was found to have optimal activity at pH 7.5 and temperature of 70 °C. The enzyme showed ˃55% activity at 5.0–15% (w/v) NaCl, and ˃93% of the initial activity after incubation at 37 °C for 60 min. Trypsin and proteinase K had no effect on MnCBS39. The enzyme showed ˃80% activity in up to 3 M urea. The N-terminal amino acid sequence, ALKGDGX, did not show identity with reported mannanases, which suggests the novelty of our enzyme. Activation energy for galactomannan hydrolysis was 26.85 kJmol⁻¹ with a K_cat of 142.58 × 10⁴ s⁻¹. MnCSB39 had K_m and V_max values of 0.082 mg/mL and 1099 ± 1.0 Umg⁻¹, respectively. Thermodynamic parameters such as ΔH, ΔG, ΔS, Q₁₀, ΔG_E-S, and ΔG_E-T supported the spontaneous formation of products and the high hydrolytic efficiency and feasibility of the enzymatic reaction, which strengthen its novelty. MnCSB39 activity was affected by metal ions, modulators, chelators, and detergents. Mannobiose was the principal end-product of hydrolysis. Bacillus subtilis CSB39 produced a maximum of 1524.44 U mannanase from solid state fermentation of 1 g wheat bran. MnCSB39 was simple to purify, was active at a wide pH and temperature range, multi-stress tolerant and catalyzes a thermodynamically possible reaction, characteristics that suggests its suitability for application as an industrial biocatalyst.