Production of pectinases by Polyporus squamosus in aqueous two-phase system

The ability of Polyporus squamosus to grow and produce pectinases in an aqueous two-phase medium composed of polyethylene glycol and crude dextran is reported. Fungal growth was restricted to the bottom phase leaving the top phase cell free. Amounts of produced biomass and endo and exo-pectinase activities were superior or equal to those obtained in homogeneous medium. The partition coefficient for the endo-pectinase was 1.52 followed by a top phase yield of 70.86%. Although the phase system composition favours partition of a greater part of exo-pectinase activity to the bottom phase (K(exo) was 0.6 and yield in top phase 48.56%) the partitioned activity in the top phase was equal to that produced in homogeneous cultivation.     

Constitutive exo-pectinase produced byAspergillus sp. CH-Y-1043 on different carbon source

Summary The production of a constitutive exo-pectinase byAspergillus sp. CH-Y-1043 grown on glucose, sucrose, fructose, glycerol and galacturonic acid is reported. The specific activity was found to be in the range of 26% to 75% of that produced with pectin or poly-galacturonic acid. The production of this exo-pectinase is strictly correlated to the exponential growth phase and it is highly sensitive to the pH of the culture medium     

Production of pectinase from deseeded sunflower head by Aspergillus niger in submerged and solid-state conditions

Studies were carried out on the production of pectinases using deseeded sunflower head by Aspergillus niger DMF 27 and DMF 45 in submerged fermentation (SmF) and solid-state fermentation (SSF). Higher titres of endo- and exo-pectinases were observed when medium was supplemented with carbon (4% glucose for SmF and 6% sucrose for SSF) and nitrogen (ammonium sulphate, 0.3% for both SmF and SSF) sources. Green gram husk proved to be relatively a better supplement to attain higher yield of endo-pectinase (11.7 U/g) and exo-pectinase (30.0 U/g) in solid-state conditions. Maximum production of endo-pectinase (19.8 U/g) and exo-pectinase (45.9 U/g) by DMF 45 were recorded in SSF when compared to endo-pectinase (18.9 U/ml) and exo-pectinase (30.3 U/ml) by DMF 27 in SmF under optimum process conditions.     

Conidial and mycelial-bound exo-pectinase of Aspergillus sp.

Abstract Intact conidia of Aspergillus sp. were able to degrade pectin 'in vitro' even when protein synthesis was inhibited, thus indicating the presence of cell bound pectinases. At least an exo-pectinase was found and this enzyme was also present in the mycelium of Aspergillus sp. Its presence was not dependent on the carbon source used for growth, suggesting its constitutive nature. This exo-pectinase could be released from conidia or mycelium by incubation at different pH values and the amount of enzyme released could be increased by treatments with chemical agents and hydrolytic enzymes.     

Rapid method for detecting low basal activity of exo-pectinase of Polyporus squamosus

Three-phase partitioning system consisting of 56% (w/v) (NH ) SO in water/10% (v/v) tert-butanol, was used to isolate exo-pectinase from Polyporus squamosus. The low activity of the constitutive exo-pectinase produced in submerged culture was brought to a detectable activity and was then purified 3-fold.     

Effect of water activity on exo-pectinase production byAspergillus niger CH4 on solid state fermentation

Summary Ethylene glycol, sorbitol and glycerol were used as water activity depressors to study the effect of water activity on pectinase production byAspergillus niger CH4. Ethylene glycol depressed a_w without supporting growth nor strongly affecting pectinase production in petri dish cultures. This depressor was used to evaluate the influence of water activity on exo-pectinase production by SSF. It was found that although pectinase production decreased at low a_w values, this activity was present at a_w values as low as 0.90. The specific activity increased up to 4.5 fold by reducing a_w from 0.98 to 0.90. The reducing groups accumulated extracellularly suggesting sugar transport limitation as a consequence of a_w depression.     

Optimization of process for the production of fungal pectinases from deseeded sunflower head in submerged and solid-state conditions

Pectinase production studies were carried out in submerged and solid-state conditions from deseeded sunflower head employing Aspergillus niger. The two potential strains of A. niger, DMF 27 for submerged and DMF 45 for solid-state were isolated by multi-step screening technique based on coefficient of pectolysis and capability of pectinase production. Process variables such as size of inoculum, pH, temperature, particle size and moisture content were optimized with an aim to achieve the maximum production of pectinases. The increased level of pectinase production was recorded at pH 5.0 and temperature 34 degrees C in submerged and solid-state conditions. The optimum inoculum size was 1x10(5)ml(-1) for submerged and 1x10(7)g(-1) for solid-state conditions. Five hundred micrometer particle size and 65% moisture content of the substrate were optimum for the maximum production of pectinases in solid-state condition. Under optimum conditions, maximum production of exo-pectinase was 34.2U/g in SSF and endo-pectinase was 12.6U/ml in SmF.     

Reverse micellar extraction for downstream processing of lipase: Effect of various parameters on extraction

Reverse micellar extraction of lipase using cationic surfactant cetyltrimethylammonium bromide (CTAB) was investigated. The effect of various process parameters on both forward and backward extraction of lipase from crude extract was studied to optimize its yield and purity. Forward extraction of lipase was found to be maximum using Tris buffer at pH 9.0 containing 0.10 M NaCl in aqueous phase and 0.20 M CTAB in organic phase consisting of isooctane, butanol and hexanol. In case of backward extraction, lipase was extracted from the organic phase to a fresh aqueous phase in 0.05 M potassium phosphate buffer (pH 7.0) containing 1.0 M KCl. The activity recovery, extraction efficiency and purification factor of lipase were found to be 82.72%, 40.27% and 4.09-fold, respectively. The studies also indicated that the organic phase recovered after back extraction could be reused for the extraction of lipase from crude extract.     

Purification of nattokinase by reverse micelles extraction from fermentation broth: effect of temperature and phase volume ratio

Nattokinase is a novel fibrinolytic enzyme that is considered to be a promising agent for thrombosis therapy. In this study, reverse micelles extraction was applied to purify and concentrate nattokinase from fermentation broth. The effects of temperature and phase volume ratio used for the forward and backward extraction on the extraction process were examined. The optimal temperature for forward and backward extraction were 25°C and 35°C respectively. Nattokinase became more thermosensitive during reverse micelles extraction. And it could be enriched in the stripping phase eight times during backward extraction. It was found that nattokinase could be purified by AOT reverse micelles with up to 80% activity recovery and with a purification factor of 3.9.     

Effects of different carbon sources on the synthesis of pectinase by Aspergillus niger in submerged and solid state fermentations

A study was made to compare the production of pectinase by Aspergillus niger CH4 in solid-state (SSF) and submerged (SmF) fermentations. Production of endo- (endo-p) and exo-pectinase (exo-p) by SSF was not reduced when glucose, sucrose or galacturonic acid (up to 10%) were added to a culture medium containing pectin. Moreover, both activities increased when concentrations of the carbon sources were also increased. In SmF, these activities were strongly decreased when glucose or sucrose (3%) was added to culture medium containing pectin. The addition of galacturonic acid affected endo-p activity production to a lesser extend than exo-p. Final endo-p and exo-p activities in SSF were three and 11 times higher, respectively, than those obtained in SmF. The overall productivities of SSF were 18.8 and 4.9 times higher for endo-p and exo-p, respectively, than those in SmF. These results indicate that regulatory phenomena, such as induction-repression or activation-inhibition, related to pectinase synthesis by A. niger CH4 are different in the two types of fermentation. Correspondence to: E. Favela-Torres     

Integrated separation process for isolation and purification of biosuccinic acid

Biotechnologically produced succinic acid has the potential to displace maleic acid and its uses. Therefore, it is of high interest for the chemical, pharmaceutical, and food industry.In addition to optimized production strains and fermentation processes, an efficient separation of succinic acid from the aqueous fermentation broth is indispensable to compete with the current petrochemical production of succinic acid. Isolation and purification of succinic acid from an Escherichia coli fermentation broth were studied with two amine-based reactive extraction systems: (i) trihexylamine in 1-octanol and (ii) diisooctylamine and dihexylamine in a mixture of 1-octanol and 1-hexanol. Back extraction of succinic acid from the organic phase was carried out using an aqueous trimethylamine solution. The trimethylammonium succinate generated after back extraction was split with an evaporation-based crystallization.The focus was on process integration, for example, reuse of the applied amines for extraction and back extraction. It was shown that the maximum trimethylamine concentration for back extraction should not exceed the stoichiometric amount (2 mol trimethylamine/mol the succinic acid in the organic phase) to ensure maximal extraction yields with the reused organic phase in subsequent extractions. Moreover, mixer-settler extraction and back extraction of succinic acid were scaled up from the milliliter- to the liter-scale making use of liquid–liquid centrifuges. The overall yield was 83.5% of the succinic acid from thefermentation supernatant. The final purity of the succinic acid crystals was 99.5%. Organic phase and amines can easily be recycled and reused.     

The effects of pH and salts on nucleic acid partitioning during phenol extraction

The partitioning of nucleic acids is sensitive to pH during phenol extraction. However, the exact effects of pH on phenol extraction had not been systematically investigated, and the mechanism of which were not fully elucidated. In this paper, we showed that the partitioning of nucleic acids was determined neither solely by the pH of the aqueous buffer being used, nor by the “pH of the phenol”; the latter is a completely wrong conception. We demonstrated that a key determinant for nucleic acid partitioning during phenol extraction was the equilibrated pH of the aqueous phase, which should be defined as the pH of phenol extraction. For example, when 50?mM NaAc-HAc buffer at pH of 3.47 was mixed with an equal volume of water-saturated phenol, the equilibrated pH of aqueous phase would be raised to ～3.84. At this pH, almost all of genomic DNA partitioned into the phenol phase, and genomic DNA-free total RNA was retained in the aqueous phase. Several salts were found affecting the partitioning of nucleic acids during phenol extraction in different manners. Based on these results, a low-cost and efficient method for genomic DNA-free total RNA extraction was developed.     

Improvement of pectinase production by interspecific hybrids of Aspergillus strains

Protoplast fusion induced by polyethylene glycol and Ca²⁺, was performed between auxotrophic mutants of pectinolytic fungi Aspergillus sp. CH-Y-1043 (A13) ade ⁻ and Aspergillus flavipes ATCC-16795 (F7) lys ⁻. Prototrophic colonies were developed on minimal medium with a fusion frequency of 1·0×10⁻². The reversion frequency of the mutation in spores and protoplasts was low and ranged from 2·0 to 4·0×10⁻⁷. Four prototrophic hybrids (HH, HE, HF and HJ) exhibited enhanced production of endo-pectinase and pectin-lyase. The highest production was observed in HJ ; maximum activities were 150 and 160% respectively, whereas the exo-pectinase production was similar to the wild-type strain Aspergillus sp. CH-Y-1043. Hybrid HJ showed the greatest growth ; nevertheless, specific endo-pectinase and pectin-lyase activities were higher in all hybrids than those produced by the wild-type strains.     

Study of the factors affecting the extraction of soybean protein by reverse micelles

In this work, the forward and back extraction of soybean protein by reverse micelles was studied. The reverse micellar systems were formed by anionic surfactant sodium bis(2-ethyl hexyl) sulfosuccinate (AOT), isooctane and KCl solution. The effects of AOT concentration, aqueous pH, KCl concentration and phase volume ratio on the extraction efficiency of soybean protein were tested. Suitability of reverse micelles of AOT and Triton-X-100/AOT mixture in organic solvent toluene for soybean protein extraction was also investigated. The experimental results lead to complete forward extraction at the AOT concentration 120 mmol l⁻¹, aqueous pH 5.5 and KCl concentration 0.8 mol l⁻¹. The backward extraction with aqueous phase (pH 5.5) resulted in 100% extraction of soybean protein from the organic phase.     

Downstream processing of soy hull peroxidase employing reverse micellar extraction

Phase transfer studies were conducted to evaluate the solubilization of soy hull peroxidase (SHP) in reverse micelles formed in isooctane/butanol/hexanol using the cationic surfactant cetyltrimethylammonium bromide (CTAB). The effect of various parameters such as pH, ionic strength, surfactant concentration of the initial aqueous phase for forward extraction and buffer pH, type and concentration of salt, concentration of isopropyl alcohol and volume ratio for back extraction was studied to improve the efficiency of reverse micellar extraction. The active SHP was recovered after a complete cycle of forward and back extraction. A forward extraction efficiency of 100%, back extraction efficiency of 36%, overall activity recovery of 90% and purification fold of 4.72 were obtained under optimised conditions. Anionic surfactant sodium bis (2-ethylhexyl) sulfosuccinate (AOT) did not yield good results under the conditions studied. The phase transfer of soy hull peroxidase was found to be controlled by electrostatic and hydrophobic interactions during forward and back extraction respectively.     

Enzyme mass-transfer coefficient in aqueous two-phase systems using static mixer extraction column

Recent technical advances in aqueous two-phase systems (ATPS) have made this a sound technique for the extraction of biomacromolecules. The extraction of alpha-amylase was investigated using aqueous two-phase systems formed by sodium sulphate-polyethylene glycol (PEG) in water in a 47-mm inner diameter spray column packed with three types of static mixers. The effects of dispersed-phase flow rate, phase composition, column height and diameter were studied. The extraction column was operated in a semi-batch manner. It was found that the hold-up and volumetric mass transfer coefficients increased with an increase in dispersed (PEG-rich) phase velocity and decreased with increasing phase composition. Empirical correlations were developed for fractional dispersed-phase hold-up and volumetric mass transfer coefficients.     

Enhanced extraction of proteins using cholinium‐based ionic liquids as phase‐forming components of aqueous biphasic systems

Aqueous biphasic systems (ABS) composed of ionic liquids (ILs) are promising platforms for the extraction and purification of proteins. In this work, a series of alternative and biocompatible ABS composed of cholinium‐based ILs and polypropylene glycol were investigated. The respective ternary phase diagrams, tie‐lines, tie‐line lengths and critical points were determined at 25°C. The extraction performance of these systems for commercial bovine serum albumin (BSA) was then evaluated. The stability of BSA at the IL‐rich phase was ascertained by size exclusion high‐performance liquid chromatography and Fourier transform infrared spectroscopy. Appropriate ILs lead to the complete extraction of BSA for the IL‐rich phase, in a single step, while maintaining the protein's native conformation. Furthermore, to evaluate the performance of these systems when applied to real matrices, the extraction of BSA from bovine serum was additionally carried out, revealing that the complete extraction of BSA was maintained and achieved in a single step. The remarkable extraction efficiencies obtained are far superior to those observed with typical polymer‐based ABS. Therefore, the proposed ABS may be envisaged as a more effective and biocompatible approach for the separation and purification of other value‐added proteins.     

Pilot-scale extraction of an intracellular recombinant cutinase from E. coli cell homogenate using a thermoseparating aqueous two-phase system

A thermoseparating aqueous two-phase system for extraction of a recombinant cutinase fusion protein from Escherichia coli homogenate has been scaled up to pilot scale. The target protein ZZ-cutinase-(WP)(4) was produced in a fed batch process at 500 l to a concentration of 12% of the total protein and at a cell concentration of 19.7 g l(-1). After harvest and high-pressure homogenisation a first extraction step was performed in an EO(50)PO(50) (50% (w/w) ethylene oxide and 50% (w/w) propylene oxide) thermopolymer/amylopectin rich Waxy barley starch system. The (WP)(4) tag was used for enhanced target protein partitioning to the EO(50)PO(50) phase while the cell debris was collected in the starch phase. A second extraction step followed where the recovered EO(50)PO(50) phase from the first step was supplemented with a non-ionic detergent (C(12-18)EO(5)) and heated to the cloud point (CP) temperature (45 degrees C). One polymer-rich liquid phase and one almost pure aqueous phase were formed. The target protein could be obtained in a water phase after the thermal phase separation at a total recovery over the extraction steps of 71% and a purification factor of 2.5. We were able to demonstrate that a disk-stack centrifugal separator could be adapted for rapid separation of both primary and thermoseparated phase systems.     

Novel biphasic separations utilising highly selective molecularly imprinted polymers as biorecognition solvent extraction agents

Molecularly imprinted polymers (MIPs) represent a class of artificial receptors that promise an environmentally robust alternative to naturally occurring biorecognition elements of biosensing devices and systems. However, in general, the performance of conventional MIPs in aqueous environments is poor. In the study reported here, this limitation has been addressed by the novel application of MIPs as a solvent extraction solid phase in a biphasic solvent system. This paper describes a previously unreported use of MIPs as solvent extraction reagents, their successful application to aqueous sample media and the opportunities for utilisation of this unique system in novel biosensing and separation procedures. This study demonstrates the development of a novel biphasic solvent system utilising MIP in the extracting phase to enhance both efficiency and selectivity of a simple two phase liquid extraction. Monodisperse propranolol imprinted polymer microspheres [p(divinylbenzene-co-methacrylic acid)] were prepared by precipitation polymerisation. Initially, the affinity of the polymers for (R,S)-propranolol was assessed by established techniques whereby the MIP demonstrated greater affinity for the template than did the non-imprinted control polymer (NIP). Importantly, MIP performance was also assessed using the novel dual solvent system. The depletion of (R,S)-propranolol from the aqueous phase into the polymer containing organic phase was determined. When compared to control extractions containing no polymer the presence of MIP in the extracting solvent phase resulted in an increased extraction of (R,S)-propranolol from the aqueous phase. Importantly, this extraction was significantly greater in the presence of MIP when compared to NIP. This unique principle generates opportunities for MIP based extractions and chemical enrichments in industrial applications, offering commercial, ecological and practical advantages to traditional solvent extraction techniques. The technique is readily transferable to analytical microsystems utilising MIP recognition elements generating promising opportunities for MIP based sensing of aqueous sample media.     

High-throughput liquid chromatography for drug analysis in biological fluids: investigation of extraction column life

A novel method was developed and assessed to extend the lifetime of extraction columns of high-throughput liquid chromatography (HTLC) for bioanalysis of human plasma samples. In this method, a 15% acetic acid solution and 90% THF were respectively used as mobile phases to clean up the proteins in human plasma samples and residual lipids from the extraction and analytical columns. The 15% acetic acid solution weakens the interactions between proteins and the stationary phase of the extraction column and increases the protein solubility in the mobile phase. The 90% THF mobile phase prevents the accumulation of lipids and thus reduces the potential damage on the columns. Using this novel method, the extraction column lifetime has been extended to about 2000 direct plasma injections, and this is the first time that high concentration acetic acid and THF are used in HTLC for on-line cleanup and extraction column lifetime extension.