The potential of enzyme recycling during the hydrolysis of a mixed softwood feedstock

Despite recent improvement in cellulase enzymes properties, the high cost associated with the hydrolysis step remains a major impediment to the commercialization of full-scale lignocellulose-to-ethanol bioconversion process. As part of a research effort to develop a commercial process for bioconversion of softwood residues, we have examined the potential for recycling enzymes during the hydrolysis of mixed softwood substrate pretreated by organosolv process. We have used response surface methodology to determine the optimal temperature, pH, ionic strength, and surfactant (Tween 80) concentration for maximizing the recovery of bound protein and enzyme activity from the residual substrates after hydrolysis. Data analysis showed that the temperature, pH and surfactant concentration were the major factors governing enzyme desorption from residual substrate. The optimized conditions were temperature 44.4 °C, pH 5.3 and 0.5% Tween 80. The optimal conditions significantly increased the hydrolysis yield by 25% after three rounds of hydrolysis. This bound enzyme desorption combining with free enzyme re-adsorption is a potential method to recover cellulase enzymes and reduce the cost of enzymatic hydrolysis.     

Characterization of an extracellular alkaline serine protease from marine <Emphasis Type="Italic">Engyodontium album</Emphasis> BTMFS10

An alkaline protease from marine Engyodontium album was characterized for its physicochemical properties towards evaluation of its suitability for potential industrial applications. Molecular mass of the enzyme by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis was calculated as 28.6 kDa. Isoelectric focusing yielded pI of 3–4. Enzyme inhibition by phenylmethylsulfonyl fluoride (PMSF) and aprotinin confirmed the serine protease nature of the enzyme. K _m, V _max, and K _cat of the enzyme were 4.727 × 10⁻² mg/ml, 394.68 U, and 4.2175 × 10⁻² s⁻¹, respectively. Enzyme was noted to be active over a broad range of pH (6–12) and temperature (15–65°C), with maximum activity at pH 11 and 60°C. CaCl₂ (1 mM), starch (1%), and sucrose (1%) imparted thermal stability at 65°C. Hg²⁺, Cu²⁺, Fe³⁺, Zn²⁺, Cd⁺, and Al³⁺ inhibited enzyme activity, while 1 mM Co²⁺ enhanced enzyme activity. Reducing agents enhanced enzyme activity at lower concentrations. The enzyme showed considerable storage stability, and retained its activity in the presence of hydrocarbons, natural oils, surfactants, and most of the organic solvents tested. Results indicate that the marine protease holds potential for use in the detergent industry and for varied applications.     

Phosphorus fixation in lake sediments using LaCl3-modified clays

Fourteen types of clay, including soils and sediments from Lake Taihu, were modified by LaCl₃, and their abilities of phosphorus (P) sorption (the total P up-taken) and fixation (the unleachable P retained in modified clays) were investigated. Results showed that P sorption rates of LaCl₃-modified clays were all higher than 90%, while the fixation rates were raised from 3–14% to 52–95%. Kaolinite was selected to study the pH effect on P sorption and desorption of La³⁺ from the modified clays. In the pH range of 4–8, P sorption on LaCl₃-modified kaolinite could reach over 80%, with a maximum of 97% at pH 5. The desorption rate of La³⁺ decreased with the increase of pH, which was lower than <0.006% at pH > 6.12. This study may provide a solution for internal P pollution from lake sediments.     

红壤中镉在有机酸作用下的解吸行为

采用平衡批处理法,研究了3种有机酸及其两两混合液在序列pH值梯度下（pH 3.0～7.0）对华南山地红壤Cd解吸行为的影响.结果表明,草酸与苹果酸不利于Cd的解吸,反而促进了吸附,其中草酸只是在较高浓度(20 mmol·L^-1)且土壤溶液pH＞5.0时促进解吸.随着pH值升高,草s酸、苹果酸以及不含有机酸的对照溶液对红壤中Cd的解吸率都快速下降.柠檬酸在pH＜5.0时不利于Cd解吸;在pH＞5.0时显著促进Cd解吸,但两种浓度柠檬酸解吸特征有所不同,在低浓度(2 mmol·L^-1)下对镉的解吸率呈降低-升高-降低变化,在高浓度(20 mmol·L^-1)下呈降低-升高变化.在低pH条件下（pH 3.0、4.0）,苹果酸最有利于Cd的解吸,但3种酸对Cd解吸率差别不大,在较高pH条件下(pH 5.0～7.0),柠檬酸最有利于解吸,且解吸率大大高于草酸与苹果酸.有机酸混合没有明显的交互作用,对Cd的解吸率介于相应单独有机酸之间.     

Purification and partial characterisation of tentatively classified acid phosphatase from the earthworm Eisenia veneta

In order to use leakage of lysosomal acid phosphatase (AP) as a biomarker of stress to earthworms, more information about AP’s in earthworms are needed. This paper describes the details about tentatively classified APs in the earthworm Eisenia veneta. Two isoenzymes (enzyme I and II) of acid phosphatase (AP) and one alkaline phosphatase (enzyme III) from the earthworm E. veneta were separated by gel filtration. All three enzymes were further purified and concentrated on a Con A Sepharose 4B column. Enzyme I was inhibited by tartrate, showed an optimal pH range between 4.0 and 5.0 and was assumed to be of lysosomal origin. Enzyme II was the major enzyme showing the highest activity of the three enzymes. It was expected to be a lysosomal AP under physiological conditions. Enzyme II had a molecular mass 113 kDa and was composed of apparently identical polypeptide chains of 36 kDa each. This enzyme was inhibited by tartrate, showed an optimal pH in the range 6.0–7.5 and was slowly degraded at temperatures above 40°C. Enzyme III is not inhibited by tartrate and has a pH-optimum >9. The subcellular location under physiological conditions was assumed to be the cytosol.     

Fast separation of bromelain by polyacrylic acid-bound iron oxide magnetic nanoparticles

The adsorption of bromelain from an aqueous solution by polyacrylic acid (PAA)-bound iron oxide magnetic nanoparticles was studied. The magnetic composite nanoparticles were shown to be efficient for the separation of bromelain. Except at pH <3, the adsorption of bromelain increased with the decrease in solution pH and reached almost 100% at pH 3–5. The adsorbed bromelain could be desorbed by the addition of KCl and complete desorption was achieved at pH 7 when [KCl]>0.6 M. The adsorption behaviour followed the Langmuir isotherm with a maximum adsorption amount of 0.476 mg/mg and a Langmuir adsorption equilibrium constant of 58.4 ml/mg at pH 4 and 0.1 M phosphate. In addition, it was notable that both the adsorption and desorption of bromelain were quite fast and could be completed in about 1 min due to the absence of internal diffusion resistance. Bromelain retained 87.4% activity after adsorption/desorption.     

Bacterial desorption from food container and food processing surfaces

The desorption ofStaphylococcus aureus, Acinetobacter calcoaceticus, and a coryneform from the surfaces of materials used for manufacturing food containers (glass, tin plate, and polypropylene) or postprocess canning factory conveyor belts (stainless steel and nylon) was investigated. The effect of time, pH, temperature, and adsorbed organic layers on desorption was studied.S. aureus did not detach from the substrata at any pH investigated (between pH 5 and 9).A. calcoaceticus and the coryneform in some cases detached, depending upon pH and substratum composition. The degree of bacterial detachment from the substrata was not related to bacterial respiration at experimental pH values. Bacterial desorption was not affected by temperature (4–30°C) nor by an adsorbed layer of peptone and yeast extract on the substrata. The results indicate that bacterial desorption, hence bacterial removal during cleaning or their transfer via liquids flowing over colonized surfaces, is likely to vary with the surface composition and the bacterial species colonizing the surfaces.     

Purification,structural characterization and biotechnological potential of tannase enzyme produced by Enterobacter cloacae strain 41

Tannase production by Enterobacter cloacae strain 41 was investigated under submerged fermentation which was optimized at various circumstances such as pH, temperature, substrate, and agitation, carbon, and nitrogen sources. Tannase was purified by a two-step approach comprising of ion exchange and size exclusion chromatography, respectively. The maximum tannase production was achieved at 1.0% tannic acid concentration, incubation temperature of 50 °C, and initial pH 6.0. The molecular weight of purified tannase was 45 kDa on 10% SDS-PAGE, and it was confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). The enzymatic products of purified tannase were characterized by HPLC, TLC and FT-IR spectroscopy which showed the functional groups such as OH, CO, and CC. The purified tannase retained the activity up to 90% under the condition at 50 °C and pH 6.0 after 1 h incubation. Enzyme kinetics and inhibition studies were also investigated. Cytotoxicity studies showed that the tannase has no cytotoxic effects on Vero cell line. The results indicated the E. cloacae strain 41 would give a potential source for the efficient production of tannase and can be used in tannery effluent degradation, food, and pharmaceutical industrial applications.     

Biobleaching effect of xylanase preparation from an alkalophilic Bacillus sp. on ramie fibers

Cellulase-free xylanase from an alkalophilic Bacillussp. was maximally active at pH 10 and 60 °C. Enzyme treatment of ramie fibers removed 40% of its hemicellulose and some chromophoric material which resulted in a brightness increment of 5.2% and boosted the effect of H₂O₂bleaching. Enzyme-treated ramie fibers were increased by 3.9% in elongation and retained appropriate tenacity. X-ray and scanning electron micrograph studies revealed some changes in fiber structure.     

Continuous Production of Thermostable β-Amylase with Clostridium thermosulfurogenes: Effect of Culture Conditions and Metabolite Levels on Enzyme Synthesis and Activity

A β-amylase-overproducing mutant of Clostridium thermosulfurogenes was grown in continuous culture on soluble starch to produce thermostable β-amylase. Enzyme productivity was reasonably stable over periods of weeks to months. The pH and temperature optima for β-amylase production were pH 6.0 and 60°C, respectively. Enzyme concentration was maximized by increasing biomass concentration by using high substrate concentrations and by maintaining a low growth rate. β-Amylase concentration reached 90 U ml⁻¹ at a dilution rate of 0.07 h⁻¹ in a 3% starch medium. A further increase in enzyme activity levels was limited by acetic acid inhibition of growth and low β-amylase productivity at low growth rates.     

Desorption of Copper from Polyvinyl Alcohol‐Immobilized Seaweed Biomass

The desorption characteristics of copper on biomass of a marine macroalga, Sargassum baccularia, immobilized in polyvinyl alcohol (PVA) gel beads, were investigated using HCl eluting solutions. Both the extent and the rate of desorption were affected by the pH of the eluent. Nearly 91% of the copper initially adsorbed was released back into an HCl solution at pH 1.0 after 40 min of contact time when apparent desorption equilibrium was achieved. When the pH was raised to 2.0, about 81% of the bound copper was desorbed within 120 min of contact time. Apparent desorption rate constants were determined using first‐order desorption models. Very high concentrations of copper in the eluate could be obtained by using small amounts of the HCl eluent. However, this was achieved at the expense of the desorption efficiency. The PVA‐immobilized seaweed biomass beads could be regenerated with HCl solution at pH 1.0 or 2.0 in multiple cycles of copper biosorption‐desorption. Following desorption at pH 1.0 in the first cycle, about 55% of the biosorption capacity of the virgin biomass could be reused in subsequent cycles; in the case of desorption at pH 2.0, about 67% of the original uptake capacity was reusable.     

Online recovery of nisin during fermentation and its effect on nisin production in biofilm reactor

An online removal of nisin by silicic acid coupled with a micro-filter module was proposed as an alternative to reduce detrimental effects caused by adsorption of nisin onto producer, enzymatic degradation by protease, and product inhibition during fermentation. In this study, silicic acid was successfully used to recover nisin from the fermentation broth of Lactococcus lactis subsp. lactis NIZO 22186. The effect of pH (at 6.8 and 3.0) during adsorption process and several eluents (deionized water, 20% ethanol, 1 M NaCl, and 1 M NaCl + 20% ethanol) for desorption were evaluated in a small batch scale. Higher nisin adsorption onto silicic acid was achieved when the adsorption was carried out at pH 6.8 (67% adsorption) than at pH 3.0 (54% adsorption). The maximum recovery was achieved (47% of nisin was harvested) when the adsorption was carried out at pH 6.8 and 1 M NaCl + 20% ethanol was used as an eluent for desorption. Most importantly, nisin production was significantly enhanced (7,445 IU/ml) when compared with the batch fermentation without the online recovery (1,897 IU/ml). This may possibly be attributed to preventing the loss of nisin due the detrimental effects and a higher biomass density achieved during online recovery process, which stimulated production of nisin during fermentation.     

Recombinant maize 9-lipoxygenase: Expression,purification, and properties

Expression of maize 9-lipoxygenase was performed and optimized in Escherichia coli Rosetta(DE3)pLysS. The purity of recombinant protein obtained during Q-Sepharose and Octyl-Sepharose chromatographies in an LP system at 4°C was >95%. Maximum activity of the lipoxygenase reaction was observed at pH 7.5. Enzyme stability was studied at pH 4.5 to 9.5 and in the presence of different compounds: phenylmethanesulfonyl fluoride, β-mercaptoethanol, ammonium sulfate, and glycerol. HPLC and GC-MS analysis showed that enzyme produced 99% 9S-hydroperoxide from linoleic acid. 13-Hydroperoxide (less than 1%) consisted of S- and R-enantiomers in ratio 2 : 3.     

In vitro fibrinolytic activity of an enzyme purified from Bacillus amyloliquefaciens strain KJ10 isolated from soybean paste

A fibrinolytic protease secreting producing Bacillus amyloliquefaciens strain KJ10 was initially screened from the fermented soybean. Maximum productivity was obtained in the culture medium after 40 h incubation, 34 °C incubation temperature at pH 8.0. Fibrinolytic protease production was enhanced in the culture medium with 1% sucrose (3712 ± 52 U/mL), 1% (w/v) yeast extract (3940 ± 28 U/mL) and 0.1% MgSO₄ (3687 ± 38 U/mL). Enzyme was purified up to 22.9-fold with 26%recovery after Q-Sepharose HP column chromatography. After three steps purification, enzyme activity was 1606U/mg and SDS-PAGE analysis revealed 29 kDa protein and enzyme band was detected by zymograpy. Enzyme was highly active at pH 8.0, at wide temperature ranges (40 °C ? 55 °C) and was activated by Mn²⁺ (102 ± 3.1%) and Mg²⁺ (101.4 ± 2.9%) ions. The purified fibrinolytic enzyme was highly specific against N-Suc-Ala-Ala-Pro-Phe-pNA (189 mmol/min/mL) and clot lytic activity reached 28 ± 1.8% within 60 minin vitro. The purified fibrinolytic enzyme showed least erythrocytic lysis activity confirmed safety to prevent various health risks, including hemolytic anemia. Based on this study, administration of fibrinolytic enzyme from B. amyloliquefaciens strain KJ10 is safe for clinical applications.     

Enzyme production in the traps of aquatic <Emphasis Type="Italic">Utricularia</Emphasis> species

We studied the influence of habitat and increased mineral phosphorus and nitrogen loading on the extracellular activity of five selected hydrolases and pH in the trap fluid of the aquatic carnivorous plants, Utricularia vulgaris, U. australis, and U. foliosa (Lentibulariaceae). Enzyme activities in the trap fluid were determined using fluorometry. Phosphatase exhibited the highest activities in the traps of the European species as well as field-grown tropical U. foliosa. Trap enzyme production appeared to be uninfluenced by elevated dissolved mineral N or P concentrations both in the trap and ambient environment and thus, it seems to be constitutive. Enzyme activity in the trap fluid was determined by species and environmental conditions and varied significantly among sites within a single species. Trap fluid pH was between 4.2–5.1 in U. vulgaris and U. australis but between 5.7–7.3 in U. foliosa and seems to be regulated by the traps.     

Rice Hull Ash and Silicic Acid as Adsorbents for Concentration of Bacteriocins

A model procedure has been developed for the rapid extraction of five bacteriocins (nisin, pediocin RS2, leucocin BC2, lactocin GI3, and enterocin CS1) from concentrated freeze-dried crude culture supernatants by adsorption onto acid or alkaline rice hull ash (RHA) or silicic acid (SA). Bacteriocins were adsorbed onto RHA or SA by a pH-dependent method and desorbed by decreasing the pH to 2.5 or 3.0 and heating at 90°C for 5 min. The maximum adsorption and optimal pH range for different bacteriocins were as follows: nisin, 97% at pH 7.0; lactocin GI3, 94% at pH 6.0; pediocin RS2, 97% at pH 8.0 to 9.0; leucocin BC2, 88% at pH 9.0; and enterocin CS1, 94% at pH 5.0. The desorption level of lactocin GI3 or enterocin CS1 from the surfaces of both RHA and SA was 94%, while the desorption level of pediocin RS2 and leucocin BC2 was 50% or less. Nisin was desorbed readily from SA (91%) but not from RHA (50% or less). The adsorption of bacteriocins onto RHA and SA increased with the increasing concentration of bacteriocins. Analysis of the desorbed bacteriocins after dialysis and sodium dodecyl sulfate–16% polyacrylamide gel electrophoresis showed a single band that gave a single inhibition zone when overlaid with Lactobacillus plantarum for detection of lactocin GI3, enterocin CS1, and nisin. RHA appears useful for extraction, concentration, and partial purification of the five bacteriocins.     

Characterization of diphtheria toxin's catalytic domain interaction with lipid membranes

In response to a low environmental pH and with the help of the B fragment (DTB) the catalytic domain of diphtheria toxin (DTA) crosses the endosomal membrane to inhibit protein synthesis. In this study, we investigated the interaction of DTA with lipid membranes by biochemical and biophysical approaches. Data obtained from proteinase K and trypsin digestion experiments of membrane-inserted DTA suggested that residues 134-157 may adopt a transmembrane orientation and residues 77-100 could be membrane-associated, adopting either a surface or a transmembrane orientation. Fourier transform infrared spectroscopy analysis (FTIR) was used to characterize the secondary and tertiary structure of DTA along its pathway, from the native secreted form at pH 7.2 to the refolded structure at neutral pH after interaction with and desorption from a lipid membrane. We found that the association of DTA with lipid membranes at low pH was characterized by an increase of β-sheet structures and that the refolded structure at neutral pH after interaction with the membrane was identical to the native structure at the same pH. We also investigated the desorption of DTA from the membrane at neutral pH as a function of temperature. Although a complete desorption was observed at 37 °C, no desorption took place at 4 °C. A model of translocation involving the possibility that DTA might insert one or several transient transmembrane domains during translocation is discussed.     

Establishing suitable conditions for metals recovery from metal saturated Bacillaceae bacterium using experimental design

In this study the response surface methodology (RSM) based on the Box-Behnken design model was applied to investigate the effect of parameter interactions that are suspected to influence the metal desorption process. The experiment was based on the four-variable-three-level design of parameters such as uptake time, pH of eluant, shaking speed during desorption, and volume of eluant. The metal-removal process was designed so as to enhance desorption and regeneration of biosorbent. The results show that all four of the variables influenced metal desorption from the biomass, and the experimental design allowed better observation of the interaction level of parameters suitable for the desorption process leading to optimum recovery and regeneration of biosorbent. Calculated P values (P = 0.0233 for nickel and P = 0.0203 for lead) confirmed the fitness of the model to the experiments conducted. The desorption process was found to be dependent on the type of mechanism involved during biosorption. Parameter settings of 20 min (level 1) uptake time, pH 5 (level 0), 50 ml (level 0) eluant, and 200 r min⁻¹ (level 1) shaking speed, led to maximum response (55.72%) during metal release. X-ray elemental mapping profiles and graphical data recorded using transmission electron microscopy (TEM) showed binding of both nickel and lead to Bacillaceae bacteria, but most importantly preferential binding of lead; FTIR spectroscopy depicted involvement of active groups such as hydroxyl, amine, and carboxyl in the uptake of both metals. Biomass regenerated was more effective in the removal of lead than nickel, achieving up to 100% uptake of lead in the second cycle. Desorption of metal from biomass is very important for the recovery of metals, which is necessary for undiminished uptake in the next cycle of metal removal.     

Isolation and characterization of N-long chain acyl aminoacylase from Pseudomonas diminuta

N-Long chain acyl aminoacylase II (Enzyme II) catalyzing the hydrolysis of N-long chain acyl amino acids was purified about 2,000-fold from the cell extracts of Pseudomonas diminuta with 1.8% of activity yield. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis and the molecular weight was 220,000. Enzyme II differed from N-long chain acyl aminoacylase I (Enzyme I) in molecular weight, in substrate specificity, and in behavior toward temperature and pH. Enzyme II showed broader substrate specificity than Enzyme I and catalyzed the hydrolysis of lipoamino acids containing various amino acid residues, although Enzyme I was almost specific to the lipoamino acids containing L-glutamate. The extent of hydrolysis by Enzyme II reaction varied depending on the kinds of lipoamino acids and were: 100% for palmitoyl-L-glutamate, 91% for myristoyl-L-glutamate, 85% for lauroyl-L-glutamate, 54% for lauroyl-L-aspartate, 28% for stearoyl-L-glutamate and 17.5% for lauroyl-glycine.     

Immobilization in the presence of Triton X-100: modifications in activity and thermostability of <Emphasis Type="Italic">Geobacillus thermoleovorans</Emphasis> CCR11 lipase

A partially purified lipase produced by the thermophile Geobacillus thermoleovorans CCR11 was immobilized by adsorption on porous polypropylene (Accurel EP-100) in the presence and absence of 0.1% Triton X-100. Lipase production was induced in a 2.5% high oleic safflower oil medium and the enzyme was partially purified by diafiltration (co. 500,000 Da). Immobilization conditions were established at 25 °C, pH 6, and a protein concentration of 0.9 mg/mL in the presence and absence of 0.1% Triton X-100. Immobilization increased enzyme thermostability but there was no change in neither the optimum pH nor in pH resistance irrelevant to the presence of the detergent during immobilization. Immobilization with or without Triton X-100 allowed the reuse of the lipase preparation for 11 and 8 cycles, respectively. There was a significant difference between residual activity of immobilized and soluble enzyme after 36 days of storage at 4 °C (P < 0.05). With respect to chain length specificity, the immobilized lipase showed less activity over short chain esters than the soluble lipase. The immobilized lipase showed good resistance to desorption with phosphate buffer and NaCl; minor loses with detergents were observed (less than 50% with Triton X-100 and Tween-80), but activity was completely lost with SDS. Immobilization of G. thermoleovorans CCR11 lipase in porous polypropylene is a simple and easy method to obtain a biocatalyst with increased stability, improved performance, with the possibility for re-use, and therefore an interesting potential use in commercial conditions.