Pyrolysis production of fruit peel biochar for potential use in treatment of palm oil mill effluent.

Fruit peel, an abundant waste, represents a potential bio-resource to be converted into useful materials instead of being dumped in landfill sites. Palm oil mill effluent (POME) is a harmful waste that should also be treated before it can safely be released to the environment. In this study, pyrolysis of banana and orange peels was performed under different temperatures to produce biochar that was then examined as adsorbent in POME treatment. The pyrolysis generated 30.7-47.7 wt% yield of a dark biochar over a temperature ranging between 400 and 500 °C. The biochar contained no sulphur and possessed a hard texture, low volatile content (≤34 wt%), and high amounts of fixed carbon (≥72 wt%), showing durability in terms of high resistance to chemical reactions such as oxidation. The biochar showed a surface area of 105 m2/g and a porous structure containing mesopores, indicating its potential to provide many adsorption sites for use as an adsorbent. The use of the biochar as adsorbent to treat the POME showed a removal efficiency of up to 57% in reducing the concentration of biochemical oxygen demand (BOD), chemical oxygen demand COD, total suspended solid (TSS) and oil and grease (O&G) of POME to an acceptable level below the discharge standard. Our results indicate that pyrolysis shows promise as a technique to transform banana and orange peel into value-added biochar for use as adsorbent to treat POME. The recovery of biochar from fruit waste also shows advantage over traditional landfill approaches in disposing this waste.

A new strategy for the on-column exopeptidase cleavage of poly-histidine tagged proteins.

This paper describes a new strategy, which aims to make on-column poly-histidine tag removal more useful in the production of recombinant proteins by improving the yield and efficiency of on-column exopeptidase cleavage. This involves improvement of the on-column cleavage condition by using imidazole concentrations in the range of 100-500 mM in the cleavage buffer. At 300 mM imidazole, maximum on-column cleavage yield (in excess of 99%) was achieved in 3h of incubation. However, as a result of the increased imidazole concentration, this new strategy of on-column cleavage results in some residual uncleaved poly-histidine tagged proteins (~0.1%) and the production of cleaved dipeptides, both of which need to be further removed in a subsequent step. A method involving the recirculation of recovered proteins and peptides through the immobilized metal affinity chromatography (IMAC) column (same-column recirculation) was found to be superior to subtractive IMAC for the purpose of contaminant clearance. Recovery of the detagged target proteins was achieved using 10 column volumes of recovery buffer, which had the effect of diluting the imidazole concentration to a suitably low level for contaminant removal by same-column recirculation. This strategy was also applicable at a higher adsorbent loading of 10 mg protein/mL adsorbent with an optimal ratio of 200 mU of DAPase per mg of adsorbed tagged maltose binding protein (MBP), giving a cleavage yield of 99.1% in 3 h. Finally, on-column cleavage conditions including the effect of protease concentration and incubation time on the new strategy have been investigated and comparisons are made for different tag removal strategies.

Trends in whey protein fractionation.

Whey is a by-product of cheese manufacture that is normally treated as a waste. However, it contains a mixture of proteins with important nutritional and biological attributes. To extract these valuable proteins, whey fractionation has been developed using three main techniques; namely chromatographic (e.g., ion-exchange and hydrophobic adsorption), membrane (e.g., traditional pressure-driven and electro-separation)-, or combined methods. Recently, new promising techniques have been introduced such as aqueous two-phase separation (ATPS) and magnetic fishing. This article reviews the use of these techniques together with an evaluation of their performance regarding the yield and purity of two major proteins in whey.

Exploiting the interactions between poly-histidine fusion tags and immobilized metal ions.

Immobilized metal affinity chromatography (IMAC) of proteins containing poly-histidine fusion tags is an efficient research tool for purifying recombinant proteins from crude cellular feedstocks at laboratory scale. Nevertheless, to achieve successful purification of large amounts of the target protein for critical therapeutic applications that demand the precise removal of fusion tags, it is important to also take into consideration issues such as protein quality, efficiency, cost effectiveness, and optimal affinity tag choice and design. Despite the many considerations described in this article, it is expected that enhanced selectivity, the primary consideration in the field of protein separation, will continue to see the use of IMAC in solving new purification challenges. In addition, the platform nature of this technology makes it an ideal choice in purifying proteins with unknown properties. Finally, the unique interaction between immobilized metal ions and poly-histidine fusion tag has enabled new developments in the areas of biosensor, immunoassay, and other analytical technologies.

Adsorptive detagging of poly-histidine tagged protein using hexa-histidine tagged exopeptidase.

The ubiquitous use of poly-histidine fusion tags has made the purification of the recombinant target proteins much simpler, although the presence of residual fusion tags can generate immunogenic products or products with changed biological activities. This work presents a generic method of removing poly-histidine fusion tags from recombinant proteins through the use of a hexa-histidine tagged exopeptidase (DAPase) when both tagged species are adsorbed to the immobilized metal affinity chromatography (IMAC) adsorbent. Adsorptive detagging was performed in the presence of 50mM imidazole in order to allow the cleavage reaction by the hexa-histidine tagged DAPase to occur. The progress of batch and adsorptive detagging by DAPase of maltose binding protein (MBP) tagged with two variants of hexa-histidine fusion tag was successfully monitored using cationic exchange chromatography. A single-step, column-based detagging strategy was then optimized to maximize the recovery of native MBP. The kinetics of batch and on-column digestion for both HT6 and HT15 fusion tags were investigated. The process involved the sequential removal of dipeptides during the digestion of full-length fusion protein down to its fully detagged native form. During the course of tag digestion, 4 and 7 different intermediates were detected for HT6 and HT15 tagged MBP respectively. The characteristics of on-column cleavage of poly-histidine fusion tags by DAPase as a function of incubation temperature and amount of protease activity used were examined. It was found that the influence of fusion tag design on the batch and column-based detagging yield and efficiency was substantial. In addition, the structural difference of fusion tags affects the binding strength of the fusion protein, which can influence the resulting product purity. Despite being a longer tag, HT15 fusion tag was the preferred sequence for shortening the time needed for on-column detagging. These results can be applied to the wider use of the proposed platform protocol for the on-column cleavage of poly-histidine tagged proteins using exopeptidases.

Applications of membrane techniques for purification of natural products.

The recent development of various membrane-based techniques for the purification of valuable natural products is reviewed and covers the important research that has been conducted in the last 5 years on the utilization of microfiltration, ultrafiltration and nanofiltration techniques, either carried out on their own or coupled with other separation techniques, in order to achieve concentration and purification of natural products from their biological source. There is also a special focus on the research that has been undertaken to overcome the membrane fouling encountered in their usage.

Process intensification for the removal of poly-histidine fusion tags from recombinant proteins by an exopeptidase.

This study describes the use of a hexa-histidine tagged exopeptidase for the cleavage of hexa-histidine tags from recombinant maltose binding protein (MBP) when both tagged species are bound to an immobilized metal affinity chromatography (IMAC) matrix. On-column exopeptidase cleavage only occurred when the cleavage buffer contained an imidazole concentration of 50 mM or higher. Two strategies were tested for the on-column tag cleavage by dipeptidylaminopeptidase (DAPase): (i) a post-load wash was performed after sample loading using cleavage buffers containing varying imidazole concentrations and (ii) a post-load wash was omitted following sample loading. In the presence of 50 mM imidazole, 46% of the originally adsorbed hexa-histidine tagged MBP was cleaved, released from the column, and recovered in a sample containing 100% native (i.e., completely detagged) MBP. This strategy renders the subsequent purification steps unnecessary as any tagged contaminants remained bound to the column. At higher imidazole concentrations, binding of both hexa-histidine tagged MBP and DAPase to the column was minimized, leading to characteristics of cleavage more closely resembling that of a batch cleavage. An on-column cleavage yield of 93% was achieved in the presence of 300 mM imidazole, albeit with contamination of the detagged protein with tag fragments and partially tagged MBP. The success of the on-column exopeptidase cleavage makes the integration of the poly-histidine tag removal protocol within the IMAC protein capture step possible. The many benefits of using commercially available exopeptidases, such as DAPase, for poly-histidine tag removal can now be combined with the on-column tag cleavage operation.

Purification of the two major proteins from whey concentrate using a cation-exchange selective adsorption process.

The packed-bed adsorption and elution of aqueous solutions of whey concentrate powders were investigated at pH 3.7 using a 5-mL SP Sepharose FF column to separate and isolate two major proteins namely, alpha-lactalbumin (ALA) and beta-lactoglobulin (BLG) from these solutions. ALA displaced and eluted BLG from the column in a pure form. Pure ALA could then be eluted with good recovery. A novel consecutive two-stage separation process was developed to separate ALA and BLG from whey concentrate mixtures. Almost all of the BLG in the feed was recovered, with 78% being recovered at 95% purity and a further 20% at 86% purity. In addition, 67% of ALA was recovered, 48% at 54% purity and 19% at 60% purity.

Confocal microscopy study of uptake kinetics of alpha-lactalbumin and beta-lactoglobulin onto the cation-exchanger SP Sepharose FF.

Confocal laser scanning microscopy (CLSM) was used to study single- and two-component protein uptake for alpha-lactalbumin (ALA) and beta-lactoglobulin (BLG), as models for whey proteins, to SP Sepharose FF at pH 3.7 during batch experiments in a finite bath. By coupling a fluorescent dye with the protein molecule, the penetration into individual adsorbent particles at different times during batch uptake was visualised. In a single-component system, BLG penetrated fast into the adsorbent beads and gradually filled them in a shell-wise fashion, while adsorption of ALA was mostly confined to the outer shells of the adsorbent. For the two-component studies, the results showed that ALA was able to displace BLG despite its lower affinity to the adsorbent under the employed conditions. CLSM results were then compared both qualitatively and quantitatively to their counterparts obtained in traditional experiments by indirect measurements of the protein concentration in the fluid phase. A novel quantitative approach was undertaken by modifying the simple kinetic rate model traditionally used to determine the kinetic rate constant, k(1), for batch uptake experiments, in order to describe batch uptake kinetics based on CLSM data. Although BLG results were in good agreement, there was a discrepancy in ALA results.

Characterization and evaluation of a macroporous adsorbent for possible use in the expanded bed adsorption of flavonoids from Ginkgo biloba L.

The suitability of the use of macroporous adsorbent Amberlite XAD7HP in expanded bed adsorption processes for the isolation of flavonoids from crude extracts of Ginkgo biloba L. has been assessed. The expansion and hydrodynamic properties of expanded beds were investigated and analyzed. The bed expansion as a function of operational fluid velocity was measured and correlated with the Richardson-Zaki equation. Theoretical predictions of the correlation parameters (the terminal settling velocity u(t) and exponent n) were improved by modifying equations in the literature. Residence time distributions (RTDs) were studied using acetone as a tracer. Three measures of liquid phase dispersion (the height equivalent of theoretical plate, Bodenstein number and axial distribution coefficient) were investigated and compared to values previously obtained with commercial EBA adsorbents developed for protein purification. A suitable bed expansion ratio was found to be 1.25 times the settled bed height, which occurred at a corresponding flow velocity of 183 cm/h. For an initial settled bed height of 42 cm, the mean residence time of liquid in the expanded bed was around 28 min. Under these flow conditions, the axial mixing coefficient D(ax) was 7.54 x 10(-6) m(2)/s and the Bodenstein number was 28; the number of theoretical plates (N) was 19 and the height equivalent of a theoretical plate (HETP) was 2.77 cm. Rutin trihydrate was used as a model flavonoid for the characterization of the adsorption properties of Amberlite XAD7HP. Adsorption was observed to reach equilibrium within 3 h with 70% of the adsorption capacity being achieved within 30 min. The estimated maximum equilibrium adsorption capacity for rutin was estimated to be 43.0 mg/(gresin) when the results were fitted to Langmuir isotherms. The adsorption performance was not seriously impaired by the physical presence of G. biloba leaf powders. Assessment of the kinetics of the adsorption of rutin revealed that the rate constant for adsorption was only reduced by 15% in the presence of leaf powders at a concentration of 50 mg/mL. The results demonstrated that Amberlite XAD7HP should be suitable for expanded bed adsorption of flavonoids from crude extracts of G. biloba L.

Use of expanded bed adsorption to purify flavonoids from Ginkgo biloba L.

Three techniques (liquid-liquid extraction, packed bed adsorption and expanded bed adsorption) have been compared for the purification of flavonoids from the leaves of Ginkgo biloba L. A crude Ginkgo extract was obtained by refluxing with ethanol for 3h. The yield of flavonoids achieved by this crude extraction was about 19% (w/w) and the purity of flavonoids in the concentrated extract was between 1.9 and 2.3% (w/w). The crude extract was then dissolved in deionized water and centrifuged where necessary to prepare clarified feedstock for further purification. For the method using liquid-liquid extraction with ethyl acetate, the purity, concentration ratio and yield of flavonoids were 25.4-31.0%, 16-18 and >98%, respectively. For the method using packed bed adsorption, Amberlite XAD7HP was selected as the adsorbent and clarified extract was used as the feedstock. The dynamic adsorption breakthrough curves and elution profiles were measured. For a feedstock containing flavonoids at a concentration of 0.25mg/mL, the appropriate loading volume to reach a 5% breakthrough point during the adsorption stage was estimated to be 550-600 mL for a packed bed of volume 53 mL and a flow rate of 183 cm/h. The results from the elution stage indicated that the majority of impurities were eluted by ethanol concentrations of 40% (v/v) or below and efficient separation of flavonoids from the impurities could be achieved by elution of the flavonoids with 50-80% ethanol reaching an average purity of approximately 25%. The recovery yield of flavonoids using the packed bed purification method was about 60% of the flavonoids present in the clarified feedstock (corresponding to around 30% for the total flavonoids in the unclarified crude extract). For the method using expanded bed adsorption also conducted with Amberlite XAD7HP as the adsorbent, the optimal operation conditions scouted during the packed bed experiments were used but unclarified crude extract could be loaded directly into the column. For an expanded bed with a settled bed height of 30 cm, the loss of flavonoids in the column flow-through was about 30%. The two-step elution protocol again proved to be effective in separating the adsorbed impurities and flavonoids. More than 96% of the bound impurities were completely removed by 40% ethanol in the first elution stage and less than 4% remained in the final product eluted by 90% ethanol in the second elution stage. Also, approximately 74% of the adsorbed flavonoids on column (corresponding to 51% of the total flavonoids in the unclarified feedstock) were recovered in the product. In addition to higher recovery yield, the average process time to obtain the same amount of product was decreased in the expanded bed adsorption (EBA) process. The results suggest that the adoption of EBA procedures can greatly simplify the process flow sheet and in addition reduce the cost and time to purify flavonoids from Ginkgo biloba. These results clearly demonstrate the potential for the use of EBA to purify pharmaceuticals from plant sources.

Single and two-component cation-exchange adsorption of the two pure major whey proteins.

Adsorption of pure alpha-lactalbumin (ALA) and beta-lactoglobulin (BLG) to the cation exchanger SP Sepharose FF was studied at pH 3.7 with the purpose of developing a process for isolating them from whey. Measurement of Langmuir parameters describing adsorption equilibrium in batch experiments and protein breakthrough time values in 1-ml packed-beds at a linear velocity of 158 cm/h and initial concentrations of 3 mg/ml for BLG and 1.5 mg/ml for ALA suggested the feasibility of using this adsorbent to separate the two proteins when present in a mixture. Subsequent experiments with 5-ml columns at the above concentrations and a linear velocity of 30 cm/h confirmed this and showed evidence of competitive adsorption as ALA displaced and eluted all BLG from the column in a pure form, and the remaining ALA could be eluted thereafter at high purity and with 91% recovery.

Novel bioreactors for the culture and expansion of aggregative neural stem cells.

Neural stem cells have been cultured as three-dimensional aggregates in a number of different types of bioreactors. The design and configuration of the bioreactor are shown to be crucial factors for the successful propagation of the cells. A novel bioreactor with liquid re-circulation and a working volume of 200 ml has been designed, tested and shown to be able to produce a higher cell vitality compared to those produced in multi-well plates, shake flasks and stirred flasks. The novel reactor was able to produce a total density of cells of 3.5 x 10(6) cells/ml consisting of a larger number of smaller and proliferative aggregates, compared to only 1.8 x 10(6) cells/ml produced in a multi-well plate. Shake flasks and stirred flasks commonly used for facilitating mass transfer in the culture of micro-organisms are shown to be unsuitable for the propagation of neural stem cells.

Separation and enrichment of neural stem cells using segregation in an expanded bed.

An expanded bed system has been developed for a novel application in which the separation and enrichment of neural stem cells from a sample containing a mixture of stem and progenitor cells is achieved based on the difference in the sizes of the aggregates of these types of cells. Inert Sephadex beads and flocculated yeast cells were used as experimental controls and references. The characteristics of the separation of neural stem cell aggregates based on size are similar to those achieved with flocculated yeast where cell-to-cell aggregation controls the pattern of size separation different from those of inert Sephadex beads.

Intensified process for the purification of an enzyme from inclusion bodies using integrated expanded bed adsorption and refolding.

This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations in an intensified process used to recover purified and biologically active proteins from inclusion bodies expressed in E. coli. Delta(5)-3-Ketosteroid isomerase with a C-terminal hexahistidine tag was expressed as inclusion bodies in the cytoplasm of E. coli. Chemical extraction was used to disrupt the host cells and simultaneously solubilize the inclusion bodies, after which EBA utilizing immobilized metal affinity interactions was used to purify the polyhistidine-tagged protein. Adsorptive refolding was then initiated in the column by changing the denaturant concentration in the feed stream from 8 to 0 M urea. Three strategies were tested for performing the refolding step in the EBA column: (i) the denaturant was removed using a step change in feed-buffer composition, (ii) the denaturant was gradually removed using a gradient change in feed-buffer composition, and (iii) the liquid flow direction through the column was reversed and adsorptive refolding performed in the packed bed. Buoyancy-induced mixing disrupted the operation of the expanded bed when adsorptive refolding was performed using either a step change or a rapid gradient change in feed-buffer composition. A shallow gradient reduction in denaturant concentration of the feed stream over 30 min maintained the stability of the expanded bed during adsorptive refolding. In a separate experiment, buoyancy-induced mixing was completely avoided by performing refolding in a settled bed, which achieved comparable yields to refolding in an expanded bed but required a slightly more complex process. A total of 10% of the available KSI-(His(6)) was recovered as biologically active and purified protein using the described purification and refolding process, and the yield was further increased to 19% by performing a second iteration of the on-column refolding operation. This process should be applicable for other polyhistidine tagged proteins and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution.

Production of enzymatically active ketosteroid isomerase following insoluble expression in Escherichia coli.

Enzymatically active Delta(5)-3-ketosteroid isomerase (KSI) protein with a C-terminus his(6)-tag was produced following insoluble expression using Escherichia coli. A simple, integrated process was used to extract and purify the target protein. Chemical extraction was shown to be as effective as homogenization at releasing the inclusion body proteins from the bacterial cells, with complete release taking less than 20 min. An expanded bed adsorption (EBA) column utilizing immobilized metal affinity chromatography (IMAC) was then used to purify the denatured KSI-(His(6)) protein directly from the chemical extract. This integrated process greatly simplifies the recovery and purification of inclusion body proteins by removing the need for mechanical cell disruption, repeated inclusion body centrifugation, and difficult clarification operations. The integrated chemical extraction and EBA process achieved a very high purity (99%) and recovery (89%) of the KSI-(His(6)), with efficient utilization of the adsorbent matrix (9.74 mg KSI-(His(6))/mL adsorbent). Following purification the protein was refolded by dilution to obtain the biologically active protein. Seventy-nine percent of the expressed KSI-(His(6)) protein was recovered as enzymatically active protein with the described extraction, purification, and refolding process. In addition to demonstrating the operation of this intensified inclusion body process, a plate-based concentration assay detecting KSI-(His(6)) is validated. The intensified process in this work requires minimal optimization for recovering novel his-tagged proteins, and further improves the economic advantage of E. coli as a host organism.

Adsorptive refolding of histidine-tagged glutathione S-transferase using metal affinity chromatography.

Column-based protein refolding strategies are often advantageous due to their ease of integration with purification operations, improved refolding yields, and the high concentrations at which the refolded protein can be recovered. His6-tagged glutathione S-transferase (GST-(His6)) was refolded while it was adsorbed in a metal affinity chromatography column. The redox environment could be controlled during the refolding reaction by the addition of reduced and oxidized glutathione without reducing the immobilized nickel metal ions. Adsorptive refolding limited the interaction of refolding intermediates at elevated protein concentrations, and thus improved the yield compared to experiments performed using dilution refolding techniques. The protein concentration during refolding was increased by a factor of 6.8 without reducing the yield achieved compared to dilution refolding. The ability of GST-(His6) to refold to the correct tertiary structure was not significantly affected by the interaction between the poly-histidine-tag and the adsorbent. Decreased refolding yields were achieved at elevated adsorbed protein concentrations, which indicated that at high concentrations the refolding intermediates aggregated despite immobilization. Following adsorptive refolding it was observed that only correctly folded protein could be eluted with imidazole, while the misfolded and aggregated proteins were retained in the column via non-specific interactions with the adsorbent matrix. An iterative refolding strategy was therefore used to re-denature the retained proteins and repeat the adsorptive refolding step, which increased the adsorptive refolding yield that could be achieved at elevated protein concentrations. The yield of correctly folded GST-(His6) from an iterative refolding process was comparable to dilution refolding performed at a 10-fold lower protein concentration. Selective elution and iterative refolding is likely to improve the yields achieved for other poly-histidine-tagged proteins refolded in metal affinity chromatography columns.

Refolding strategies for ketosteroid isomerase following insoluble expression in Escherichia coli.

Dilution and column-based protein refolding techniques are compared for refolding Delta 5-3-ketosteroid isomerase (KSI) with a C-terminus his6-tag. Column refolding was performed by removing the denaturant while the protein was adsorbed in an immobilized metal affinity chromatography column. Both dilution refolding and a single-step column-based refolding strategy were optimized to maximize the recovery of KSI enzyme activity, and achieved refolding yields of 87% and 70% respectively. It was found that the column-based refolding yield was reduced at higher adsorbed protein concentrations. An elution gradient with increasing imidazole concentration was used to selectively elute the biologically active KSI protein following column refolding, with high molecular weight KSI aggregates retained in the column. An iterative column-refolding process was then developed to denature and refold protein retained in the column, which significantly increased the refolding yield at high-adsorbed protein concentrations. Repetition of the column refolding operation increased the refolding yield from 50% to 75% for protein adsorbed at a concentration of 2.9 mg/mL of adsorbent. Although for the KSI protein column-based refolding did not improve the overall refolding yield compared to dilution refolding, it may still be advantageous due to the ease of integration with purification operations, increased control over the refolding conditions, and the ability to segregate refolded protein from inactive aggregates during elution.

A critical assessment of the impact of mixing on dilution refolding.

Refolding often presents a bottleneck in the generation of recombinant protein expressed as inclusion bodies. Few studies have looked at the effect of physical factors on the yield from refolding steps. Refold reactors typically operate in fed-batch mode with a slow injection rate. This paper characterizes mixing in a novel reactor, and seeks to relate the conditions of mixing to native lysozyme yields after refolding. A novel twin-impeller system incorporating a mini-paddle impeller located in the vicinity of the injection point was used to increase the local levels of energy dissipation experienced by the injected material, and to improve refolding yields. Mixing only affected yields during and immediately after denatured protein addition. Analysis of lysozyme refolding yield, under a variety of conditions, revealed that dispersive mixing affected the yield. The beneficial effect of the mini-paddle impeller in providing a source of localized energy dissipation was limited to conditions where the bulk impeller intensity was low. The effects appeared to become more significant when injection times were longer, because of increased exposure of the injected material to the energy dissipation of the mini-impeller. The results suggest that for fed-batch protein refolding systems, where mixing has been shown to be a critical factor, the local energy dissipation experienced in the vicinity of the injection point is critical to the refolding yields.