Biochemical Engineering Journal (v.46, #3)

BEJ Keywords (II).

Recent metabolic engineering were reviewed for the production of useful metabolites. Although much attention has been paid to metabolic engineering with some practical success, without profound insight into metabolic regulation, the modification of pathways based on the knockout of corresponding genes and/or amplification by plasmids does not necessarily lead to a significant improvement in cell growth and/or the production of specific metabolites. Although much information is available on the genetic regulation, biochemistry, and physiology of cellular metabolism, we are still far from understanding their overall regulation. Metabolic flux distribution is the manifestation of regulation at the enzyme level of the concentrations of intracellular metabolites. Moreover, proteins (including enzymes) are encoded by the corresponding metabolic pathway genes, which are under control of global regulators that respond to the environment. It is strongly desired that different levels of (omics) information be integrated by comparison of the data from different mutants and/or the different cultural environments using a systems biology approach. In particular, the roles of global regulators are quite important for metabolic regulation.
Keywords: Metabolic engineering; Systems biology; Metabolic regulation;

An attempt was made to use cyanobacterial biomass of water bloom, groundnut shell (GNS) and dye effluent as culture medium for laccase enzyme production by Coriolus versicolor. Laccase production was found to be 10.15 ± 2.21 U/ml in the medium containing groundnut shell and cyanobacterial bloom in a ratio of 9:1 (dry weight basis) in submerged fermentation at initial pH 5.0 and 28 ± 2 °C temperature. Half life of enzyme was found to be 74 min at 60 °C. Kinetic analysis of laccase when made with substrate ABTS, K m and V max were found to be 0.29 mM and 9.49 μmol/min respectively. Azide and hydroxylamine were found to exert significant inhibition on thermostable laccase. Inhibitor constant (k i) for azide and hydroxylamine were 1.33 and 0.18 mM respectively. This study forms the first report on the potential application of waste water cyanobacterial bloom and dyeing effluent as a medium for laccase production by C. versicolor MTCC138.
Keywords: Laccase; Cyanobacterial biomass; Dyeing effluent; Groundnut shell; Kinetic study;

Production of structured triacylglycerols by acidolysis catalyzed by lipases immobilized in a packed bed reactor by Estrella Hita; Alfonso Robles; Belén Camacho; Pedro A. González; Luis Esteban; María J. Jiménez; María M. Muñío; Emilio Molina (257-264).
The aim of this work was to produce structured triacylglycerols (STAGs), with caprylic acid located at positions 1 and 3 of the glycerol backbone and docosohexaenoic acid (DHA) at position 2, by acidolysis of tuna oil and caprylic acid (CA) catalyzed by lipases Rd, from Rhizopus delemar, and Palatase 20000L from Mucor miehei immobilized on Accurel MP1000 in a packed bed reactor (PBR), working in continuous and recirculation modes. First, different lipase/support ratios were tested for the immobilization of lipases and the best results were obtained with ratios of 0.67 (w/w) for lipase Rd and 6.67 (w/w) for Palatase. Both lipases were stable for at least 4 days in the operational conditions. In the storage conditions (5 °C) lipases Rd and Palatase maintained constant activity for 5 months and 1 month, respectively.These catalysts have been used to obtain STAGs by acidolysis of tuna oil and CA in a PBR operating with recirculation of the reaction mixture through the lipase bed. Thus, STAGs with 52–53% CA and 14–15% DHA were obtained. These results were the basis for establishing the operational conditions to obtain STAGs operating in continuous mode. These new conditions were established maintaining constant intensity of treatment (IOT, lipase amount × reaction time/oil amount). In this way STAGs with 44–50% CA and 17–24% DHA were obtained operating in continuous mode. Although the compositions of STAGs obtained with both lipases were similar, Palatase required an IOT about four times higher than lipase Rd.To separate the acidolysis products (free fatty acids, FFAs, and STAGs) an extraction method of FFAs by water–ethanol solutions was tested. The following variables were optimized: water/ethanol ratio (the best results were attained with a water/ethanol ratio of 30:70, w/w), the solvent/FFA–STAG mixture ratio (3:1, w/w) and the number of extraction steps (3–5). In these conditions highly pure STAGs (93–96%) were obtained with a yield of 85%. The residual FFAs can be eliminated by neutralization with a hydroethanolic KOH solution to obtain pure STAGs. The positional analysis of these STAGs, carried out by alcoholysis catalyzed by lipase Novozym 435, has shown that CA represents 55% of fatty acids located at positions 1 and 3 and DHA represents 42% of fatty acids at position 2.
Keywords: Structured lipids; Structured triacylglycerols; Acidolysis; Docosohexaenoic acid (DHA); Caprylic acid; Immobilized lipase; Packed bed reactor; Tuna oil; Lipase from Rhizopus delemar; Palatase 20000L;

A novel low-temperature resistant alkaline lipase from a soda lake fungus strain Fusarium solani N4-2 for detergent formulation by Ruizhi Liu; Xiaolu Jiang; Haijin Mou; Huashi Guan; HueyMin Hwang; Xiaoxia Li (265-270).
The effects of temperature, pH, and various components in a detergent on the activity and stability of a lipase produced by a soda lake fungus strain Fusarium solani N4-2 were studied in a preliminary evaluation for use in detergent formulation. The molecular mass of the lipase was 31.6 kDa by SDS-PAGE after purification using acetone fractionation and Q-sepharose ion exchange technique. In aqueous solutions, the lipase showed maximal activity at pH 9.0 in a glycine–NaOH buffer at 30 °C. At 0 °C and 10 °C, 52.3% and 82.6% of its maximum activities were detained, respectively. Among the metal ions tested, additions of Cu2+, Ca2+, Mg2+, Al3+ and Mn2+ ions were found to enhance the enzyme activity while Zn2+, Ba2+ and Hg2+ ions showed inhibitory effects. Compared with the commercial lipase Lipolase®, the enzyme showed better stability towards selected surfactants, commercial detergents, oxidizing agents and proteases. The remarkable resistance capability of the lipase makes it a potential additive for better detergent formulation.
Keywords: Enzyme; Microbial; Chromatography; Biocatalyst preparation; Detergent; Alkaline lipase;

Recombinant Candida rugosa LIP2 expression in Pichia pastoris under the control of the AOX1 promoter by Pau Ferrer; Manuel Alarcón; Ramón Ramón; María Dolors Benaiges; Francisco Valero (271-277).
The LIP2 isoenzyme gene from Candida rugosa has been completely synthesised and functionally expressed under the AOX1 promoter control in Pichia pastoris. The on-line monitoring and control of methanol, the key inducer carbon source in fed-batch cultures, has enhanced the yield product/biomass 7.8-fold and the productivity 12.8-fold compared to the best batch cultivation with the Pichia system and, 10-fold compared to the fed-batch cultivation process using the native C. rugosa strain.Nevertheless, the high ionic strength of culture broth favoured aggregation of Lip2, leading to total loss of lipolytic activity. After cultivation, a diaultrafiltration process was implemented to diminish ionic strength, allowing for the recovery of lipolytic activity in the diaultrafiltrate. The developed bioprocess resulted into a reproducible product in terms of quality and productivity.
Keywords: Lip2; Candida rugosa; Pichia pastoris; AOX1 promoter; Enzyme; On-line fed-batch; Stirred tanks; Protein engineering;

Cyclodextrin glucanotransferase production by cell biocatalysts of alkaliphilic bacilli by Nikolina Atanasova; Tsvetina Kitayska; Dragomir Yankov; Miroslava Safarikova; Alexandra Tonkova (278-285).
Cells of obligated alkaliphiles Bacillus pseudalcaliphilus 20RF and Bacillus pseudalcaliphilus 8SB isolated from Bulgarian habitats, producers of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19), were immobilized by three different techniques: on two types of polysulphone membranes; entrapped in agar-gel beads containing magnetite and by nano-particles of silanized magnetite covalently bound on the cell surface. The biocatalysts obtained demonstrated the opportunity for a significantly enhanced CGTase production compared to free cells for a long period of time (10 days semicontinuous cultivation) without impact on their mechanical stability. The cell membrane-biocatalysts exhibited the highest enzyme activity after 240 h repeated batch cultivation and retained 1.3–2.3-fold increase of the CGTase yield compared to free cells at the end of the process. Membrane biocatalysts were applied for a direct cyclodextrin (CD) production. The results obtained demonstrated the possibility of starch conversion into cyclodextrins by immobilized cells without using of crude or purified enzyme. The membrane biocatalysts of both obligated alkaliphiles formed mainly β- and γ-CDs after 6 h enzyme reaction at pH 9.0 of the reaction mixture. Under these conditions, the quantity of γ-CDs was a relative high, to 35–37% of the total CD amount.
Keywords: Alkaliphilic bacilli; Cell immobilization; Cyclodextrin glucanotransferase; Cyclodextrins; Magnetite; Semicontinuous cultivation;

The effects of cell entrapment on the growth rate and metabolic activity of major groups of bacteria (betaproteobacteria and gammaproteobacteria) in biological municipal wastewater treatment were investigated. Three different cell entrapment media (alginate, carrageenan and polyvinyl alcohol) and three cell-to-matrix ratios (0.1%, 0.2% and 0.6%, w v−1) were examined. Representative species of betaproteobacteria were Alcaligenes faecalis and Comamonas testosteroni whereas Pseudomonas putida was a gammaproteobacteria species studied. Free (non-entrapped) cells were included in the study for comparative purpose. Results indicated that the entrapment, type of entrapment media, and cell-to-matrix ratio had significant effects on the growth and metabolic activity of major groups of bacteria in wastewater treatment. Polyvinyl alcohol entrapped cells had the highest specific growth and specific substrate utilization rates. Increase of cell-to-matrix ratio (from 0.1% to 0.2% or 0.6%) did not improve the specific growth and specific substrate utilization rates. The relative performances provided by different entrapment media of the three species studied were quite consistent. This study showed that the suitable choices of entrapment media and cell-to-matrix ratio are important but similar for major groups of bacteria in wastewater treatment.
Keywords: Cell entrapment; Activity; Pseudomonas putida; Comamonas testosteroni; Alcaligenes faecalis; Wastewater treatment;

Biosorption behavior of heavy metals in bioleaching process of MSWI fly ash by Aspergillus niger by Jie Yang; Qunhui Wang; Qishi Luo; Qi Wang; Tingji Wu (294-299).
In this study, it was considered that the biosorption of heavy metals by biomass might occur during the bioleaching of fly ash. This work is focused on the biosorption behavior of Al, Fe, Pb and Zn by Aspergillus niger during the bioleaching process. The fungal biomass was contacted with heavy metals solution which extracted from fly ash by using gluconic acid as leaching agent. The equilibrium time for biosorption was about 120 min. The biosorption experiment data at initial pH 6.5 was used to fit the biosorption kinetics and isotherm models. The results indicated that the biosorption of Al, Fe and Zn by A. niger biomass were well described by the pseudo-first order kinetic model. The pseudo-second order kinetic model was more suitable for that of Pb. The Langmuir isotherm model could well describe the biosorption of Fe, Pb and Zn while the Freundlich model could well describe the biosorption of Al. Furthermore, the biosorption of metal ions decreased evidently in the presence of fly ash as compared to that in the absence of fly ash. This research showed that although the biomass sorption occurred during the bioleaching process, it did not inhibit the removal of Al, Fe, Pb and Zn evidently from fly ash.
Keywords: Biosorption; Heavy metals; Biomass; Waste treatment; Aspergillus niger; Bioleaching;

An open tank photobioreactor containing transparent rectangular chambers (TRCs) was developed to improve the photosynthetic efficiency of microalgal cultivation. The TRCs, made of transparent acrylic, conducted light deep into the photobioreactor, especially at high cell concentrations. The average irradiance, I av , was calculated by Lambert–Beer's law, and was used to determine the light conditions in the cultivation system. The photobioreactor provided large areas of illumination that improved the effective utilization of light energy for microalgae growth and created a good artificial environment for a high rate of cell growth, even at low I av . The biomass concentration of Chlorella sp. reached 3.745 g L−1 on the 13th day, with biomass productivity of 0.340 g L−1  d−1. The total biomass obtained was 56% more than that of similar culture systems without TRCs.
Keywords: Photobioreactor; Microalgae; Chlorella sp.; Average irradiance; Lambert–Beer's law; Light utilization;

Purification of CBS 819.72 α-amylase by aqueous two-phase systems: Modelling using Response Surface Methodology by Radhouane Kammoun; Hichem Chouayekh; Hajeur Abid; Belgacem Naili; Samir Bejar (306-312).
The partition and purification of α-amylase from a culture supernatant of Aspergillus oryzae CBS 819.72 was made in aqueous two-phase system (ATPS). According to bibliography and preliminary studies, the factors polyethylene glycol (PEG) molecular weight (M PEG) and concentration (C PEG), buffer type (BU) and concentration (C BU), temperature (T), salt nature (SALT) and concentration (C SALT), bioligand (BL) and concentration (C BL) and pH were investigated using a Plackett–Burman design to identify the factors affecting separation. Taking into consideration a simultaneous increase in enzyme recovery (R Y) and purification factor (P F), the best performance of the system was obtained at 4 °C and pH 6 using PEG 8000 g/mol, citrate buffer, KCl and sucrose. Experimental Box–Behnken design together with the Response Surface Methodology (RSM) have been used to find optimum C PEG, C Citrate and C SALT. Quadratic models were predicted for P F and R Y in the top phase and a better compromise between these two parameters can be found by superimposing the contour plots of P F and R Y for 8% citrate. A region in the experimental space can be defined where the purification factor is always higher than 3 with yields exceeding 65%.
Keywords: Purification; Aqueous two-phase system; Aspergillus oryzae; α-Amylase; Modelling; Response Surface Methodology; PEG/citrate;

Role of liposome on recognition and folding of oxidized and fragmented superoxide dismutase for its re-activation by Hiroshi Umakoshi; Le Quoc Tuan; Toshinori Shimanocuhi; Ryoichi Kuboi (313-319).
The behaviors of adsorption and folding of oxidized superoxide dismutase (SOD) fragment on the liposome surface have been studied by varying the type of liposomes. The recognition of the SOD fragment by the liposome was found to be performed, depending on the membrane fluidity, surface charge density and, also, instability of the hydrogen bonds of the liposome surface. The conformational change of the SOD fragment on the liposome membrane was dependent on its adsorption although the variation of their secondary structures was observed. The SOD LIPOzyme (liposome with enzyme-like activity) with high SOD-like activity was obtained by the appropriate control of the recognition and folding of SOD fragment through the suitably-regulated interactions between the liposome and the peptide.
Keywords: Membranome; Membrane stress biotechnology; LIPOzyme; Liposome; Antioxidative enzyme; Recognition;

The kinetics of metal uptake by gel and dry calcium alginate beads was analysed using solutions of copper or lead ions. Gel beads sorbed metal ions faster than the dry ones and larger diffusivities of metal ions were calculated for gel beads: approximately 10−4  cm2/min vs. 10−6  cm2/min for dry beads. In accordance, scanning electron microscopy and nitrogen adsorption data revealed a low porosity of dry alginate particles. However, dry beads showed higher sorption capacities and a mechanical stability more suitable for large-scale use. Two sorption models were fitted to the kinetic results: the Lagergren pseudo-first order and the Ho and McKay pseudo-second order equations. The former was found to be the most adequate to model metal uptake by dry alginate beads and kinetic constants in the orders of 10−3 and 10−2  min−1 were obtained for lead solutions with concentrations up to 100 g/m3. The pseudo-first order model was also found to be valid to describe biosorbent operation with a real wastewater indicating that it can be used to design processes of metal sorption with alginate-based materials.
Keywords: Biosorption; Biopolymer; Heavy metals; Biomaterial; Effluent; Water treatment;

Rapid decolourization of azo dyes by a new isolated higher manganese peroxidase producer: Phanerochaete sp. HSD by Wang Hailei; Li Ping; Pang Min; Zhai Zhijun; Yu Guangli; Liu Guosheng; Yao Jianming (327-333).
In the present paper, a strain of higher MnP producer, Phanerochaete sp. HSD, was screened and the important medium components influencing MnP production were optimized using fractional factorial design and central composite experimental design; statistical analysis suggested diammonium tartrate and Mn2+ were the important factors and under the optimum conditions, MnP activity reached 2613 ± 22 U/l, accorded with the predicted value from response surface analysis. The feasibility of using this fungus to decolourize azo dyes was examined too. Results indicated that crude enzyme solution of it could decolourize three azo dyes efficiently and speedily: for 120 and 350 mg/l of Congo red, 95% decolourization rate was observed at the 5th and 8th hour; for 200, 350 and 600 mg/l methyl orange, 95% decolourization rate was obtained at the 5th, 6th and 9th hour; furthermore, the decolourization rates of 150 and 300 mg/l of Eriochrome black T were up to 97.1% and 91.4% at the 7th and 13th hour, respectively. In addition, MnP played a crucial role in the decolourization process.
Keywords: Shake-flask; Colourants; Optimization; Manganese peroxidase; Decolourization; Azo dyes;

The densities of aqueous 1,4-dioxane solutions of tris(hydroxymethyl)aminomethane (TRIS), N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS), N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid (TAPSO), and N-tris[hydroxymethyl]-4-amino-butanesulfonic acid (TABS) were measured at T  = 298.15, 303.15, 310.15, and 318.15 K, using a high precision vibrating tube digital densitometer. On the basis of these data, the isobaric thermal expansibilities, α(m, T), have been evaluated. The solubilities of these buffers in water and at several concentrations of 1,4-dioxane at 298.15 K have also been determined from the experimental results of density measurements. Two-liquid phase behavior was found from the binary system containing the TABS buffer over a given composition range. The solubility data were further used to calculate the free energy of transfer ( Δ G ′ tr ) of these buffers from water to aqueous 1,4-dioxane solutions at 298.15 K. We observed positive values of ( Δ G ′ tr ) for the buffers from water to 1,4-dioxane, indicating that the interactions between these biological buffers and 1,4-dioxane are unfavorable. The contribution of transfer free energies ( Δ g ′ tr ) of various functional groups from water to the aqueous 1,4-dioxane solutions, have been estimated.
Keywords: Enzyme; Protein engineering; Cell engineering; Biomaterials engineering; Biological buffers interactions; Transfer free energy;

A new high-throughput screening method for determining active and enantioselective hydrolases by Bo Wang; Xiaoling Tang; Gangfeng Ren; Ji Liu; Hongwei Yu (345-349).
A new high-throughput screening method using fluorescein sodium salt as an indicator to obtain hydrolases with high enantioselectivity is developed, which is demonstrated to be sensitive and reliable. The results determined by the method correlate well with those from GC analysis. This method can be applied to determine activity and enantioselectivity of not only lipase and esterase, but also other enzymes which catalyze hydrolysis reaction releasing proton, such as the protease or amidase. Because of the application of small amount of optically pure enantiomers, screening large libraries of enzymes is allowed at low cost and in short time.
Keywords: Biocatalyst preparation; Enzyme; Microbial; Protein engineering; High-throughput screening; Fluorescein sodium salt;