Stability and release mechanism of double emulsification (W1/O/W2) for biodegradable pH-responsive polyhydroxybutyrate/cellulose acetate phthalate microbeads loaded with the water-soluble bioactive compound niacinamide.

Microbeads of biodegradable polyhydroxybutyrate (PHB) offer environmental benefits and economic competitiveness. The aim of this study was to encapsulate a water-soluble bioactive compound, niacinamide (NIA), in a pH-responsive natural matrix composed of PHB and cellulose acetate phthalate (CAP) by double emulsification (W1/O/W2) to improve the encapsulation efficiency (%EE) and loading capacity (%LC). PHB was produced in-house by Escherichia coli JM109 pUC19-23119phaCABA-04 without the inducing agent isopropyl ß-D-1-thiogalactopyranoside (IPTG). The influences of PHB and polyvinyl alcohol (PVA) concentrations, stirring rate, PHB/CAP ratio and initial NIA concentration on the properties of NIA-loaded pH-responsive microbeads were studied. The NIA-loaded pH-responsive PHB/CAP microbeads exhibited a spherical core-shell structure. The average size of the NIA-loaded pH-responsive microbeads was 1243.3 ± 11.5 µm. The EE and LC were 33.3 ± 0.5 % and 28.5 ± 0.4 %, respectively. The release profiles of NIA showed pH-responsive properties, as 94.2 ± 3.5 % of NIA was released at pH 5.5, whereas 99.3 ± 2.4 % of NIA was released at pH 7.0. The NIA-loaded pH-responsive PHB/CAP microbeads were stable for >90 days at 4 °C under darkness, with NIA remaining at 73.65 ± 1.86 %. A cytotoxicity assay in PSVK1 cells confirmed that the NIA-loaded pH-responsive PHB/CAP microbeads were nontoxic at concentrations lower than 31.3 µg/mL, in accordance with ISO 10993-5.

Fabrication, characterization and release behavior of α-tocopherol acetate-loaded pH-responsive polyhydroxybutyrate/cellulose acetate phthalate microbeads.

Microbeads are used in personal care and cosmetic products (PCCPs) but are produced from nondegradable materials. Biodegradable polyhydroxybutyrate (PHB) has been recognized as a promising alternative material for use in PCCPs; however, utilizing PHB to encapsulate PCCPs is challenging because PCCPs need to be protected from the environment but their release needs to be permitted under specific physiological conditions. The aim of this work was to develop and evaluate pH-responsive cellulose acetate phthalate (CAP) to formulate lipophilic α-tocopherol acetate (α-TA)-loaded pH-responsive PHB/CAP microbeads. The influences of the PHB/CAP ratio and initial α-TA loading on the microbead size, surface morphology, encapsulation efficiency (%EE), loading capacity (%LC), and α-TA release profile were studied. The microbeads exhibited a spherical shape with a size of 328.7 ± 2.9 µm. The EE and LC were 86.7 ± 2.6 % and 13.5 ± 0.4 %, respectively. The release profile exhibited pH-responsive characteristics. These α-TA-loaded pH-responsive microbeads were stable with >50 % of the α-TA remaining after 90 days at 4, 25 and 45 °C in the dark. The results from the cytotoxicity assay with PSVK1 cells demonstrated that the microbeads were nontoxic. Hence, our developed formulation has the potential to be used to encapsulate oil-based drugs to formulate lipophilic substance-loaded pH-responsive microbeads.

Preparation and characterization of astaxanthin-loaded biodegradable polyhydroxybutyrate (PHB) microbeads for personal care and cosmetic applications.

Due to its biodegradability and biocompatibility, polyhydroxybutyrate (PHB) has received attention as an alternative material for microbeads in personal care and cosmetic products (PCCPs). Here, PHB was produced from crude glycerol by an Escherichia coli JM109 strain harboring pUC19-23,119-phaCABA-04 without isopropyl ß-D-1-thiogalactopyranoside (IPTG), an inducing agent. Astaxanthin-loaded PHB microbeads were prepared through emulsification-solvent evaporation. Studies were performed to determine how the concentration of PHB and stirring rate influence the size, surface morphology, encapsulation efficiency (EE), and astaxanthin release profile. The astaxanthin-loaded PHB microbeads exhibited a rough surface, 98.1 ± 0.7 % EE, spherical shape and 179 ± 44 µm size. In addition, <50 % astaxanthin release was observed within 240 min. Stability studies revealed that astaxanthin-loaded microbeads retained over 85.3 ± 4.2 % of astaxanthin after 90 days at 4 °C and showed a 2-fold reduction in astaxanthin degradation compared to their unencapsulated counterparts; thus, astaxanthin-loaded microbeads show promise for PCCPs applications. A cytotoxicity assay revealed that astaxanthin-loaded PHB microbeads were nontoxic to the human epidermal keratinocyte cell line, PSVK1, and EpiSkin® cells. Skin irritation and sensitization were not observed during a human repeated insult patch test (HRIPT), according to clinical practice guidelines of the Japanese dermatological association.

Valorization of agro-industrial waste from the cassava industry as esterified cellulose butyrate for polyhydroxybutyrate-based biocomposites.

The aim of this study was to utilize cassava pulp to prepare biocomposites comprising microcrystalline cellulose from cassava pulp (CP-MCC) as a filler and polyhydroxybutyrate (PHB) synthesized in-house by Cupriavidus necator strain A-04. The CP-MCC was extracted from fresh cassava pulp. Next, the CP-MCC surface was modified with butyryl chloride (esterified to CP-MCC butyrate) to improve dissolution and compatibility with the PHB. FTIR results confirmed that the esterified CP-MCC butyrate had aliphatic chains replacing the hydroxyl groups; this substitution increased the solubilities in acetone, chloroform, and tetrahydrofuran. Biocomposite films were prepared by varying the composition of esterified CP-MCC butyrate as a filler in the PHB matrix at 0, 5, 10, 15, 20 and 100 wt%. The results for the 95:5 and 90:10 CP-MCC butyrate biocomposite films showed that esterification led to improvements in the thermal properties and increased tensile strengths and elongations at break. All prepared biocomposite films maintained full biodegradability.

Valorization of organic wastes using bioreactors for polyhydroxyalkanoate production: Recent advancement, sustainable approaches, challenges, and future perspectives.

Microbial polyhydroxyalkanoates (PHA) are encouraging biodegradable polymers, which may ease the environmental problems caused by petroleum-derived plastics. However, there is a growing waste removal problem and the high price of pure feedstocks for PHA biosynthesis. This has directed to the forthcoming requirement to upgrade waste streams from various industries as feedstocks for PHA production. This review covers the state-of-the-art progress in utilizing low-cost carbon substrates, effective upstream and downstream processes, and waste stream recycling to sustain entire process circularity. This review also enlightens the use of various batch, fed-batch, continuous, and semi-continuous bioreactor systems with flexible results to enhance the productivity and simultaneously cost reduction. The life-cycle and techno-economic analyses, advanced tools and strategies for microbial PHA biosynthesis, and numerous factors affecting PHA commercialization were also covered. The review includes the ongoing and upcoming strategies viz. metabolic engineering, synthetic biology, morphology engineering, and automation to expand PHA diversity, diminish production costs, and improve PHA production with an objective of "zero-waste" and "circular bioeconomy" for a sustainable future.

Green composites made of polyhydroxybutyrate and long-chain fatty acid esterified microcrystalline cellulose from pineapple leaf.

Pineapple leaf fibres are an abundant agricultural waste product that contains 26.9% cellulose. The objective of this study was to prepare fully degradable green biocomposites made of polyhydroxybutyrate (PHB) and microcrystalline cellulose from pineapple leaf fibres (PALF-MCC). To improve compatibility with PHB, the PALF-MCC was surface modified using lauroyl chloride as an esterifying agent. The influence of the esterified PALF-MCC laurate content and changes in the film surface morphology on biocomposite properties was studied. The thermal properties obtained by differential scanning calorimetry revealed a decrease in crystallinity for all biocomposites, with 100 wt% PHB displaying the highest values, whereas 100 wt% esterified PALF-MCC laurate showed no crystallinity. The addition of esterified PALF-MCC laurate increased the degradation temperature. The maximum tensile strength and elongation at break were exhibited when adding 5% of PALF-MCC. The results demonstrated that adding esterified PALF-MCC laurate as a filler in the biocomposite film could retain a pleasant value of tensile strength and elastic modulus whereas a slight increase in elongation can help to enhance flexibility. For soil burial testing, PHB/ esterified PALF-MCC laurate films with 5-20% (w/w) PALF-MCC laurate ester had higher degradation than films consisting of 100% PHB or 100% esterified PALF-MCC laurate. PHB and esterified PALF-MCC laurate derived from pineapple agricultural wastes are particularly suitable for the production of relatively low-cost biocomposite films that are 100% compostable in soil.

Microwave-assisted cassava pulp hydrolysis as food waste biorefinery for biodegradable polyhydroxybutyrate production.

Cassava pulp is one of the most abundant agricultural residues that can cause serious disposal problems. This study aimed to apply a biorefinery approach by examining the feasibility of microwave-assisted cassava pulp hydrolysis to attain sustainable management and efficient use of natural resources. Four factors, namely, the liquid-to-solid ratio (20 mL/g, 10 mL/g, 7.5 mL/g, and 5 mL/g), types of acids (H2SO4 and H3PO4), watt power (600 W, 700 W, and 800 W) and time (3, 5 and 8 min), were carefully investigated. The highest fermentable sugar content of 88.1 g/L ± 0.7 g/L (0.88 g fermentable sugars/g dry cassava pulp) was achieved when 20 mL/g cassava pulp was hydrolyzed with 2.5% (v/v) H2SO4 under microwave irradiation at 800 W for 8 min. Glucose was a major product (82.0 g/L ± 5.2 g/L). The inhibitor concentration was 5.17 g/L ± 0.01 g/L, and the levulinic acid concentration was 5.15 g/L ± 0.01 g/L. The results indicated that the liquid-to-solid ratio, diluted acid concentration, irradiation watt power and time were important factors in producing fermentable sugars from acid hydrolysis under microwave irradiation. The crude hydrolysate was used for PHB production by Cupriavidus necator strain A-04. The hydrolysate to nutrients ratio of 30:70 (v/v) yielded a cell dry weight of 7.5 g/L ± 0.1 g/L containing PHB content of 66.8% ± 0.3% (w/w), resulting in a yield Y P / S (g-PHB/g- S P H B ) of 0.35 g/g. This study demonstrated that the microwave-assisted cassava pulp hydrolysate developed in this study provided a high amount of glucose (88.1% conversion) and resulted in a low concentration of inhibitors without xylose; this was successfully achieved without pregelatinization, alkaline pretreatment or detoxification.

Optimization for the efficient recovery of poly(3-hydroxybutyrate) using the green solvent 1,3-dioxolane.

In this study, a simple non-toxic recovery process of biodegradable poly(3-hydroxybutyrate) (PHB) using the green solvent 1,3-dioxolane and water was successfully developed. The critical parameters were optimized, and the process platform was scaled up from 2 ml to 1,000 ml for the efficient recovery of PHB. The physical parameters including continuous shaking, ultrasonication, extraction using the Soxhlet extractor, diluted 1,3-dioxolane, reused 1,3-dioxolane, and cell rupture by steam explosion prior to solvent extraction were carefully investigated. The results showed that continuous shaking played a major role in increasing the recovery efficiency during the scale-up process. The PHB extraction at 2 ml from dried cells at 80°C with 100 rpm of shaking speed for 5 h resulted in a recovery yield of 96.6 ± 0.1% with purity up to 99.1 ± 0.6% and that from wet cells under the same condition resulted in a recovery yield of 94.6 ± 4.8% and purity of 97.0 ± 0.1%. It should be noted that the PHB extracted from wet cells at room temperature with 150 rpm of shaking speed for 36 h resulted in a recovery yield of 93.5 ± 0.7% and purity of 97.7 ± 1.3% and had an MW of 3.1×105, MN of 2.7×105, and polydispersity index of 1.1. The direct scale-up process at 1,000 ml showed comparable results in purity, recovery yield, molecular weight distribution, thermal properties, and mechanical properties. The PHB extraction from dried cells gave the highest purity of 99.3 ± 0.5% and recovery of 94.0 ± 0.3%, whereas the PHB extraction from wet cells gave a purity of 90.3 ± 1.5% and recovery of 92.6 ± 1.0%. The novel recovery process showed its feasibility to be applied on an industrial scale.

Strategies for Poly(3-hydroxybutyrate) Production Using a Cold-Shock Promoter in Escherichia coli.

The present study attempted to increase poly(3-hydroxybutyrate) (PHB) production by improving expression of PHB biosynthesis operon derived from Cupriavidus necator strain A-04 using various types of promoters. The intact PHB biosynthesis operon of C. necator A-04, an alkaline tolerant strain isolated in Thailand with a high degree of 16S rRNA sequence similarity with C. necator H16, was subcloned into pGEX-6P-1, pColdI, pColdTF, pBAD/Thio-TOPO, and pUC19 (native promoter) and transformed into Escherichia coli JM109. While the phaC A-04 gene was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that the cold-shock cspA promoter enhanced phaCA-04 protein expression and the chaperone function of TF play critical roles in increasing soluble phaCA-04 protein. Induction strategies and parameters in flask experiments were optimized to obtain high expression of soluble PhaCA-04 protein with high YP/S and PHB productivity. Soluble phaCA-04 was purified through immobilized metal affinity chromatography (IMAC). The results demonstrated that the soluble phaCA-04 from pColdTF-phaCAB A-04 was expressed at a level of as high as 47.4 ± 2.4% of total protein and pColdTF-phaCAB A-04 enhanced soluble protein formation to approximately 3.09-4.1 times higher than that from pColdI-phaCAB A-04 by both conventional method and short induction method developed in this study. Cultivation in a 5-L fermenter led to PHB production of 89.8 ± 2.3% PHB content, a YP/S value of 0.38 g PHB/g glucose and a productivity of 0.43 g PHB/(L.h) using pColdTF-phaCAB A-04. The PHB film exhibited high optical transparency and possessed Mw 5.79 × 105 Da, Mn 1.86 × 105 Da, and PDI 3.11 with normal melting temperature and mechanical properties.

Polyhydroxybutyrate (PHB) Production Using an Arabinose-Inducible Expression System in Comparison With Cold Shock Inducible Expression System in Escherichia coli.

Cupriavidus necator strain A-04 has shown 16S rRNA gene identity to the well-known industrial strain C. necator H16. Nevertheless, the cell characteristics and polyhydroxyalkanoate (PHA) production ability of C. necator strain A-04 were different from those of C. necator H16. This study aimed to express PHA biosynthesis genes of C. necator strain A-04 in Escherichia coli via an arabinose-inducible expression system. In this study, the PHA biosynthesis operon of C. necator strain A-04, consisting of three genes encoding acetyl-CoA acetyltransferase (phaA A-04, 1182 bp, 40.6 kDa), acetoacetyl-CoA reductase (phaB A-04, 741 bp, 26.4 kDa) and PHB synthase Class I (phaC A-04, 1770 bp), was identified. Sequence analysis of the phaA A-04, phaB A-04, and phaCA-04 genes revealed that phaC A-04 was 99% similar to phaC H16 from C. necator H16. The difference in amino acid residue situated at position 122 of phaC A-04 was proline, whereas that of C. necator H16 was leucine. The intact phaCAB A-04 operon was cloned into the arabinose-inducible araBAD promoter and transformed into E. coli strains Top 10, JM109 and XL-1 blue. The results showed that optimal conditions obtained from shaken flask experiments yielded 6.1 ± 1.1 g/L cell dry mass (CDM), a PHB content of 93.3 ± 0.9% (w/w) and a productivity of 0.24 g/(L⋅h), whereas the wild-type C. necator strain A-04 accumulated 78% (w/w) PHB with a productivity of 0.09 g/(L⋅h). Finally, for the scaled-up studies, fed-batch cultivations by pH-stat control in a 5-L fermenter of E. coli strains XL1-Blue harboring pBAD/Thio-TOPO-phaCAB A-04 and pColdTF-phaCAB A-04 in MR or LB medium, leading to a PHB production of 31.4 ± 0.9 g/L at 54 h with a PHB content of 83.0 ± 3.8% (w/w), a CDM of 37.8 ± 1.2 g/L, a Y P/S value of 0.39 g PHB/g glucose and a productivity of 0.6 g PHB/(L⋅h) using pColdTF-phaCAB A-04 in MR medium. In addition, PHB production was 29.0 ± 1.1 g/L with 60.2 ± 2.3% PHB content in the CDM of 53.1 ± 1.0 g/L, a Y P/S value of 0.21 g PHB/g glucose and a productivity of 0.4 g PHB/(L⋅h) using pBAD/Thio-TOPO-phaCAB A-04 in LB medium. Thus, a relatively high PHB concentration and productivity were achieved, which demonstrated the possibility of industrial production of PHB.

Use of agro-industrial residue from the canned pineapple industry for polyhydroxybutyrate production by Cupriavidus necator strain A-04.

BACKGROUND: Pineapple is the third most important tropical fruit produced worldwide, and approximately 24.8 million tons of this fruit are produced annually throughout the world, including in Thailand, which is the fourth largest pineapple producer in the world. Pineapple wastes (peel and core) are generated in a large amount equal to approximately 59.36% based on raw material. In general, the anaerobic digestion of pineapple wastes is associated with a high biochemical oxygen demand and high chemical oxygen demand, and this process generates methane and can cause greenhouse gas emissions if good waste management practices are not enforced. This study aims to fill the research gap by examining the feasibility of pineapple wastes for promoting the high-value-added production of biodegradable polyhydroxybutyrate (PHB) from the available domestic raw materials. The objective of this study was to use agro-industrial residue from the canned pineapple industry for biodegradable PHB production. RESULTS: The results indicated that pretreatment with an alkaline reagent is not necessary. Pineapple core was sized to - 20/+ 40 mesh particle and then hydrolyzed with 1.5% (v/v) H2SO4 produced the highest concentration of fermentable sugars, equal to 0.81 g/g dry pineapple core, whereas pineapple core with a + 20 mesh particle size and hydrolyzed with 1.5% (v/v) H3PO4 yielded the highest concentration of PHB substrates (57.2 ± 1.0 g/L). The production of PHB from core hydrolysate totaled 35.6 ± 0.1% (w/w) PHB content and 5.88 ± 0.25 g/L cell dry weight. The use of crude aqueous extract (CAE) of pineapple waste products (peel and core) as a culture medium was investigated. CAE showed very promising results, producing the highest PHB content of 60.00 ± 0.5% (w/w), a cell dry weight of 13.6 ± 0.2 g/L, a yield ( YP/S ) of 0.45 g PHB/g PHB substrate, and a productivity of 0.160 g/(L h). CONCLUSIONS: This study demonstrated the feasibility of utilizing pineapple waste products from the canned pineapple industry as lignocellulosic feedstocks for PHB production. C. necator strain A-04 was able to grow on various sugars and tolerate levulinic acid and 5-hydroxymethyl furfural, and a detoxification step was not required prior to the conversion of cellulose hydrolysate to PHB. In addition to acid hydrolysis, CAE was identified as a potential carbon source and offers a novel method for the low-cost production of PHB from a realistic lignocellulosic biomass feedstock.

Medium chain length polyhydroxyalkanoates consisting primarily of unsaturated 3-hydroxy-5-cis-dodecanoate synthesized by newly isolated bacteria using crude glycerol.

BACKGROUND: Our study aimed to search for novel bacteria capable of producing polyhydroxyalkanoates (PHAs) using crude glycerol residue obtained from biodiesel production in which used cooking oils were the substrates. RESULTS: Newly isolated bacteria from soils in Thailand were screened for the efficient production of PHAs from crude glycerol. The bacterial strains were cultivated on glucose, refined glycerol, crude glycerol, or various cooking oils (canola oil, palm oil, soybean oil, sunflower oil, corn oil, grape seed oil, olive oil, rice bran oil, camellia seed oil) for growth and PHA production. The effects of the total organic carbon (TOC) concentration and the mole ratio of carbon to nitrogen were investigated in batch cultivation. (1)H NMR, two dimensional-(1)H-correlation spectroscopy (2D-(1)H-COSY) and (13)C NMR analyses confirmed four bacterial strains were capable of producing medium-chain-length PHAs (mcl-PHAs), consisting of 3-hydroxyoctanoate (3HO) and 3-hydroxy-5-cis-dodecanoate (3H5DD), from crude glycerol. On the basis of phenotypic features and genotypic investigations, the bacterial strains were assigned as: ASC1, Acinetobacter genus (94.9% similarity); ASC2, Pseudomonas genus (99.2% similarity); ASC3, Enterobacter genus (99.2% similarity); ASC4, Bacillus genus (98.4% similarity). The highest amount of mcl-PHAs, 17.5 ± 0.8 g/L (content 61.8 ± 3.3% wt), with 3HO (14.7 ± 2.2 mol %), 3H5DD (85.3 ± 2.2 mol%), and a total biomass of 32.3 ± 0.3 g/L, was obtained from Pseudomonas sp. ASC2 in batch cultivation after 36 h. The mcl-PHAs recovered had a number-average molecular weight (M N) of 3.6 × 10(4) Da. Homopolymeric 3H5DD was obtained when the cultivation time was prolonged to 96 h. CONCLUSIONS: Novel PHA-producing strains were isolated and identified. These bacterial strains are able to produce mcl-PHAs from crude glycerol. The mcl-PHAs produced contained a high percentage of 3H5DD, which suggests their future application as softeners mixed with other biomaterials. The unsaturated side chain of 3H5DD monomers containing double bounds offers additional potential for improving the properties of the mcl-PHAs or extending their applications to the food industry.

Biocompatibilities and biodegradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)s produced by a model metabolic reaction-based system.

BACKGROUND: This study evaluated the biocompatibilities of random and putative block poly(3-hydroxybutyrate-co-3-hydroxyvalerate)s (PHBVs) produced by a metabolic reaction-based system. The produced PHBVs were fractionated, and the copolymer sequence distributions were analyzed using (1)H and (13)C NMR spectroscopy. The thermal properties were analyzed using differential scanning calorimetry (DSC). Mechanical tests were conducted using a universal testing machine. The in vitro cytotoxicities of films composed of random PHBVs and putative block PHBVs were investigated against three types of mammalian cells. The surfaces of the copolymer films and the morphologies of the cells were qualitatively monitored using scanning electron microscopy (SEM) and atomic force microscopy (AFM). RESULTS: Films composed of poly(3-hydroxybutyrate) (PHB), random PHBVs, putative block PHBVs, polystyrene and polyvinylchloride were prepared and characterized. The diad and triad sequence distributions indicated that the PHBVs produced via the fed-batch cultivation using two different feed systems resulted in two types of copolymers: random PHBVs and putative block PHBVs. The monomer compositions and sequence distributions strongly affected the thermal and mechanical properties. The mechanical integrity and characteristics of the film surfaces changed with the HV content. Notably, the random PHBVs possessed different mechanical properties than the putative block PHBVs. The biocompatibilities of these films were evaluated in vitro against three types of mammalian cells: L292 mouse connective tissue, human dermal fibroblast and Saos-2 human osteosarcoma cells. None of the PHBV films exhibited cytotoxic responses to the three types of mammalian cells. Erosion of the PHA film surfaces was observed by scanning electron microscopy and atomic force microscopy. The production of transforming growth factor-ß-1 and interleukin-8 was also examined with regards to the usefulness of PHB and PHBV as biomaterials for regenerative tissue. The production of IL-8, which is induced by PHB and PHBVs, may be used to improve and enhance the wound-healing process because of deficiencies of IL-8 in the wound area, particularly in problematic wounds. CONCLUSION: Taken together, the results support the use of PHB and the random and putative block PHBVs produced in this study as potential biomaterials in tissue engineering applications for connective tissue, bone and dermal fibroblast reconstruction.

High expression of fusion proteins consisting of a single-chain variable fragment antibody against a tumor-associated antigen and interleukin-2 in Escherichia coli.

BACKGROUND/AIM: The aim of this study was to establish a strategy for high-level production of single-chain variable fragment (scFv) antibodies fused with interleukin-2 (IL-2) in Escherichia coli. MATERIALS AND METHODS: We constructed two fusion sequences consisting of a scFv gene derived from a mouse monoclonal antibody against a tumor-associated antigen (MK-1) and human Interleukin-2(IL-2) gene, ligated the fusions into pET15b and transformed into three different E. coli strains. The effects of temperature, isopropyl-ß-D-thiogalactopyranoside (IPTG) concentration and duration of IPTG induction were investigated. RESULTS: Employing E. coli strain Rosetta-gami B, which has an oxidizing cytoplasm that facilitates cytoplasmic disulfide bond formation, improved the level of soluble protein expression. Under optimal conditions, the highest levels of fusion protein expression and high percentages of the proteins were found in their soluble form. Specifically, 89.29% (0.28 g/l) of one fusion protein was soluble after a 10-h induction and 84.61% (0.26 g/l) of the other fusion protein was soluble after a separate 10-h induction. When analyzed by enzyme-linked immunosorbent assay, the partially-purified fusion proteins retained a specific binding activity to the cell lysate of Chinese hamster ovary (CHO) cells expressing MK-1. CONCLUSION: Taken together, the methods described herein permit the production of substantial amounts of the fusion proteins for conducting functional studies on the biological role of these fusion proteins.

Optimal production of a fusion protein consisting of a single-chain variable fragment antibody against a tumor-associated antigen and interleukin-2 in fed-batch culture of Pichia pastoris.

BACKGROUND/AIM: The aim of the present study was to establish the strategy for producing a single-chain variable fragment (scFv) antibody fused with interleulin-2 (IL2) by Pichia pastoris and to optimize production during fed-batch cultivation in a 5-l fermenter. MATERIALS AND METHODS: We constructed a fusion sequence consisting of an scFv gene derived from a mouse monoclonal antibody against a tumor-associated antigen (designated MK-1 antigen) and human interleulin-2 (IL-2) gene, ligated the sequences to expression vector pPICZα-A and separately transformed the constructs into Pichia pastoris strains GS115 and KM71H. RESULTS: The highest concentration of secreted fusion protein, 738 ± 44 mg/l, was obtained after a 60-h induction. To investigate the specific binding activity of the partially purified fusion protein, we used an enzyme-linked immunosorbent assay and antigen from a whole-cell lysate. Student's t-test showed that the specific binding activity of the partially-purified fusion protein to the lysate of Chinese hamster ovary cell lines expressing the MK-1 antigen was significantly higher than that of the lysate of CHO cell lines that do not express MK-1. CONCLUSIONS: The method described here permits the production of substantial amounts of the fusion protein for conducting functional studies on the biological role of these fusion proteins.