Applied Biochemistry and Biotechnology (v.169, #7)

In this study, the native acid protease gene in Yarrowia lipolytica 22a-2 with high content of protein was disrupted, and the disruptant 3-13-10 obtained had very low acid protease activity. Then, the acid protease gene (AP1 gene) from Saccharomycopsis fibuligera A11 was actively expressed in the disruptant 3-13-10, and the transformant 43 carrying the AP1 gene had high specific acid protease activity (46.7 U/mg). The recombinant acid protease produced by the transformant 43 was found to be able to actively clot milk, and the transformant 43 still kept high content of protein. The hydrolysis products of κ-casein under catalysis of the recombinant acid protease and the commercial calf rennet had the same molecular mass, suggesting that the recombinant acid protease and its producer can be used both in cheese manufacturing and as protein source in food industry.
Keywords: Acid protease gene; Yarrowia lipolytica ; Cheese making; Single cell protein; Saccharomycopsis fibuligera

A reliable in vitro regeneration system for the economical and medicinally important Piper nigrum L. has been established. Callus and shoot regeneration was encouraged from leaf portions on Murashige and Skoog (MS) medium augmented with varied concentrations of plant growth regulators. A higher callus production (90 %) was observed in explants incubated on MS medium incorporated with 1.0 mg L−1 6-benzyladenine (BA) along with 0.5 mg L−1 gibberellic acid after 4 weeks of culture. Moreover, a callogenic response of 85 % was also recorded for 1.0 mg L−1 BA in combination with 0.25 mg L−1 α-naphthalene acetic acid (NAA) and 0.25 mg L−1 2,4-dichlorophenoxyacetic acid or 0.5 mg L−1 indole butyric acid (IBA) along with 0.25 mg L−1 NAA and indole acetic acid. Subsequent sub-culturing of callus after 4 weeks of culture onto MS medium supplemented with 1.5 mg L−1 thiodiazoran or 1.5 mg L−1 IBA induced 100 % shoot response. Rooted plantlets were achieved on medium containing varied concentrations of auxins. The antioxidative enzyme activities [superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX)] revealed that significantly higher SOD was observed in regenerated plantlets than in other tissues. However, POD, CAT, and APX were higher in callus than in other tissues. A high-performance liquid chromatography (HPLC) fingerprint analysis protocol was established for quality control in different in vitro-regenerated tissues of P. nigrum L. During analysis, most of the common peaks represent the active principle “piperine.” The chemical contents, especially piperine, showed variation from callus culture to whole plantlet regeneration. Based on the deviation in chromatographic peaks, the in vitro-regenerated plantlets exhibit a nearly similar piperine profile to acclimated plantlets. The in vitro regeneration system and HPLC fingerprint analysis established here brought a novel approach to the quality control of in vitro plantlets, producing metabolites of interest with substantial applications for the conservation of germplasm.
Keywords: Piper nigrum L.; In vitro regeneration; Antioxidant enzyme activities; HPLC; Piperine

Pretreatment of Chicken Feather Waste for Improved Biogas Production by Gergely Forgács; Magnus Lundin; Mohammad J. Taherzadeh; Ilona Sárvári Horváth (2016-2028).
This study deals with the utilization of chicken feather waste as a substrate for anaerobic digestion and improving biogas production by degradation of the compact structure of the feather keratin. In order to increase the digestibility of the feather, different pretreatments were investigated, including thermal pretreatment at 120 °C for 10 min, enzymatic hydrolysis with an alkaline endopeptidase [0.53–2.66 mL/g volatile solids (VS) feathers] for 0, 2, or 24 h at 55 °C, as well as a combination of these pretreatments. The effects of the treatments were then evaluated by anaerobic batch digestion assays at 55 °C. The enzymatic pretreatment increased the methane yield to 0.40 Nm3/kg VSadded, which is 122 % improvement compared to the yield of the untreated feathers. The other treatment conditions were less effective, increasing the methane yield by 11–50 %. The long-term effects of anaerobic digestion of feathers were examined by co-digestion of the feather with organic fraction of municipal solid waste performed with and without the addition of enzyme. When enzyme was added together with the feed, CH4 yield of 0.485 Nm3/kg VS−1 d−1 was achieved together with a stable reactor performance, while in the control reactor, a decrease in methane production, together with accumulation of undegraded feather, was observed.
Keywords: Anaerobic digestion; Feather waste; Pretreatment; Municipal solid waste; Co-digestion

Biodegradation of Phenol by the Yeast Candida tropicalis: An Investigation by NMR Spectroscopy by U. D. Phalgune; P. R. Rajamohanan; B. G. Gaikwad; R. J. Varma; S. George (2029-2037).
The process of phenol biodegradation by the yeast Candida tropicalis NCIM 3556 in aqueous medium was studied by 1H, 13C, and DOSY NMR techniques. Samples at regular intervals were centrifuged to separate the cells, and 1H spectral data were collected at 400 MHz. Though a gradual decrease in the concentration of phenol was observed, after an incubation period of ~8 h, formation of any intermediate products could not be detected. Experiments carried out with uniformly 13C-labeled phenol also failed to detect formation of any carboxylic acid intermediates during degradation. The studies indicated that the phenol was completely degraded to carbon dioxide and water in approximately 20 h. Self-diffusion coefficient measurements showed that the lifetime of phenol in the bound form is too small to impart any change in its diffusion behavior and the intermediates formed are converted to carbon dioxide and water at a very fast rate.
Keywords: Enzyme; NMR spectroscopy; Carboxylic acid; Biocatalysis; Diffusion; Phenol

Use of Multi-parameter Flow Cytometry as Tool to Monitor the Impact of Formic Acid on Saccharomyces carlsbergensis Batch Ethanol Fermentations by Cláudia Freitas; Elisabete Neves; Alberto Reis; Paula C. Passarinho; Teresa Lopes da Silva (2038-2048).
The use of lignocellulosic materials as substrate for bioethanol production is considered a cost-effective approach to make the biofuel production process economically sustainable. However, lignocellulosic hydrolysis releases toxic compounds such as weak acids which inhibit microorganism growth and ethanol production. In order to understand the physiological response of Saccharomyces carlsbergensis when fermenting glucose in the presence of formic acid (HF), the yeast growth was monitored by multi-parameter flow cytometry. Cytoplasmic membrane potential decreased as the HF concentration increased and as the yeast culture reached the stationary phase. However, the proportion of cells with permeabilized membrane did not increase with the HF concentration increase. The accumulation of reactive oxygen species was also monitored. Control and fermentations at low HF concentrations (<1 g/L) resulted in a high proportion of highly oxidized cells at the stationary phase. The multi-parameter flow cytometry approach proved to be a useful tool to monitor the physiological stress response of S. carlsbergensis growth and ethanol production in the presence of HF, an inhibitor present in lignocellulosic hydrolysates. The information here obtained at near real time can be used to enhance second-generation bioethanol production process efficiency.
Keywords: Lignocellulosic materials; Bioethanol; Saccharomyces carlsbergensis ; Formic acid; Flow cytometry

Flocculation Optimization of Microalga Nannochloropsis oculata by Y. Shen; Y. Cui; W. Yuan (2049-2063).
The objective of this work was to understand and optimize the flocculation of a marine alga Nannochloropsis oculata with two cationic salts, aluminum sulfate (AS), and ferric chloride (FC). Based on single-factor and response-surface-methodology experiments, second-order polynomial models were developed to examine the effect of initial algal biomass concentration (IABC), pH, and flocculant dose (FD) on final solid concentration of algae (SCA). The experimental and modeling results showed that SCA favored low pH, which however was undesirable to biomass recovery rate. There existed a positive stoichiometric relationship between FD and IABC; higher IABC required higher FD, and vice versa, for higher SCA. Optimum flocculation conditions were predicted at IABC of 1.7 g/l, pH 8.3, and FD of 383.5 μM for AS, and IABC of 2.2 g/l, pH 7.9, and FD of 438.1 μM for FC, under which the predicted maximum SCA were 32.98 and 30.10 g/l using AS and FC, respectively. The predictions were close to validation experimental results, indicating that the models can be used to guide and optimize the flocculation of N. oculata using AS and FC as the flocculants.
Keywords: Algae; Aluminum sulfate; Ferric chloride; Nannochloropsis oculata; Flocculation; RSM

Multistep Conversion of para-Substituted Phenols by Phenol Hydroxylase and 2,3-Dihydroxybiphenyl 1,2-Dioxygenase by Yuanyuan Qu; Shengnan Shi; Qiao Ma; Chunlei Kong; Hao Zhou; Xuwang Zhang; Jiti Zhou (2064-2075).
A multistep conversion system of para-substituted phenols by recombinant phenol hydroxylase (PHIND) and 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphCLA-4) was constructed in this study. Docking studies with different para-substituted phenols and corresponding catechols inside of the active site of PHIND and BphCLA-4 predicted that all the substrates should be transformed. High-performance liquid chromatography–mass spectrometry analysis showed that the products of multistep conversion were the corresponding para-substituted catechols and semialdehydes. For the first-step conversion, the formation rate of 4-fluorocatechol (0.39 μM/min/mg dry weight) by strain PHIND hydroxylation was 1.15, 6.50, 3.00, and 1.18-fold higher than the formation of 4-chlorocatechol, 4-bromocatechol, 4-nitrocatechol, and 4-methylcatechol, respectively. For the second-step conversion, the formation rates of semialdehydes by strain BphCLA-4 were as follows: 5-fluoro-HODA > 5-chloro-HODA > 2-hydroxy-5-nitro-ODA > 5-bromo-HODA > 2-hydroxy-5-methyl-ODA. The present study suggested that the multistep conversion by both ring hydroxylase and cleavage dioxygenase should be potential in the synthesis of industrial precursors and provide a novel avenue in the wastewater recycling treatment.
Keywords: Multistep conversion; para-Substituted phenols; Phenol hydroxylase; 2,3-Dihydroxybiphenyl 1,2-dioxygenase; Docking study

β-Glucosidase hydrolyzes cellobiose to glucose and is an important enzyme in the consortium used for hydrolysis of cellulosic and lignocellulosic feedstocks. In the present work, β-glucosidase was covalently immobilized on non-porous magnetic particles to enable re-use of the enzyme. It was found that particles activated with cyanuric chloride and polyglutaraldehyde gave the highest bead-related immobilized enzyme activity when tested with p-nitrophenyl-β-D-glucopyranoside (104.7 and 82.2 U/g particles, respectively). Furthermore, the purified β-glucosidase preparation from Megazyme gave higher bead-related enzyme activities compared to Novozym 188 (79.0 and 9.8 U/g particles, respectively). A significant improvement in thermal stability was observed for immobilized enzyme compared to free enzyme; after 5 h (at 65 °C), 36 % of activity remained for the former, while there was no activity in the latter. The performance and recyclability of immobilized β-glucosidase on more complex substrate (pretreated spruce) was also studied. It was shown that adding immobilized β-glucosidase (16 U/g dry matter) to free cellulases (8 FPU/g dry matter) increased the hydrolysis yield of pretreated spruce from ca. 44 % to ca. 65 %. In addition, it was possible to re-use the immobilized β-glucosidase in the spruce and retain activity for at least four cycles. The immobilized enzyme thus shows promise for lignocellulose hydrolysis.
Keywords: Lignocellulose hydrolysis; Immobilization; Enzymes; Magnetic particles; Pretreated spruce

The increase of multidrug-resistant pathogens and the restriction on the use antibiotics due to its side effects have drawn attention to the search for possible alternatives. Bacteriocins are small antimicrobial peptides produced by numerous bacteria. Much interest has been focused on bacteriocins because they exhibit inhibitory activity against pathogens. Lactic acid bacteria possess the ability to synthesize antimicrobial compounds (like bacteriocin) during their growth. In this study, an antibacterial substance (bacteriocin PJ4) produced by Lactobacillus helveticus PJ4, isolated from rat gut microflora, was identified as bacteriocin. It was effective against wide assay of both Gram-positive and Gram-negative bacteria involved in various diseases, including Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Enterococcus faecalis, and Staphylococcus aureus. The antimicrobial peptide was relatively heat-resistant and also active over a wide pH range of 2–10. It has been partially purified to homogeneity using ammonium sulfate precipitation and size exclusion chromatography and checked on reverse-phase high-performance liquid chromatography. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of bacteriocin PJ4 purified through size exclusion chromatography resolved ~6.5 kDa protein with bacteriocin activity. The peptide is inactivated by proteolytic enzymes, trypsin, and lipase but not when treated with catalase, α-amylase, and pepsin. It showed a bactericidal mode of action against the indicator strains E. coli MTCC443, Lactobacillus casei MTCC1423, and E. faecalis DT48. Such characteristics indicate that this bacteriocin may be a potential candidate for alternative agents to control important pathogens.
Keywords: Lactobacillus helveticus ; Bacteriocin; Purification; Characterization; Mode of action; Pathogen

The present paper studies the biotechnological production of xylitol using sugarcane bagasse hydrolysate in a repeated batch fermentation system with immobilized cells of Candida guilliermondii FTI20037. Immobilized cell system is considered as an attractive alternative to reuse the well-grown and adapted yeast cells in a new fresh fermentation media, without the need of the inoculum stage. In this work, seven repeated batches were performed in a fluidized bed bioreactor using immobilized cells in calcium alginate beads. According to the obtained results it was observed that the immobilized cells of C. guilliermondii can be reused for six successive batches maintaining an average xylitol yield (Y p/s) of 0.7 g/L and a volumetric productivity (Q p) of 0.42 g/L h at the end of 432 h of fermentation. On the other hand, in the seventh batch (504 h), a decrease of 44 % in the final concentration of xylitol was observed. This reduction can be explained by the possible diffusion and accumulation of insoluble substances, found in the hemicellulosic hydrolysate, in the interior of the immobilization support resulting in substrate mass transfer limitations.
Keywords: Xylitol; Immobilized cells; Repeated batch fermentation; Fluidized bed reactor; Lignocellulosic hydrolysate

Genome-Wide Identification of the Maize Calcium-Dependent Protein Kinase Gene Family by Pengda Ma; Jingying Liu; Xiangdong Yang; Rui Ma (2111-2125).
In higher plants, calcium is a ubiquitous second messenger in eukaryotic signal transduction cascades. The plant-specific calcium-dependent protein kinases (CDPKs) play important roles regulating downstream components of calcium signaling. We conducted a genome-wide analysis of maize (Zea mays) CDPKs and identified 35 CDPK genes. Maize CDPKs were found to be similar to their counterparts in rice in gene structure, GC content and subgroup classification. Divergence time estimation suggested that maize–rice orthologs were largely consistent with the time when these two species diverged from the last common ancestor. Semiquantitative RT-PCR revealed that the 29 of total 35 maize CDPK genes were expressed in all tissues, including root, stem, leaf, tassel, ear, and kernel. Our genomic and bioinformatics analyses will provide an important foundation for further functional dissection of the maize CDPK gene family.
Keywords: Calcium-dependent protein kinase; Maize; Gene family; Gene expression

Improving the Performance of Membrane Bioreactors by Adding a Metabolic Uncoupler, 4-Nitrophenol by Tang Qiong; Lin Song; Ying Cheng; Jun-Ru Xiong (2126-2137).
Effects of 4-nitrophenol (4NP) on the overall performance of membrane bioreactors (MBRs) were investigated in two bench-scale submerged MBRs. Positive impacts of 4NP on activated sludge production and membrane fouling were demonstrated over 45 days of stable operational period. After addition of 4-nitrophenol, the sludge production could be reduced effectively, but only a slight reduction in chemical oxygen demand removal was obtained. The effluent NH4 +-N concentrations were almost the same in two MBRs. The transmembrane pressures (TMPs) and resistance R increased with increasing mixed liquor suspended solid concentration at each MBR. The average daily TMP increase rates in the control MBR reactor remained at about 0.23 kPa day−1 and dropped to about 0.12 kPa day−1 in the 4NP-MBR. Compared with the control MBR, a wider dispersion and lower peak of floc size, a lower zeta potential, and a lower extracellular polymeric substance concentration were observed in the 4NP-MBR.
Keywords: Membrane fouling; 4-Nitrophenol; Metabolic uncoupler; Membrane bioreactor

White-rot fungus Inonotus obliquus grown in submerged culture produces antioxidative phenolic compounds. In this study, addition of lignocellulosic materials into the liquid culture increased the production and antioxidant activity of extra- and intra-cellular phenolic compounds (EPC and IPC, respectively). The production of EPC and IPC was significantly enhanced by wheat straw (by 151.2 and 45.3 %), sugarcane bagasse (by 106.9 and 26.1 %), and rice straw (by 67.6 and 38.9 %). Both of the EPC and IPC extracts from the three substrates showed a higher hydroxyl and 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity than those from the control medium. The highly active polyphenols such as tea catechins of epicatechin-3-gallate (ECG) and epigallocatechin-3-gallate (EGCG), and phelligridin G in the EPC extracts increased by 113.1, 75.0, and 86.3 % in the sugarcane bagasse medium. Davallialactone and inoscavin B in the EPC extracts were generated in large amounts in the lignocellulose media but not found in the control medium. The IPC extract from the wheat straw medium had the highest production of EGCG and ECG (17.6 and 18.1 mg/l). The different enhancement among the materials was attributed to the content and degradation rate of cellulose, hemicellulose, and lignin. The different antioxidant activity of the EPC and IPC extracts was related to their phenolic compositions.
Keywords: Inonotus Obliquus; Microbial Growth; Lignocellulose Degradation; Phenolic Compounds; Submerged Culture

Purification of Alcohol Dehydrogenase from Saccharomyces cerevisiae Using Magnetic Dye-Ligand Affinity Nanostructures by Nazife Kaya; Deniz Aktaş Uygun; Sinan Akgöl; Adil Denizli (2153-2164).
Reactive Green 19 was covalently immobilized onto magnetic nanostructures for purification of alcohol dehydrogenase from Saccharomyces cerevisiae. The Reactive Green 19 immobilized magnetic nanostructures were characterized by Fourier transform infrared spectroscopy, electron spin resonance, atomic force microscope, and energy dispersive X-ray analysis. Particle size of nanostructures was found to be roughly 70 nm. Alcohol dehydrogenase adsorption experiments were investigated under different conditions in batch system (i.e., medium pH, alcohol dehydrogenase concentration, temperature, and ionic strength). Maximum alcohol dehydrogenase adsorption capacity was found to be 176.09 mg/g polymer while nonspecific alcohol dehydrogenase adsorption onto plain magnetic nanostructures was negligible (19.4 mg/g polymer). Alcohol dehydrogenase molecules were desorbed by using 1.0 M NaCl with 98.4 % recovery. Alcohol dehydrogenase from S. cerevisiae was purified 45.63-fold in single step with dye-immobilized magnetic nanostructures, and purity of alcohol dehydrogenase was shown by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Keywords: Alcohol dehydrogenase; Reactive Green 19; Dye-ligand; Magnetic nanostructures

A monomeric 9.4-kDa peptide with antifungal activity was isolated from seeds of Phaseolus vulgaris cv Legumi secchi by using a protocol that involved affinity chromatography on Blue-Sepharose, ion exchange chromatography on Q-Sepharose, and gel filtration on Superdex 75. It was adsorbed on Blue-Sepharose and unadsorbed on Q-Sepharose. Its N-terminal sequence resembled those of other leguminous defensins. It impeded mycelial growth in the fungi Helminthosporium maydis, Rhizoctonia solani, Mycosphaerella arachidicola, and Fusarium oxysporum with an IC50 value of 9.5, 3.5, 1, and 9.2 μM, respectively, but there was no effect on Valsa mali. SYTOX Green uptake by R. solani indicated that the antifungal peptide induced fungal membrane permeabilization. In contrast to the majority of previously reported defensins/defensin-like peptides, Legumi secchi antifungal peptide did not reduce the viability of MCF-7 breast cancer cells and HepG2 hepatoma cells or inhibit HIV-1 reverse transcriptase, indicating a dissociation between antifungal, antiproliferative and HIV-1 reverse transcriptase inhibitory activities.
Keywords: Phaseolus vulgaris cv Legumi secchi; Antifungal; Antifungal peptide

Biotechnological Production of Phenyllactic Acid and Biosurfactants from Trimming Vine Shoot Hydrolyzates by Microbial Coculture Fermentation by Noelia Rodríguez-Pazo; José Manuel Salgado; Sandra Cortés-Diéguez; José Manuel Domínguez (2175-2188).
Coculture fermentations show advantages for producing food additives from agroindustrial wastes, considering that different specified microbial strains are combined to improve the consumption of mixed sugars obtained by hydrolysis. This technology dovetails with both the growing interest of consumers towards the use of natural food additives and with stricter legislations and concern in developed countries towards the management of wastes. The use of this technology allows valorization of both cellulosic and hemicellulosic fractions of trimming vine shoots for the production of lactic acid (LA), phenyllactic acid (PLA), and biosurfactants (BS). This work compares the study of the potential of hemicellulosic and cellulosic fractions of trimming vine shoots as cheaper and renewable carbon sources for PLA and BS production by independent or coculture fermentations. The highest LA and PLA concentrations, 43.0 g/L and 1.58 mM, respectively, were obtained after 144 h during the fermentation of hemicellulosic sugars and simultaneous saccharification and fermentation (SSF) carried out by cocultures of Lactobacillus plantarum and Lactobacillus pentosus. Additionally, cell-bond BS decreased the surface tension (ST) in 17.2 U; meanwhile, cell-free supernatants (CFS) showed antimicrobial activity against Salmonella enterica and Listeria monocytogenes with inhibition halos of 12.1 ± 0.6 mm and 11.5 ± 0.9 mm, respectively.
Keywords: Coculture; Trimming vine wastes; Phenyllactic acid; Biosurfactants; Lactic acid; Lactic acid bacteria

In order to illustrate the underlining mechanism of the effect of high pressure on lipases from different resources, the influence of compressed carbon dioxide treatment on the esterification activities and conformation of the three lipases Candida rugosa lipase (CRL), Pseudomonas fluorescens lipase, and Rhizopus oryzae lipase was investigated in the present work. The results showed that the lipases activities were significantly enhanced in most of high-pressure treatments, except the pressure had a negative effect on CRL activity in supercritical condition. Mild depressurization rate could remain the lipase’s activity by protecting its rigid structure under supercritical fluid. Conformational analysis by Fourier transform-infrared spectrometry and fluorescence emission spectra revealed that the variances of lipase activity after high-pressure treatment were correlated with the changes of its α-helix content and fluorescence intensity. Additionally, transesterification catalyzed by three lipases in supercritical carbon dioxide were conducted, and 87.2 % biodiesel conversion was obtained by CRL after 3 h, resulting in a great reduction of reaction time.
Keywords: Lipases; Compressed carbon dioxide; Conformational analysis; Fourier transform-infrared spectrometry (FT-IR); Fluorescence spectra (FP)

The main aim of this study was to compare different materials for Y. lipolytica immobilization that could be used in the production of γ-decalactone (a peach-like aroma) in order to prevent the toxic effect both of the substrate and the aroma upon the cells. Therefore, cells adsorption onto pieces of methyl polymethacrylate and of DupUM® was studied and further used in the biotransformation of castor oil into γ-decalactone. The highest aroma concentration was obtained with immobilized cells in DupUM®, where reconsumption of the aroma by the cells was prevented, contrarily to what happens with free cells. This is a very promising result for γ-decalactone production, with potential to be used at an industrial level since the use of immobilized cells system will facilitate the conversion of a batch process into a continuous mode keeping high cell density and allowing easier recovery of metabolic products.
Keywords: γ-decalactone; DupUM®; Immobilized cells; Methyl polymethacrylate; Yarrowia lipolytica

Layer-by-Layer Self-Assembly Immobilization of Catalases on Wool Fabrics by J. Liu; Q. Wang; X. R. Fan; X. J. Sun; P. H. Huang (2212-2222).
A new immobilization strategy of catalases on natural fibers was reported in this paper. Catalase (CAT) from Bacillus subtilis was assembled into multiple layers together with poly(diallyldimethylammonium chloride) (PDDA) on wool fabrics via layer-by-layer (LBL) electrostatic self-assembly deposition. The mechanism and structural evaluation of LBL electrostatic self-assembly were studied in terms of scanning electron microscopy (SEM), surface zeta potential, and apparent color depth (K/S). The SEM pictures showed obvious deposits absorbed on the wool surfaces after LBL self-assembly. The surface zeta potential and dyeing depth of CAT/PDDA-assembled wool fabrics presented a regular layer-by-layer alternating trend along with the change of deposited materials, revealing the multilayer structure of the wool fiber immobilized catalases. The V max values were found to be 2,500 ± 238 U/mg protein for the free catalase and 1,000 ± 102 U/mg protein for the immobilized catalase. The K m value of free catalase (11.25 ± 2.3 mM) was found to be lower than that of the immobilized catalase (222.2 ± 36.5 mM). The immobilized catalase remained high enzymatic activity and showed a measureable amount of reusability, which proved that LBL electrostatic self-assembly deposition is a promising approach to immobilize catalases.
Keywords: Immobilization; Catalase; Wool; Layer-by-layer electrostatic self-assembly

Prior studies disclosed that Aeromonas hydrophila NIU01 was a biodecolorization and bioelectricity bacterium which was isolated from a cross-strait of Taiwan. However, enzymatic function, laccase, involved in this strain had never been reported. This first attempt is to explore its laccase activity, the molecular cloning and heterologous recombinant expression in Escherichia coli. A full-length novel gene of 1,647 bp, LacA, encoding of 549 amino acids was successfully cloned by polymerase chain reaction. The recombinant pET-15b(+)-NIU-LacA expression was compared in different E. coli strains. By applying Taguchi’s L9 in culture optimization, the soluble laccase increased to 22.7 %, in which the conditions were obtained at 22 °C with initial shaking speed at 200 rpm, addition of lactose of 0.2 mM and CuSO4 of 0.5 mM to the medium, and shaking off while cell mass reached to OD600nm of 1.5. NIU-LacA was strongly inhibited by chloride ion. The optimal temperature was 60 °C and the optimum pH for ABTS (2,2′-azino-bis (3-ethylbenzthiazolinesulfonic acid) and 2,6-DMP (2,6-dimethoxyphenol) were pH 2.1 and pH 7.5 which enzymatic activity was 274.6 and 44.8 U/L, respectively. Further study in structural modeling of NIU-LacA showed the C terminal domain was the major variance in the three most closely A. hydrophila strains.
Keywords: Aeromonas hydrophila ; Laccase; Expression strategy; Escherichia coli ; Optimization; Simulation modeling