Purification and in situ immobilization of lipase from of a mutant of Trichosporon laibacchii using aqueous two-phase systems

The crude intracellular lipase (cell homogenate) from Trichosporon laibacchii was subjected to partial purification by aqueous two-phase system (ATPS) and then in situ immobilization by directly adding diatomites as carrier to the top PEG-rich phase of ATPS. A partition study of lipase in the ATPS formed by polyethylene glycol–potassium phosphate has been performed. The influence of system parameters such as molecular weight of PEG, system phase composition and system pH on the partitioning behaviour of lipase was evaluated. The ATPS consisting of PEG 4000 (12%) and potassium phosphate (K₂HPO₄, 13%) resulted in partition of lipase to the PEG-rich phase with partition coefficient 7.61, activity recovery 80.4%, and purification factor of 5.84 at pH of 7.0 and 2.0% NaCl. Moreover, the in situ immobilization of lipase in PEG phase resulted in a highest immobilized lipase activity of 1114.6 U g^?1. The above results show that this novel lipase immobilization procedure which couples ATPS extract and enzyme immobilization is cost-effective as well as time-saving. It could be potentially useful technique for the purification and immobilization of lipase.     

PEG/NaPA aqueous two-phase systems for the purification of proteases expressed by Penicillium restrictum from Brazilian Savanna

The partitioning of proteases expressed by Penicillium restrictum from Brazilian Savanna in an inexpensive aqueous two-phase system composed of poly (ethylene glycol) (PEG) and sodium polyacrylate (NaPA) was studied. The effects of PEG molecular weight and concentration, as well as NaPA concentration and the concentration of fermented broth on protease partitioning were studied. Partitioning into the top PEG-rich phase was increased in systems with smaller PEG-molecular weight, higher NaPA concentration and lower PEG concentration. For most systems studied, purification has been achieved by directing the biomolecule partition to the opposite phase of the other proteins, providing the enzyme purification. The highest partition coefficient was obtained using 20 wt% NaPA, 4 wt% PEG 2000 g mol⁻¹ and 45 wt% fermented broth, leading to a purification factor of 1.98 and partition coefficient of 37.73. The system showed high mass balances and yield, indicating enzyme stability and applicability for industrial processes. The partitioning results using the PEG/NaPA/NaCl system show that this method could be used to purify or concentrate protease from fermented broth.     

Multi-stage laccase extraction and separation using aqueous two-phase systems: Experiment and model

This work presents results of experimental and model investigation of continuous multi-stage enzyme extraction using aqueous two-phase systems for the first time. The aqueous two-phase system comprised polyethylene glycol 3000 and phosphate with additional sodium chloride buffered to pH 7. Two different laccases served as model enzymes. One of the laccases was directly taken from fungal culture supernatant, while the other laccase was solubilized lyophilisate. The modeling is based on an equilibrium stage approach. Equilibrium data were taken from single-stage experiments and approximated by different correlation equations. The model describes densities, phase equilibrium, enzyme activity partitioning between the phases. Moreover it allows to consider activity changes due to the aqueous two-phase system. Eight multi-stage mixer-settler experiments under varying operation conditions were performed to validate the proposed model; whereas the total throughput of all multi-stage extraction experiments was about 350 g h⁻¹. The average relative deviation of modeled activities from experimentally measured activities was 23%. Therefore, the model is able to calculate the behavior of the phases as well as the partitioning of the two enzymes between the two phases for a multi-stage process based on single-stage data.     

Enzymatic transformation of tyrosol by Trametes trogii laccases: Identification of the product and study of its biological activities

Oxidative transformation of tyrosol catalysed by Trametes trogii laccases in aqueous solution was investigated. LC–MS analysis shows that tyrosol was converted to its dimer. The enzymatic reaction was also investigated by ¹H and ¹³C nuclear magnetic resonance, and the product formed was identified as a dimeric tetracyclic ketone. The bactericidal and fungicidal properties of tyrosol dimer were investigated using the NCCLS broth dilution and EN 1276 standard methods. High bactericidal and fungicidal effect of concentrations ranged between 1–0.5 g L⁻¹ and 8–4 g L⁻¹ were obtained. Dimer concentrations of 33 g L⁻¹ and 66 g L⁻¹ allowed reductions in viability higher than 5 log units per mL for Pseudomonas aeruginosa ATCC 15442, Escherichia coli ATCC 10536 and Enterococcus hirae ATCC 10541, Staphylococcus aureus ATCC 9144 respectively, within a contact time of 5 min under dirty conditions. The effect of this product on Tuta absoluta, a harmful pest of tomato in the world, was also evaluated. The results showed high insecticidal activity against this insect at a concentration of 16.5 g L⁻¹. Germinability experiments on Lycopersicum esculetum were conducted in order to evaluate the potential of a laccase treatment in removing tyrosol phytotoxicity. The results showed that tyrosol dimer was nonphytotoxic. This study presents the first comprehensive results of biological characterisation of the product obtained by the action of laccase on tyrosol transformation with T. trogii laccases.     

Laccase mediator system for activation of agarose gel: Application for immobilization of proteins

Cross-linked Sepharose beads were treated with laccase–TEMPO system for oxidation of the primary alcohol groups on the sugar moieties. Optimal activation conditions using Trametes versicolor laccase were at pH 5 and 22 °C, giving an aldehyde content of 55 μmol g⁻¹ Sepharose with 28 units g⁻¹ of laccase and 12.5 mM TEMPO. The activated Sepharose was used for immobilization of trypsin as model protein. Highest degree of immobilization was obtained at pH 10.5 but the activity yield was only 31% of that loaded on the gel. The yield of gel bound trypsin activity was increased to 76% (corresponding to about 43 U g⁻¹ Sepharose) when the immobilization was performed in the presence of trypsin inhibitor, benzamidine. The immobilization yields were comparable to that obtained on the matrix activated using sodium periodate (containing 72 μmol aldehyde per g Sepharose). Recycling and storage of the immobilized trypsin preparations showed high stability of the enzyme bound to laccase–TEMPO activated gel.     

Protocorm culture of Dendrobium candidum in balloon type bubble bioreactors

Protocorm cultures of Dendrobium candidum were established in balloon type bubble bioreactors using Murashige and Skoog (MS) medium with 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), 2.5% (w/v) sucrose, 5:25 mM NH₄:NO₃ and 1% (v/v) banana homogenate for the production of biomass and bioactive compounds. In 3 l bioreactor containing 2 l medium, a maximum protocorm biomass (21.0 g l⁻¹ dry biomass) and also optimum quantities of total polysaccharides (389.3 mg g⁻¹ DW), coumarins (18.0 mg g⁻¹ DW), polyphenolics (11.9 mg g⁻¹ DW), and flavonoids (4.5 mg g⁻¹ DW) were achieved after 7 weeks of culture. Based on these studies, 5 and 10 l bioreactor cultures were established to harvest 80 g and 160 g dry biomass. In 10 l bioreactors, the protocorms grown were accumulated with optimal levels of polysaccharides (424.1 mg g⁻¹ DW), coumarins (15.8 mg g⁻¹ DW), polyphenols (9.03 mg g⁻¹ DW) and flavonoids (4.7 mg g⁻¹ DW). The bioreactor technology developed here will be useful for the production of important bioactive compounds from D. candidum.     

Bench-scale fermentation of Trichoderma viride on wastewater sludge: Rheology,lytic enzymes and biocontrol activity

Conidiation and lytic enzyme production by Trichoderma viride at different solids concentration of pre-treated municipal wastewater sludge was examined in a 15-L fermenter. The maximum conidia concentration (5.94 × 10⁷ CFU mL⁻¹ at 96 h) was obtained at 30 g L⁻¹ suspended solids. The maximum lytic enzyme activities were achieved around 12–30 h of fermentation. Bioassay against a fungal phytopathogen, Fusarium sp. showed maximum activity in the sample drawn around 96 h of fermentation at 30 g L⁻¹ suspended solids concentration. Entomotoxicity against spruce budworm larvae showed maximum value ≈17290 SBU μL⁻¹ at 30 g L⁻¹ suspended solids concentration at the end of fermentation (96 h). Plant bioassay showed dual action of T. viride, i.e., disease prevention and growth promotion. The rheological analyses of fermentation sludges showed the pseudoplastic behaviour. In order to maintain required dissolved oxygen concentration ≥30%, the agitation and aeration requirements significantly increased at 35 g L⁻¹ compared to 30 and 25 g L⁻¹. The oxygen uptake rate and volumetric oxygen mass transfer coefficient, k_La at 35 g L⁻¹ did not increase in comparison to 30 g L⁻¹ due to rheological complexity of the broth during fermentation. Thus, the successful fermentation operation of the biocontrol fungus T. viride is a rational indication of its potential for mass-scale production for agriculture and forest sector as a biocontrol agent.     

Continuous succinic acid production by Actinobacillus succinogenes in a biofilm reactor: Steady-state metabolic flux variation

Continuous anaerobic fermentations were performed in a novel external-recycle, biofilm reactor using d-glucose and CO₂ as carbon substrates. Succinic acid (SA) yields were found to be an increasing function of glucose consumption with the succinic acid to acetic acid ratio increasing from 2.4 g g⁻¹ at a glucose consumption of 10 g L⁻¹, to 5.7 g g⁻¹ at a glucose consumption of 50 g L⁻¹. The formic acid to acetic acid ratio decreased from an equimolar value (0.77 g g⁻¹) at a glucose consumption of 10 g L⁻¹ to a value close to zero at 50 g L⁻¹. The highest SA yield on glucose and highest SA titre obtained were 0.91 g g⁻¹ and 48.5 g L⁻¹ respectively. Metabolic flux analysis based on the established C₃ and C₄ metabolic pathways of Actinobacillus succinogenes revealed that the increase in the succinate to acetate ratio could not be attributed to the decrease in formic acid and that an additional source of NADH was present. The fraction of unaccounted NADH increased with glucose consumption, suggesting that additional reducing power is present in the medium or is provided by the activation of an alternative metabolic pathway.     

Comparison of salinity tolerance of three Atriplex spp. in well-watered and drying soils

Members of the Chenopodiaceae are well adapted to both salt and drought stress and can serve as model species to understand the mechanisms of tolerance in plants. We grew Atriplex hortensis (ATHO), A. canescens (ATCA), and A. lentiformis (ATLE) along a NaCL salinity gradient under non-water-limited conditions and in drying soils in greenhouse experiments. The species differed in photosynthetic carbon fixation pathway, capacity for sodium uptake, and habitat preferences. Under non-water-limited conditions, ATLE (C4) maintained high growth rates up to 30 g L⁻¹ NaCl. ATHO (C3) had lower growth than ATLE at high salinities, while ATCA (C4) grew more slowly than either ATLE or ATHO and showed no net growth above 20 g L⁻¹ NaCl. ATHO and ATLE accumulated twice as much sodium in their shoots as ATCA, but all three species had increasing sodium levels at higher salinities. Potassium, magnesium and calcium levels were relatively constant over the salinity gradient. All three species showed marked accumulation of chloride across the salinity gradient, whereas nitrate, phosphorous and sulfate decreased with salinity. The effect of drought was simulated by growing plants in sealed pots with an initial charge of water plus NaCl, and allowing them to grow to the end point at which they no longer were able to extract water from the soil solution. Drought and salinity were not additive stress factors for Atriplex spp. in this experiment. NaCl increased their ability to extract water from the soil solution compared to fresh water controls. ATLE showed increased shoot dry matter production and increased water use efficiency (WUE) as initial salinity levels increased from 0 to 30 g L⁻¹ NaCl, whereas dry matter production and WUE peaked at 5 g L⁻¹ for ATHO and ATCA. Final soil moisture salinities tolerated by species were 85 g L⁻¹, 55 g L⁻¹ and 160 g L⁻¹ NaCl for ATHO, ATCA and ATLE, respectively. C4 photosynthesis and sodium accumulation in shoots were associated with high drought and salt tolerance.     

Partitioning of protease from stomach of albacore tuna (Thunnus alalunga) by aqueous two-phase systems

Partitioning of protease from stomach of albacore tuna using an aqueous two-phase system (ATPS) was investigated. The best ATPS conditions for protease partitioning from stomach extract (SE) and acidified counterpart (ASE) were 25% PEG1000–20% MgSO₄ and 15% PEG2000–15% MgSO₄, which increased the purity by 7.2-fold and 2.4-fold with the recovered activity of 85.7% and 89.1%, respectively. Electrophoretic study revealed that SE had a major protein with a molecular weight (MW) of 40.6 kDa, while protein with MW of 32.7 kDa was predominant in ASE and ATPS fractions. Pepsinogen in SE might be activated to pepsin by acidification and partitioning process. SE was quite stable at 0 and 4 °C up to 14 days. The loss in protease activity in ASE and selected ATPS fractions was more pronounced when storage time and temperature increased. Therefore, ATPS can be effectively used to recover and purify protease from albacore tuna stomach.     

Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysate

 Enzymatic hydrolysis of corncob and ethanol fermentation from cellulosic hydrolysate were investigated. After corncob was pretreated by 1% H₂SO₄ at 108 °C for 3 h, the cellulosic residue was hydrolyzed by cellulase from Trichoderma reesei ZU-02 and the hydrolysis yield was 67.5%. Poor cellobiase activity in T. reesei cellulase restricted the conversion of cellobiose to glucose, and the accumulation of cellobiose caused severe feedback inhibition to the activities of β-1,4-endoglucanase and β-1,4-exoglucanase in cellulase system. Supplementing cellobiase from Aspergillus niger ZU-07 greatly reduced the inhibitory effect caused by cellobiose, and the hydrolysis yield was improved to 83.9% with enhanced cellobiase activity of 6.5 CBU g⁻¹ substrate. Fed-batch hydrolysis process was started with a batch hydrolysis containing 100 g l⁻¹ substrate, with cellulosic residue added at 6 and 12 h twice to get a final substrate concentration of 200 g l⁻¹. After 60 h of reaction, the reducing sugar concentration reached 116.3 g l⁻¹ with a hydrolysis yield of 79.5%. Further fermentation of cellulosic hydrolysate containing 95.3 g l⁻¹ glucose was performed using Saccharomyces cerevisiae 316, and 45.7 g l⁻¹ ethanol was obtained within 18 h. The research results are meaningful in fuel ethanol production from agricultural residue instead of grain starch.     

Effects of feed sugar concentration on continuous ethanol fermentation of cheese whey powder solution (CWP)

Cheese whey powder (CWP) solution with different CWP or sugar concentrations was fermented to ethanol in a continuous fermenter using pure culture of Kluyveromyces marxianus (DSMZ 7239). Sugar concentration of the feed CWP solution varied between 55 and 200 g l⁻¹ while the hydraulic residence time (HRT) was kept constant at 54 h. Ethanol formation, sugar utilization and biomass formation were investigated as functions of the feed sugar concentration. Percent sugar utilization and biomass concentrations decreased and the effluent sugar concentration increased with increasing feed sugar concentrations especially for the feed sugar contents above 100 g l⁻¹. Ethanol concentration and productivity (DP) increased with increasing feed sugar up to 100 g l⁻¹ and then decreased with further increases in the feed sugar content. The highest ethanol concentration (3.7%, v v⁻¹) and productivity (0.54 gE l⁻¹ h⁻¹) were obtained with the feed sugar content of 100 g l⁻¹ or 125 g l⁻¹. The ethanol yield coefficient (Y_P/S) was also maximum (0.49 gE gS⁻¹) when the feed sugar was between 100 and 125 g l⁻¹. The growth yield coefficient (Y_X/S) decreased steadily from 0.123 to 0.063 gX gS⁻¹ when the feed sugar increased from 55 to 200 g l⁻¹ due to adverse effects of high sugar contents on yeast growth. The optimal feed sugar concentration maximizing the ethanol productivity and sugar utilization was between 100 and 125 g l⁻¹ under the specified experimental conditions.     

The effects of NH4+ and NO3− on growth,resource allocation and nitrogen uptake kinetics of Phragmites australis and Glyceria maxima

The effects of NH₄⁺ or NO₃⁻ on growth, resource allocation and nitrogen (N) uptake kinetics of two common helophytes Phragmites australis (Cav.) Trin. ex Steudel and Glyceria maxima (Hartm.) Holmb. were studied in semi steady-state hydroponic cultures. At a steady-state nitrogen availability of 34 μM the growth rate of Phragmites was not affected by the N form (mean RGR = 35.4 mg g⁻¹ d⁻¹), whereas the growth rate of Glyceria was 16% higher in NH₄⁺-N cultures than in NO₃⁻-N cultures (mean = 66.7 and 57.4 mg g⁻¹ d⁻¹ of NH₄⁺ and NO₃⁻ treated plants, respectively). Phragmites and Glyceria had higher S/R ratio in NH₄⁺ cultures than in NO₃⁻ cultures, 123.5 and 129.7%, respectively.Species differed in the nitrogen utilisation. In Glyceria, the relative tissue N content was higher than in Phragmites and was increased in NH₄⁺ treated plants by 16%. The tissue NH₄⁺ concentration (mean = 1.6 μmol g fresh wt⁻¹) was not affected by N treatment, whereas NO₃⁻ contents were higher in NO₃⁻ (mean = 1.5 μmol g fresh wt⁻¹) than in NH₄⁺ (mean = 0.4 μmol g fresh wt⁻¹) treated plants. In Phragmites, NH₄⁺ (mean = 1.6 μmol g fresh wt⁻¹) and NO₃⁻ (mean = 0.2 μmol g fresh wt⁻¹) contents were not affected by the N regime. Species did not differ in NH₄⁺ (mean = 56.5 μmol g⁻¹ root dry wt h⁻¹) and NO₃⁻ (mean = 34.5 μmol g⁻¹ root dry wt h⁻¹) maximum uptake rates (V_max), and V_max for NH₄⁺ uptake was not affected by N treatment. The uptake rate of NO₃⁻ was low in NH₄⁺ treated plants, and an induction phase for NO₃⁻ was observed in NH₄⁺ treated Phragmites but not in Glyceria. Phragmites had low K_m (mean = 4.5 μM) and high affinity (10.3 l g⁻¹ root dry wt h⁻¹) for both ions compared to Glyceria (K_m = 6.3 μM, affinity = 8.0 l g⁻¹ root dry wt h⁻¹). The results showed different plasticity of Phragmites and Glyceria toward N source. The positive response to NH₄⁺-N source may participates in the observed success of Glyceria at NH₄⁺ rich sites, although other factors have to be considered. Higher plasticity of Phragmites toward low nutrient availability may favour this species at oligotrophic sites.     

Uptake and allocation of 13C by Enhalus acoroides at sites differing in light availability

Carbon fixation and allocation were studied using ¹³C incubation and leaf marking techniques in mature monospecific stands of Enhalus acoroides L.f. Royle in August 1998 and January 1999 in Banten Bay, Indonesia. The highest rate of ¹³C uptake (>0.008 g ¹³C g C⁻¹ d⁻¹) was found in the middle to distal parts of leaves of E. acoroides. Young and senescing leaves numbers had lower ¹³C incorporation compared to mature leaves. The incorporation of ¹³C by epiphytes on the leaves was higher than that of the leaves themselves (>0.01 g ¹³C g C⁻¹ d⁻¹). The results showed that turbidity of the water influenced the leaf growth, productivity and Relative Growth Rate of E. acoroides, which were lower at Kepuh Island, the more turbid site. However, at Kepuh Island, where the water column was turbid, the plant could still harvest sufficient light for an uptake rate of ¹³C, higher than the uptake rates at Kubur and Panjang Islands, stations with a much more transparent water column (on average 0.0047 g ¹³C g C⁻¹ d⁻¹ at Kepuh Island, versus 0.0045 g ¹³C g C⁻¹ d⁻¹ at Panjang Island and 0.0034 g ¹³C g C⁻¹ d⁻¹ at Kubur Island). There was evidence that ¹³C was exported from the incubated shoots to the roots and rhizomes and to neighboring shoots of E. acoroides in clear water, but not in turbid water. We suggest that single shoots of E. acoroides are able to grow in turbid water under low light conditions. They assimilate sufficient carbon for their own maintenance but are not able to export to neighboring plant parts.     

Evaluation of distant carbon sources in biosurfactant production by a gamma ray-induced Pseudomonas putida mutant

Pseudomonas putida 33 wild strain, subjected to gamma ray mutagenesis and designated as P. putida 300-B mutant was used as microbial rhamnolipid-producer by using distant carbon sources (viz. hydrocarbons, waste frying oils ‘WFOs’, vegetable oil refinery wastes and molasses) in the minimal media under shake flask conditions. The behavior of glucose as co-substrate and growth initiator was examined. The 300-B mutant strain showed its ability to grow on all the substrates tested and produced rhamnolipid surfactants to different extents however; soybean and corn WFOs were observed to be preferred carbon sources followed by kerosene and paraffin oils, respectively. The best cell biomass (3.5 g l⁻¹) and rhamnolipids yield (4.1 g l⁻¹) were obtained with soybean WFO as carbon source and glucose as growth initiator under fed-batch cultivation showing an optimum specific growth rate (μ) of 0.272 h⁻¹, specific product yield (q_p) of 0.318 g g⁻¹ h and volumetric productivity (P_V) of 0.024 g l⁻¹ h. The critical micelle concentration of its culture supernatant was observed to be 91 mg rhamnolipids l⁻¹ and surface tension as 31.2 mN m⁻¹.     

d- and l-lactic acid production from fresh sweet potato through simultaneous saccharification and fermentation

The aim of this study was to develop a bioprocess for l- and d-lactic acid production from raw sweet potato through simultaneous saccharification and fermentation by Lactobacillus paracasei and Lactobacillus coryniformis, respectively. The effects of enzyme and nitrogen source concentrations as well as of the ratio of raw material to medium were investigated. At dried material concentrations of 136.36–219.51 g L⁻¹, yields of 90.13–91.17% (w/w) and productivities of 3.41–3.83 g L⁻¹ h⁻¹ were obtained with lactic acid concentrations as high as 198.32 g L⁻¹ for l-lactic acid production. In addition, d-lactic acid was produced with yields of 90.11–84.92% (w/w) and productivities of 2.55–3.11 g L⁻¹ h⁻¹ with a maximum concentration of 186.40 g L⁻¹ at the same concentrations of dried material. The simple and efficient process described in this study will benefit the tuber and root-based lactic acid industries without requiring alterations in plant equipment.     

Evaluating aqueous two-phase systems for Yarrowia lipolytica extracellular lipase purification

In this study an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) and potassium phosphate was tested for the purification of lipase from Yarrowia lipolytica IMUFRJ 50682. Ultrafiltration and precipitation with acetone and kaolin were also used as traditional comparison methods Ultrafiltration was a good method with a purification factor of 6.55, but protease was also purified in this extract. For the precipitation with acetone and kaolin lower values of lipase and protease activity were found in relation to the original crude enzyme extract. Under the best conditions of ATPS (pH 6 and 4 °C), the purification fold was greater than 40 and selectivity was almost 500. Lipase was recovered in the salty phase which makes it easier to purify it. The optimum pH and temperature ranges for purified lipase with this system was 6–7 and 35–40 °C, respectively. Lipase thermostability was increased in relation to crude extract after the purification with the PEG/phosphate buffer system for temperatures lower than 50 °C. All enzyme extracts showed good stability to a wide pH range. Y. lipolytca lipase was successfully purified by using ATPS in a single downstream processing step and presented good process characteristics after this treatment.     

Poly-β-hydroxybutyrate/ectoine co-production by ectoine-excreting strain Halomonas salina

The ectoine-excreting bacterial strain of Halomonas salina was employed in the co-production of poly-β-hydroxybutyrate (PHB) and ectoine (Ect) during a fermentation process (PHB/Ect co-production). An efficient PHB/Ect co-production process was carried out at low NaCl concentration (30 g L⁻¹). It was established using ¹H Nuclear Magnetic Resonance spectroscopy that H. salina produces PHB. The effects of the NaCl concentration, the initial C/N ratio, the phosphate concentration and mixed carbon sources were investigated with respect to PHB/Ect co-production. The PHB/Ect co-production system comprised growing and non-growing cell phases and was developed with NaCl concentration of 30 g L⁻¹. The optimal conditions for PHB/Ect co-production by the ectoine-excreting strain of H. salina were 30 g L⁻¹ NaCl, with an initial C/N ratio of 15, an initial phosphate concentration of 12 g L⁻¹ and mixed carbon sources of 55 g L⁻¹ glucose and 25 g L⁻¹ monosodium glutamate. Using a PHB/Ect co-production system with growing and non-growing cell phases prevents the inhibition of PHB synthesis by high concentration of NaCl and significantly reduces ectoine degradation. PHB and ectoine concentrations as high as 35.3 g L⁻¹ and 8.6 g L⁻¹, respectively, were achieved. The efficient co-production of PHB and ectoine at a low NaCl concentration has been realised.     

Synthesis of thermo-sensitive polyacrylamide derivatives for affinity precipitation and its application in purification of lysozyme

The thermo-sensitive N-alkyl substituted polyacrylamide polymer was synthesized by radical polymerization and its lower critical solution temperature (LCST) was controlled to be 28 °C. The thermo-sensitive recovery of polymer was over 95% in the presence of 0.05 M NaClO₄. Cibacron Blue F3GA was covalently immobilized onto the polymer via the nucleophilic reaction between the active chlorine atom of its triazine ring and the hydroxyl group of the polymer. The ligands density was 30 μmol g⁻¹ polymer. The adsorption capacity of lysozyme on the polymer was 3.4 mg g⁻¹polymer in affinity precipitation process. And over 90% of adsorbed lysozyme was eluted by 0.5 M KSCN at pH 8.0. When the affinity polymer was applied in the purification of lysozyme from egg white, the purification factor was 28 and lysozyme yield was 80% or so.