Process Biochemistry (v.43, #4)

Lipase enzyme immobilization on synthetic beaded macroporous copolymers for kinetic resolution of chiral drugs intermediates by Indu Bhushan; Rajinder Parshad; Gulam Nabi Qazi; Ganesh Ingavle; C.R. Rajan; Surendera Ponrathnam; Vijay Kumar Gupta (321-330).
Lipase isolated from Arthrobacter sp. (bacterial strain, MTCC No. 5125) at RRL Jammu, being used for various process development. Arthrobacter sp. lipase (ABL) now has been immobilized on synthetic polymers and reused many a times. In this investigation number of various synthetic macroporous alkylated glycidyl epoxy copolymers with varying hydrophobicity, pore volume and surface area were prepared and used for this study. Among all the polymers prepared and used only two epoxy polymers GMA-EGDM 75-20(I) and GMA-EGDM 75-30(I) with particle size in the range of 150–450 nm, epoxy groups 80 and 70%, tertiary amino groups 20 and 30% was found suitable for immobilization of lipase (ABL). Dibutyl amine (DBA) incorporation created an internal pore radii 20–50 nm and hydrophobic microenvironment in both the polymers for binding the enzyme, which led to improvement in stability and enatioselectivity in racemic resolution process especially by binding to one of the isomers. The optimal ABL binding capacity of polymer GMA-EGDM 75-20(I) was 60 units, 34 mg protein and GMA-EGDM 75-30(I) was 36 units, 21 mg protein/g polymer. The immobilized lipase matrices displayed enhanced pH, thermal, organic solvent and long-term storage stability. Both the immobilized enzyme matrices were tested firstly for the hydrolysis of triglycerides using tributyrin as substrate. After testing, both the matrices were reused for racemic resolution of ethyl-3-hydroxy-3-phenyl propanoate (fluoxetine intermediate, an antidepressant drug) and racemic chiral auxiliary, acetyl-1-phenyl ethanol (intermediate of many chiral drugs) for 15 cycles. These immobilized lipase matrices have shown very high stability on recycling, high-enantioselectivity, high conversion and faster recovery of product compare to free enzyme, therefore these matrices may find use in kinetic resolution process developments.
Keywords: Lipase; Chiral resolution; Immobilization; Ethyl-3-hydroxy-3-phenyl propanoate; 1-Phenyl ethanol and enantioselectivity; Enantiomeric excess (ee);

This paper presents an innovative technique of immobilizing Baker's yeast invertase using polyvinyl alcohol (PVA)–alginate matrix. Previous immobilization technique was improved by adding treatment solutions such as boric acid and sodium sulphate. PVA–alginate beads with four different compositions were investigated in terms of enzyme activity within the beads, immobilization yield, diffusion coefficient and also chemical and mechanical stability. The enzyme activity within the beads was also compared with the free enzyme activity. Finally, the microstructure of the beads was analyzed using SEM. Amongst others, the results revealed that the fabricated beads remained insoluble in aqueous solution owing to the innovative technique. In addition, beads produced from 12% (w/v) PVA concentration and 5% (w/v) boric acid possess at least 10% higher enzyme activity while those produced from 12% (w/v) PVA concentration and 7% (w/v) boric acid have at least 28% higher mechanical stability compared to the other formulations.
Keywords: PVA; Alginate; Immobilized invertase; Enzyme activity; Beads stability; Boric acid;

Effect of preservatives for food grade C-PC from Spirulina platensis by Sanjiv K. Mishra; Anupama Shrivastav; Sandhya Mishra (339-345).
The effect of selected edible preservatives, citric acid, sucrose and calcium chloride on the stability of C-phycocyanin (C-PC) at 0 ± 5 °C and 35 ± 5 °C was studied in aqueous solution. While screening the edible preservatives for a protein like C-phycocyanin, the denaturation of C-PC with urea as a denaturant and thermal unfolding studies through differential scanning calorimetry (DSC) was carried out to select a stabilizing agent having Hofmeister series behaviour acting on hydrophobic interactions. While studying the efficacy of edible preservatives, citric acid (4 mg/ml) was observed to be one of the best preservative for phycocyanin at 35 ± 5 °C in aqueous solution for 45 days with negligible loss which is comparable to the stability of C-PC at 0 ± 5 °C.Calcium chloride and sucrose were also found to be effective in maintaining the stability of C-PC in aqueous phase, but at lower temperature. Citric acid was able to maintain the stability even at higher temperature lasting for more than 1 month in aqueous solution.
Keywords: Citric acid; C-phycocyanin; Degradation; DSC; Preservative; Stability;

The effects of temperature and initial pH on anaerobic xylose utilization by the thermotolerant yeast Kluyveromyces marxianus IMB2, IMB4, and IMB5 were evaluated. Ethanol yield from xylose was greatest with IMB4, a temperature of 40 °C and an initial pH of 5.5. Xylitol yield was greatest with yeast strains IMB2 and IMB5, a temperature of 45 °C and an initial pH of 4.5 or 5.0. Additional fermentations of xylose at 40 and 45 °C, pH 5.5, and 20 times more cells were done using IMB4 since it exhibited the greatest ethanol yield. Ethanol and xylitol yields from xylose were greater at 40 °C than at 45 °C and volumetric production rates for ethanol and xylitol were 0.02 and 0.08 g/(l h), respectively. IMB4 xylitol yields were comparable to other xylitol producers, but with lower productivity. These strains are not good candidates for utilization of xylose under anaerobic conditions and other strategies should be explored.
Keywords: Xylose; Fermentation; Thermotolerant; Yeast; Biofuels; Bioproducts;

DCO2 on-line measurement used in rapamycin fed-batch fermentation process by Yinliang Chen; Jeffrey Krol; Weimin Huang; Julia P. Cino; Rajul Vyas; Richard Mirro; Brian Vaillancourt (351-355).
A Streptomyces fed-batch fermentation was studied with on-line measurement of dissolved carbon dioxide (DCO2). A selected cell strain R060107 of Streptomyces hygroscopicus was used in the production of rapamycin. pH was controlled only in the production phase at the low limit of 5.1 after its value dropped from an initial 6.5 to 5.1. DCO2 was used to control the feed in the fed-batch fermentation of S. hygroscopicus R060107. The experiment investigated the relationship between rapamycin production and the evolution of DCO2. A high limit of DCO2 (hDCO2) was used to control medium feed. In order to meet the needs for the dynamic growth and metabolism of cells in the process, the hDCO2 was defined as a dynamic parameter, and was therefore calculated constantly in the process. A fermentation condition of limited nutrient supply was realized. It was observed that rapamycin was secreted when the glycerol concentration was well controlled in the fermentation condition. Experimental results showed that 500 mg/l of rapamycin was produced in 120 h of fermentation.
Keywords: DCO2; Streptomyces; Antibiotic; Rapamycin; Fed-batch; Fermentation;

The effect of acid stress on lactate production and growth kinetics in Lactobacillus rhamnosus cultures by Remedios Yáñez; Susana Marques; Francisco M. Gírio; J. Carlos Roseiro (356-361).
The present study describes the inhibitory effect on growth and lactate production by Lactobacillus rhamnosus cells exposed to acidic stress conditions. Cultivations were carried out in presence of an uncoupling agent, hexanoic acid, as a model weak acid, on several initial concentrations and at different incubation temperatures. A statistical experimental design was used showing a more marked influence of temperature than hexanoic acid on both lactate production and biomass concentration. The effect of temperature is more intense on biomass (decrease of about 50% in the production) than on lactate production (decrease of about 35% in the production).Lactate production does not follow a proportional decay to biomass in the presence of the acid, i.e. the decrease is less accentuated. It indicates that the decrease of lactate in acid toxicity conditions is a result of cell drop and not a direct effect on lactate metabolism. The dissociation between lactate production and biomass in cultures under acid stress indicates that the excess of carbon inside the cells due to the diversion of ATP to expel protons expands metabolite production as the metabolic pathways receive the excess carbon acting as a cell dissipation mechanism.
Keywords: Lactic acid; Acid stress; Hexanoic acid; Lactobacillus rhamnosus;

In this study, efficient biodegradation of glycerol-containing synthetic wastes using an immobilization bioreactor was examined. A fixed-bed bioreactor packed with polyurethane was used in semi-continuous mode for glycerol removal under mesophilic and thermophilic anaerobic conditions. The best performance was obtained from the reactor under the thermophilic (55 °C) conditions. The average removal of dissolved organic carbon was 86.7% at an organic loading rate of 1.00 g/(l-reactor d). Molecular cloning of 16S rRNA gene sequences indicated that the archaeal clones in the thermophilic reactor were affiliated with two main groups, the hydrogenotrophic Methanobacterium sp. (17 out of 20 clones) and the aceticlastic Methanosarcina sp. (3 out of 20 clones). The bacterial clones were mostly affiliated with Bacillus sp., Clostridium sp., Desulfotomaculum sp., and Ruminococcus sp. Scanning electron microscopy (SEM) observation of the main cellular morphologies present in the biofilm on the colonization indicated that the immobilized microorganism was primarily composed of coccus, diplococci-shaped Methanosarcina-like cells, rods of Methanobacterium-like cells, rods of Bacillus sp., rods and coccus of Clostridium sp. and Desulfotomaculum sp., and coccobacillus of Ruminococcus sp.-like bacteria. They presumably form a syntrophic association.
Keywords: Anaerobic digestion; Biodegradation; Glycerol; Methane production; Fixed-bed bioreactor;

Ligninolytic enzyme ability and potential biotechnology applications of the white-rot fungus Grammothele subargentea LPSC no. 436 strain by Mario C.N. Saparrat; Paulina Mocchiutti; Constanza S. Liggieri; Mónica B. Aulicino; Néstor O. Caffini; Pedro A. Balatti; María Jesús Martínez (368-375).
To get a better insight into the ligninolytic system of Grammothele subargentea, extracellular ligninolytic enzyme activities and ability to degrade synthetic dyes as well as Eucalyptus globulus wood were assayed in cultures grown on an agar medium with Cu2+ or dyes and on E. globulus wood chips. Laccase was the only ligninolytic enzyme detected. The fungus was able to decolorize different dyes, being the highest levels of laccase activity in cultures with Brilliant Green. Cultures on wood showed both ligninolytic activity and degradative ability on lipophilic extractives. An extracellular laccase with pI 3.5 and maximal activity at pH 4.0 and 50–55 °C was detected on liquid cultures containing 0.6 mM Cu2+. The enzyme extract was stable at pH 6.0–7.0 and up to 60 °C. A laccase-mediator system using a G. subargentea laccase crude extract and 1-hydroxybenzotriazole as mediator improved the tensile strength of a paper from recycled high-kappa-number pulp.
Keywords: Dye decolorization; Extracellular ligninolytic enzymes; Laccase; Paper industry; White-rot fungus; Wood degradation;

Lipids from rainbow trout (Oncorhynchus mykiss) roe were extracted by chemical and enzymatic methods. Enzymatic hydrolysis by Alcalase®, Neutrase® and Protamex® yielded three fractions after centrifugation: a light phase (oil), an intermediate fraction (water-soluble material) and a residual heavy phase. Best results were obtained with Alcalase® where the roe proteins hydrolysis degree was 7.8% compared with 19% for trout fillets. The difference was explained by the particular composition of the proteins of the fish egg chorion. The yield of oil extracted after proteolysis was 3.7% vs. 9.6% by chemical technique. The polyunsaturated fatty acids (PUFAs) amount in the oil phase was high (41.8%) with 34.7% n−3 PUFA accounting for total lipids. High-concentration of PUFA was found in the polar lipid fraction of the heavy phase where docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) accounted for 25.6 and 10.9% of total lipids, respectively.
Keywords: Trout roe; Enzymatic extraction; Lipid composition; PUFA; Cholesterol;

A mathematical model with adequate complexity integrating hydraulics, biofilm and microbial conversion processes is successfully developed for a continuously moving bed biofilter performing tertiary nitrification. The model was calibrated and validated using data from Nether Stowey pilot plant in UK. For the model, the mixing is approximated using tanks-in-series approach, the biofilm is described using a one-dimensional multi-species model, and the microbial processes are described by ASM1. A scenario analysis with the model revealed that the temperature has a significant impact on the ammonium removal efficiency, doubling nitrification capacity every 5 °C increase. However, at temperatures higher than 20 °C, the biofilm thickness starts to decrease due to increased decay rate. The influent nitrogen load was also found to be influential on the filter performance, while the hydraulic loading had relatively negligible impact. Overall, the calibrated model can now reliably be used for design and process optimization purposes.
Keywords: ASTRASAND; Biofilm; Modeling; Moving bed sand filter; Nitrification; Tertiary treatment;

Influence of culture conditions on the productivity and lutein content of the new strain Scenedesmus almeriensis by J.F. Sánchez; J.M. Fernández; F.G. Acién; A. Rueda; J. Pérez-Parra; E. Molina (398-405).
This paper presents the first characterization data of the lutein-rich microalgae Scenedesmus almeriensis, a new strain isolated within a farmer's greenhouse. The main objective is to determine the appropriate conditions for the culture of this microalgae and any factors that might enhance its lutein content. The maximum growth rate was determined first in batch cultures, resulting an initial estimate of 0.63 1/day. Then, the influence of environmental culture conditions such as temperature, pH, culture medium, external irradiance, and salinity were assessed operating in continuous mode at a dilution rate half the maximum previously determined. Finally, the possible interactions between irradiance and temperature were studied by means of a surface response analysis and the optimal conditions of irradiance and temperature were tested in a separate experiment. The results show that the medium proposed by [Mann JE, Myers J. On pigments, growth and photosynthesis of Phaeodactylum tricornutum. J Phycol 1968;4:349–55] was adequate for the growth of this strain and that increasing the initial nutrient concentration did not improve the performance of the cultures. Measurements of chlorophylls fluorescence showed that there was no photoinhibition even under the highest external irradiances tested (1700 μE/m2  s). The optimal pH was found to be 8.0. With regard to the temperature, this strain grew well in the range of 30–40 °C, being clearly distressed in the experiments carried out at 48 °C. Low to medium salinities, between 0 and 5 gNaCl/L, were appropriate to promote growth rate. A response surface analysis predicted a maximum biomass productivity of 0.7 g/L day at 33 °C and 1700 μE/m2  s, and a maximum lutein content of 0.54% d. wt. at 44 °C and 1233 μE/m2  s. The experimental confirmation of the optimal conditions for lutein resulted in a biomass productivity of 0.73 g/L day with a lutein content of 0.53% d. wt., and therefore a lutein productivity of 3.8 mg/L day. The biomass of S. almeriensis is a rich source of lutein, about ten fold richer than the commercial source of this compound, Marigold flowers. The high lutein content and growth rate and the capacity to endure harsh environmental conditions make S. almeriensis a promising source of lutein.
Keywords: Scenedesmus; Lutein; Biomass productivity; Continuous culture; Irradiance; Temperature;

Two-stage sequencing batch reactor (SBR) with independent nitrification was evaluated for simultaneous removal of COD, nitrogen, and phosphorus from a low carbon-to-nitrogen wastewater (C/N ratio ranged from 1.1 to 7.4). Independent nitrification proceeded by incorporating the contact period within the system and the nitrification period in the external reactor, which was an outsourcing resource dedicated to enhancing nitrification activity. The contact period enabled the fraction of organic substrate in the influent. Ammonia nitrogen was then able to be decanted to the external nitrification reactor, in which ammonia nitrogen was nitrified as much as 70%. During the nitrification period, dominate microbial group was an ammonia oxidizing bacteria such as Nitrosomonas sp. JL21, Nitrosomonas sp. JL2, and Nitrosolobus multiformis. During the react period, the nitrogen nitrified was not only denitrified, but organic matters were also biodegraded. It was revealed that the effective and preferential use of organic substrate enhanced the overall performance of the system without the supplement of any external carbon sources. Removal efficiencies of COD, nitrogen, and phosphorus were as high as 87% at the loading rate of 0.06–4.79 kg COD/m3  day, 81% at the loading rate of 0.05–0.84 kg N/m3  day, and 60% at the loading rate of 0.01–0.09 kg P/m3  day, respectively.
Keywords: Independent nitrification; Low carbon-to-nitrogen ratio; Sequencing batch reactor; Simultaneous removal;

PMAL1, the sigma 70-like sequences possessing promoter of the maltase gene of Hansenula polymorpha, was evaluated for its application in heterologous protein production in Escherichia coli. Levansucrases Lsc1, Lsc2 and Lsc3 of Pseudomonas syringae pv. tomato DC3000 were expressed from PMAL1 in E. coli as biotechnologically relevant model proteins. Production of soluble levansucrases with high specific activity confirmed appropriate strength of PMAL1 in a prokaryotic host. As about 90% of levansucrase activity was present in the cytoplasm of E. coli, no levan-synthesis related sucrose intolerance of bacteria was observed. All three levansucrases hydrolyzed and polymerized both, sucrose and raffinose. The raffinose-related activity of levansucrases has not been previously described in P. syringae. The PMAL1 expression system was used to produce Lsc3 protein in E. coli for purification. The purified levansucrase showed much higher affinity for sucrose cleavage (K m  = 21 mM) than the levansucrase from P. syringae pv. phaseolicola described in the literature with K m of 160 mM.
Keywords: Heterologous protein expression; Sigma 70; Levansucrase; Pseudomonas syringae; Hansenula polymorpha; Yeast promoter;

Effect of a fungal immunomodulatory protein from Ganoderma tsugae on cell cycle and interferon-gamma production through phosphatidylinositol 3-kinase signal pathway by Yi-Min Hsiao; Yu-Lu Huang; Sheau-Chung Tang; Gow-Jen Shieh; Jing-Ying Lai; Po-Hui Wang; Tsung-Ho Ying; Jiunn-Liang Ko (423-430).
A fungal immunomodulatory protein (FIP-gts) was purified from Ganoderma tsugae. Recombinant FIP-gts was expressed as glutathione S-transferase fusion protein in E. coli. In this study, recombinant FIP-gts promoted cell cycle progression from G0/G1 to S phase and signaling pathways were investigated in human peripheral mononuclear cells (HPBMCs) by PI staining and flow cytometry. FIP-gts significantly induced cytokine interferon-gamma (IFN-γ) expression. Pre-treatment of cells with phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002 abolished FIP-gts, induced increase in S phase, and inhibited IFN-γ secretion. Akt, a downstream effector of PI 3-kinase was phosphorylated and activated by FIP-gts. Results show that FIP-gts is a potent activator in PBMC and its effects are mediated via cytokine regulation of PI 3-kinase.
Keywords: Fungal immunomodulatory protein; IFN-γ; PI 3-kinase;

Previously, success in producing and extracting continuously LVV-heamorphin-7 and VV-haemorphin-7 in an aqueous/butan-2-ol–octan-1-ol biphasic medium in the course of hydrolysis of bovine haemoglobin by immobilized pepsin was achieved. In this paper, the coupling of the continuous hydrolysis of haemoglobin by pepsin immobilized on a duolite with concomitant extraction of the two haemorphins and haem by a butan-2-ol–octan-1-ol mixture, their adsorption on an aluminium oxide column and the recycling of the solvent mixture in the reactor are described. A steady-state for haemorphin concentrations in the regenerated organic phase at the outlet of the continuous-stirred-tank-reactor (CSTR) was achieved and maintained for more than 10 bed volumes. Finally, the two haemorphins were selectively eluted from the aluminium oxide column tank to a volatile ethanolamine solution.
Keywords: Haemoglobin; Opioid peptides; Triphasic system; Continuous-stirred-tank-reactor; Solvent extraction; Solvent recycling;

Fed-batch culture of Nocardia sp. for epoxysuccinate hydrolase production by Qiongying Long; Lei Huang; Zhimin Li; Qin Ye (438-444).
Fed-batch cultures of Nocardia sp. were carried out in a 5-l fermentor for production of cis-epoxysuccinate hydrolase (EH). Since EH production was significantly repressed by glucose, the cultivation process was divided into a growth phase on glucose and a production phase induced by cis-epoxysuccinate (ES). The cells of Nocardia sp. were unable to utilize exogenous tartrate, so the intracellular tartrate produced from ES was the only carbon source in the production phase, and supply of enough ES was crucial. When ES was supplied at a constant rate of 0.3 g/(l h), the specific EH activity of the culture was only 75.1 U/g wet cell weight (WCW) due to limitation of ES. When the ES concentration in the culture was maintained at about 5 g/l by adding more ES based on material balance, the specific EH activity reached 182 U/g WCW. The EH activity measured with intact cells was influenced by the ES concentration in the culture due to the effect on cell permeability, the activity in this study was measured with ultrasound-disrupted cells.
Keywords: Nocardia; Cis-epoxysuccinic acid; l(+)-Tartaric acid; Epoxysuccinate hydrolase; Fed-batch culture; Carbon source;

Two strains of Leptospirillum ferrooxidans were isolated from samples taken from La Silvita polymetallic sulphide mine, Neuquen, Argentina. Isolates were identified by 16S rRNA gene restriction enzyme analysis. The specific growth rate (μ) at different temperatures and pH values were examined. The potential of two isolates to assist the leaching process using a mineral ore from the same mine was evaluated. The tests were carried out in shake flask at 1% (w/v) pulp density. The main sulphides found in the mineralogical composition of La Silvita ore were: sphalerite, galena, pyrite and chalcopyrite.Zinc and copper recoveries with a native isolate were 100% in 20 days and 12 days, respectively. Iron solubilization was 42% after 33 days of operation. These values were greater than those obtained using a laboratory reference strain, L. ferrooxidans DSMZ 2705, or abiotic conditions.
Keywords: Leptospirillum; Isolation; Bioleaching; Polymetallic sulphide mineral;

Bisphenol A removal by a membrane bioreactor by Jianhua Chen; Xia Huang; Duujong Lee (451-456).
Endocrine disrupting chemicals (EDCs) are potentially harmful chemicals during wastewater reclamation. Bisphenol A (BPA) is a typical EDC, and its removal using a submerged membrane bioreactor (MBR) was investigated. For comparison, a conventional activated sludge reactor (CASR) was simultaneously tested using the same BPA sludge loadings as the MBR. The results showed that MBR could remove BPA a little more effectively than CASR, despite changes in sludge loadings ranging from 0.046 to 10.2 g kg−1  d−1. However, MBR could bear much higher volume loadings than CASR and still achieve the same BPA removal efficiencies. In MBR, HRT did not obviously influence the removal of BPA. The results also showed that the contributions of sludge adsorption to BPA removal were quite low in both reactors. In addition, one metabolite of BPA biodegradation, 4-hydroxy-acetophenone, was detected. These results suggested that biodegradation dominated the BPA removal process.
Keywords: BPA; Membrane bioreactor; Removal; Adsorption; Biodegradation;

Purification and identification of novel angiotensin-I-converting enzyme inhibitory peptides from shark meat hydrolysate by Hao Wu; Hai-Lun He; Xiu-Lan Chen; Cai-Yun Sun; Yu-Zhong Zhang; Bai-Cheng Zhou (457-461).
Proteins, especially the proteins of marine origin, are potential resources of natural drugs and food additives. Our previous results showed that shark meat hydrolysate obtained with protease SM98011 digestion showed high angiotensin-I-converting enzyme (ACE) inhibitory activity, with an IC50 value of 0.4 mg/mL. In this article, ACE inhibitory peptides were separated from shark meat hydrolysate and identified. By ultrafiltration, gel filtration and RP-HPLC, 4 peptides with high ACE inhibitory activity were purified. Their sequences identified by Secondary Ion Mass Spectrometry were Cys-Phe, Glu-Tyr, Met-Phe and Phe-Glu. Cys-Phe, Glu-Tyr and Phe-Glu were conformed to be novel ACE inhibitory peptides, with IC50 values of 1.96, 2.68 and 1.45 μM, respectively. They may have potential in the treatment of hypertension or in clinical nutrition. This is the first report about novel ACE inhibitory peptides from hydrolysate of shark meat. This research may provide an efficient utilization of shark meat.
Keywords: Shark meat; Hydrolysate; Angiotensin-I-converting enzyme (ACE); ACE inhibitory peptides;