Applied Biochemistry and Biotechnology (v.169, #4)

Two-Stage Acidic–Alkaline Hydrothermal Pretreatment of Lignocellulose for the High Recovery of Cellulose and Hemicellulose Sugars by Bin Guo; Yuanhui Zhang; Guo Yu; Won-Heong Lee; Yong-Su Jin; Eberhard Morgenroth (1069-1087).
The focus of this work was to develop a combined acid and alkaline hydrothermal pretreatment of lignocellulose that ensures high recovery of both hexose and pentose. Dilute sulfuric acid and lime pretreatments were employed sequentially. Process performance was optimized in terms of catalyst concentration, retention time, and temperature using response surface methodology. Medium operational conditions in the acid stage and harsh conditions in the alkaline stage were desirable with optimal performance at 0.73 wt% H2SO4, 150 °C, 6.1 min in the first stage, and 0.024 g lime/g biomass, 202 °C, 30 min in the second stage. In comparison to single-stage pretreatments with high recovery of either glucose or xylose, two-stage process showed great promises with >80 % glucose and >70 % xylose recovery. In addition, the method greatly improved ethanol fermentation with yields up to 0.145 g/g Miscanthus, due to significantly reduced formation of inhibitory by-products such as weak acids, furans, and phenols. Supplementing biomimetic acids would further increase glucose yield by up to 15 % and xylose yield by 25 %.
Keywords: Two-stage acidic–alkaline pretreatment; Miscanthus ; Combined acid hydrolysis; Response surface methodology; Lignocellulose

Biotransformation of Indole to Indigo by the Whole Cells of Phenol Hydroxylase Engineered Strain in Biphasic Systems by Shengnan Shi; Fang Ma; Tieheng Sun; Ang Li; Jiti Zhou; Yuanyuan Qu (1088-1097).
Biotransformation of indole to indigo in liquid–liquid biphasic systems was performed in Escherichia coli cells expressing phenol hydroxylase. It was suggested that indole could inhibit the cell growth even at low concentration of 0.1 g/L. The critical Log P for strain PH_IND was about 5.0. Three different solvents, i.e., decane, dodecane, and dioctyl phthalate, were selected as organic phase in biphasic media. The results showed that dodecane gave the highest yield of indigo (176.4 mg/L), which was more than that of single phase (90.5 mg/L). The optimal conditions for biotransformation evaluated by response surface methodology were as follows: 540.26 mg/L of indole concentration, 42.27 % of organic phase ratio, and 200 r/min of stirrer speed; under these conditions, the maximal production of indigo was 243.51 mg/L. This study proved that the potential application of strain PH_IND in the biotransformation of indole to indigo using liquid–liquid biphasic systems.
Keywords: Phenol hydroxylase; Indole; Indigo; Biotransformation; Biphasic systems

Chemopreventive Effect and HPTLC Fingerprinting Analysis of Jasminum sambac (L.) Ait. Extract Against DLA-Induced Lymphoma in Experimental Animals by M. Kalaiselvi; R. Narmadha; P. Ragavendran; B. Vidya; D. Gomathi; C. Arul Raj; T. Starlinraj; V. K. Gopalakrishnan; C. Uma; K. Kalaivani (1098-1108).
The anticancer activity of the ethanolic extract of Jasminum sambac against Dalton’s lymphoma ascites-induced lymphatic cancer in Swiss albino mice was investigated. The anticancer activity of J. sambac was studied against lymphoma using lipid profiles, biochemical parameters, and membrane-bound marker enzymes by standard procedures. A high-performance thin-layer chromatography fingerprinting analysis showed the presence of terpenoids and flavonoids. The levels of cholesterol, triglyceride, VLDL cholesterol, and LDL cholesterol were significantly decreased in tumor-induced mice, while HDL cholesterol showed increased levels compared with those profiles. On treatment with J. sambac, the levels were brought back to near normal. The albumin, creatinine, total protein, urea, and uric acid contents were also approaching normal values. There was s significant increase in the levels of ATPase in group II. These levels were brought back to normal upon plant extract treatment of mice. DNA fragmentation occurred in the tumor-induced group of tissue, and treatment with ethanolic extract reduced the DNA damage caused by lymphoma. Expression of lactate dehydrogenase (LDH) isoenzymes shows an increase in the levels of LDH-4 and LDH-5 in cancer-bearing animals which is brought back to near normal. Histopathological investigation showed normal sections of liver tissues in the treatment group. The results found in mice treated with ethanolic extract 100 mg kg−1 body weight quite promising and were comparable with the standard drug 5-fluorouracil. The statistically processed results support the conclusion that the ethanolic extract of J. sambac flower (100 mg kg−1) possesses a dose-dependent significant anticancer activity against lymphoma.
Keywords: Jasminum sambac ; HPTLC fingerprinting; Lymphatic cancer; Terpenoids; Flavonoids; Anticancer

Sequence Analysis of KpnI Repeat Sequences to Revisit the Phylogeny of the Genus Carthamus L. by Shweta Mehrotra; Shailendra Goel; Sarita Sharma; Soom Nath Raina; Vijay Rani Rajpal (1109-1125).
Repetitive DNA sequences constitute a significant proportion of eukaryotic genomes. Knowledge about the distribution of repetitive DNA sequences is necessary in order to gain insights into the organization, evolution and behavior of eukaryotic genomes. Therefore, we used two repetitive DNA sequences pCtKpnI-I and pCtKpnI-II, earlier reported in Carthamus tinctorius L. to study the phylogeny and to revise the taxonomic status of the taxa belonging to the genus. The study unraveled two major lines within the genus Carthamus; one line included all the diploid taxa (2n = 24) and the other line comprised the taxa with 2n = 20 and the polyploid taxa (2n = 44 and 64). The results of the present study will prove useful in molecular breeding for improving some targeted agronomic traits in genus Carthamus.
Keywords: Carthamus ; Repetitive sequences; Phylogeny; pCt KpnI repeats

In thermal printing, bisphenol A (BPA) functions chemically as a developer and reacts with white or colorless dyes in the presence of heat, converting them to a dark color. BPA can transfer readily to skin in small amounts from these papers. Its damage to environment and organisms has caused an extensive concern. In the present study, thermal paper used at the local automated teller machine counters of India were analyzed for the presence of BPA, and the capability of the paper to produce estrogenicity were assessed using a yeast two-hybrid assay experimental system. The study also focused on eliminating the endocrine-disrupting properties with partially purified laccase from newly isolated ascomycete fungi. The results indicate that these papers can produce estrogen hormone-like effect on experimental systems. It should be noted that on a daily basis, tons of such receipts are being dumped in the environment. Estrogenic properties of thermal paper were effectively removed from the reaction mixture within 3 h of incubation with the partially purified enzyme. We propose the utilization of waste thermal paper as a cheap substrate for laccase production for a safer and cleaner environment.
Keywords: Bisphenol A; Laccase; Thermal paper; Trichoderma sp; Yeast estrogen strain

Antiviral and Antibacterial Polyurethanes of Various Modalities by Daewon Park; Alyssa M. Larson; Alexander M. Klibanov; Yadong Wang (1134-1146).
We have prepared and characterized a new polyurethane-based antimicrobial material, N,N-dodecyl,methyl-polyurethane (Quat-12-PU). It exhibits strong antiviral and antibacterial activities when coated (as an organic solution or an aqueous nanosuspension) onto surfaces and antibacterial activity when electrospun into nanofibers. Quat-12-PU surfaces are able to kill airborne Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria, as well as inactivate the enveloped influenza virus (but not the non-enveloped poliovirus).
Keywords: Antiviral; Antibacterial; Polyurethane; Nanoparticles; Nanofibers

Enhanced Saccharification of Biologically Pretreated Wheat Straw for Ethanol Production by M. López-Abelairas; T. A. Lu-Chau; J. M. Lema (1147-1159).
The biological pretreatment of lignocellulosic biomass with white-rot fungi for the production of bioethanol is an alternative to the most used physico-chemical processes. After biological treatment, a solid composed of cellulose, hemicellulose, and lignin—this latter is with a composition lower than that found in the initial substrate—is obtained. On the contrary, after applying physico-chemical methods, most of the hemicellulose fraction is solubilized, while cellulose and lignin fractions remain in the solid. The optimization of the combination of cellulases and hemicellulases required to saccharify wheat straw pretreated with the white-rot fungus Irpex lacteus was carried out in this work. The application of the optimal dosage made possible the increase of the sugar yield from 33 to 54 %, and at the same time the reduction of the quantity of enzymatic mixture in 40 %, with respect to the initial dosage. The application of a pre-hydrolysis step with xylanases was also studied.
Keywords: Bioethanol; Lignocellulosic biomass pretreatment; Ligninolytic fungus; Enzymatic hydrolysis; Wheat straw

In this work, the high-level expression of the lipase r27RCL was achieved by optimization of the lipase gene copy number in the host strain Pichia pastoris. The copy number of the lipase gene proRCL from Rhizopus chinensis CCTCC M201021 was quantified by real-time quantitative polymerase chain reaction and a range of Mut+ P. pastoris strains carrying one, three, five, and six copies of proRCL were obtained. The maximum lipase activity was achieved at 12,500 U/mL by the five-copy recombinant strain after 96 h of methanol induction in the 7-L fermenter. However, the enzyme activity of the six-copy recombinant strain decreased remarkably. By transcription analysis of proRCL, ERO1, and PDI, it suggested that unfolded protein response seemed to be triggered in the highest copy recombinant strain after 24 h. Thus, elaborate optimization of foreign gene dosage was very important for the high-level expression of foreign proteins in P. pastoris.
Keywords: Lipase; Pichia pastoris ; Real-time quantitative PCR (RT-QPCR); Transcription analysis; Gene dosage

A simple and efficient protocol for Agrobacterium-mediated genetic transformation of tomato was developed using combination of non-tissue culture and micropropagation systems. Initially, ESAM region of 1-day-old germinated tomato seeds were microinjected for one to five times with Agrobacterium inoculums (OD600 = 0.2–1.0). The germinated seeds were cocultivated in the MS medium fortified with (0–200 mM) acetosyringone and minimal concentrations of (0–20 mg L−1) kanamycin, and the antibiotic concentration was doubled during the second round of selection. Bacterial concentration of OD600 = 0.6 served as an optimal concentration for infection and the transformation efficiency was significantly higher of about 46.28 %. In another set of experiment, an improved and stable regeneration system was adapted for the explants from the selection medium. Four-day-old double cotyledonary nodal explants were excised from the microinjected seedlings and cultured onto the MS medium supplemented with 1.5 mg L−1 thidiazuron, 1.5 mg L−1 indole-3-butyric acid, 30 mg L−1 kanamycin, and 0–1.5 mg L−1 adenine sulphate. Maximum of 9 out of 13 micropropagated shoots were shown positive to GUS assay. By this technique, the transformation efficiency was increased from 46.28 to 65.90 %. Thus, this paper reports the successful protocol for the mass production of transformants using microinjection and micropropagation techniques.
Keywords: Embryonic shoot apical meristem; Double cotyledonary node; Microinjection; Micropropagation; Tomato

Selective Lead Adsorption by Recombinant Escherichia coli Displaying a Lead-Binding Peptide by Thuong T. L. Nguyen; Hae Ryong Lee; Soon Ho Hong; Ji-Ryang Jang; Woo-Seok Choe; Ik-Keun Yoo (1188-1196).
A highly specific lead-binding peptide ThrAsnThrLeuSerAsnAsn was displayed on Escherichia coli, and lead adsorption characteristics of the recombinant bacteria were investigated. Cell surface-displayed peptide was expressed under the control of an arabinose promoter using outer membrane protein C (OmpCt) as an anchoring motif. The optimal induction period and arabinose concentration for the expression of peptide-fused OmpCt were determined to be 2 h and 0.001 g/L, respectively. Selective adsorption of Pb2+ onto recombinant cells was verified with individual or combinatory use of four metal ions, Pb2+, Ni2+, Co2+, and Cu2+; the amount of bound Pb2+ onto the biosorbents was significantly higher than the other metal ions. The adsorption isotherm of recombinant cells for Pb2+ followed the Langmuir isotherm with a maximum adsorption loading (q max) of 526 μmol/g dry cell weight.
Keywords: E. coli ; Lead-binding peptide; Cell surface display; Biosorbent; Biosorption

The present study dealt with the interaction of dichloromethane (DCM) with other non-chlorinated organic solvents such as methanol, acetone, toluene, and benzene, which are commonly present in the pharmaceutical wastewater, during biodegradation by mixed bacterial consortium. Non-chlorinated solvents were easily degradable even at an initial concentration of 1,000 mg/L, whereas only 20 mg/L of DCM was degraded when used as sole carbon source. The Monod Inhibition model appears to simulate the single pollutant biodegradation kinetics satisfactorily. In dual substrate systems, low concentrations (100 mg/L) of non-chlorinated solvents did not interfere with the DCM degradation. Non-interaction sum kinetics model was able to simulate the experimental results well in this case. However, high concentrations of non-chlorinated solvents (1,000 mg/L) affected the DCM degradation significantly. There was severe competition between the chlorinated and the non-chlorinated solvents. In this case, competitive inhibition model predicted the experimental results better compared to co-metabolism model. In multiple substrate system also, presence of DCM prolonged the degradation of the other non-chlorinated solvents. However, the presence of non-chlorinated compounds accelerated the degradation of DCM. The results of the present study may be helpful in optimal design of biological systems treating mixed pollutants.
Keywords: Biodegradation; Dichloromethane; Substrate interaction; Competitive inhibition model; Pharmaceutical wastewater

A decoupled input–output linearizing controller (DIOLC) was designed as an alternative advanced control strategy for controlling bioprocesses. Simulation studies of its implementation were carried out to control ethanol and biomass production in Saccharomyces cerevisiae and its performance was compared to that of a proportional–integral–derivative (PID) controller with parameters tuned according to a linear schedule. The overall performance of the DIOLC was better in the test experiments requiring the controllers to respond accurately to simultaneous changes in the trajectories of the substrate and dissolved oxygen concentration. It also exhibited better performance in perturbation experiments of the most significant parameters q S,max, q O2,max, and k s , determined through a statistical design of experiments involving 730 simulations. DIOLC exhibited a superior ability of constraining the process when implemented in extreme metabolic regimes of high oxygen demand for maximizing biomass concentration and low oxygen demand for maximizing ethanol concentration.
Keywords: Yeast fermentation; Nonlinear control; PID control; Ethanol production; Biomass maximization

Slow-Release Nutrient Capsules for Microorganism Stimulation in Oil Remediation by E. A. Reis; M. H. M. Rocha-Leão; S. G. F. Leite (1241-1249).
As the concern towards environmental deterioration grows worldwide, new technological achievements become essential for all countries. Among the technologies with great potential of bioremediation is microencapsulation of active material. Several studies have investigated the use of controlled release of active materials as a way of biostimulation and supplying the nutrients necessary for the bioremediation process. In fact, as the use of microorganisms has a great potential in degrading crude oils, this work aims to use that technology and to associate it to produce controlled-release capsules of nitrogen, phosphorus, and potassium (N, P, and K) for bioremediation purposes. For the capsule formulation, polymers of sodium alginate, Capsul®, and the commercial fertilizer NPK from Sempre Verde Inc. were used. Crude oil was the only carbon source and mineral medium for microorganism growth. Controlled-release nutrient capsules, with 4 mm in diameter, made of 3.0 % alginate (w/v) and 4.0 % Capsul® (w/v) were produced. Those capsules were used in association with a microbial consortium, in a liquid phase bioremediation process, having degraded 43.6 % of the total hydrocarbon within 240 h, evidencing thus as a promising tool for hydrocarbon bioremediation.
Keywords: Capsule; Alginate; Modified starch; Slow-release fertilizer; Bioremediation

Metabolomic Profilings of Urine and Serum from High Fat-Fed Rats via 1H NMR Spectroscopy and Pattern Recognition by Jingjing Xu; Changqin Liu; Shuhui Cai; Jiyang Dong; Xuejun Li; Jianghua Feng; Zhong Chen (1250-1261).
1H NMR spectroscopy in combination with multivariate statistical analysis was applied to explore the metabolic variability in urine and serum of high fat-fed rats relative to normal chow-fed ones. Metabolites contributing to intergroup discrimination identified by partial least squares discriminant analysis include 3-hydroxybutyrate, glutamate, glutamine, citrate, choline, hippurate, alanine, lactate, creatinine, taurine, acetate, etc. The aging effect along with long-term feeding was delineated with metabolic trajectory in principal component analysis score plot and age-related differences on metabolic profiling under different dietary intervention were recognised. The identified metabolites responsible for obesity were all imported into a web tool for network-based interpretation of compound lists to interpret their functional context, molecular mechanisms and disturbed signalling pathway globally and systematically. The results are useful for interpreting the pathology of obesity and further probing into the relationship between dietary-induced obesity and type 2 diabetes mellitus.
Keywords: High fat; NMR; Metabolomics; PLS-DA; TICL

Activation of Kraft Lignin by an Enzymatic Treatment with a Versatile Peroxidase from Bjerkandera sp. R1 by R. Taboada-Puig; T. A. Lú-Chau; M. T. Moreira; G. Feijoo; J. M. Lema (1262-1278).
Enzymatic lignin activation may be an environmentally friendly alternative to the use of chemicals in the production of wood fibers composites. Most studies on enzymatic activation of lignin for improving the adhesion of lignocellulosic products have been carried out using laccases. In this work, the use of a versatile peroxidase (VP) from the white-rot fungus Bjerkandera sp. (anamorph R1) for activating Kraft lignin was studied. The effect of enzyme dosage, incubation time, and H2O2 addition profile on lignin activation was evaluated by quantifying the phenoxy radicals formed using electron paramagnetic resonance (EPR) spectroscopy. Two alternative enzymatic systems based on the use of VP (a two-stage and an enzymatic cascade system) were also assayed. At optimal conditions (dose of 15 U g−1 and continuous addition of H2O2 (5.24 μmol h−1) during 1 h) the content of phenoxy radicals was doubled as compared with an untreated control. Moreover, using the two-stage VP system, a lignin activation similar to that found at optimal conditions could be reached in a shorter time.
Keywords: Versatile peroxidase; Electronic paramagnetic resonance; Lignin activation; Catalytic cascade; Mn(III)-malonate

Advances and Drawbacks of the Adaptation to Serum-Free Culture of CHO-K1 Cells for Monoclonal Antibody Production by Maria Elisa Rodrigues; Ana Rita Costa; Mariana Henriques; Philip Cunnah; David W. Melton; Joana Azeredo; Rosário Oliveira (1279-1291).
Currently, mammalian cell technology has become the focus of biopharmaceutical production, with strict regulatory scrutiny of the techniques employed. Major concerns about the presence of animal-derived components in the culture media led to the development of serum-free (SF) culture processes. However, cell adaptation to SF conditions is still a major challenge and limiting step of process development. Thus, this study aims to assess the impact of SF adaptation on monoclonal antibody (mAb) production, identify the most critical steps of cell adaptation to the SF EX-CELL medium, and create basic process guidelines. The success of SF adaptation was dependent on critical steps that included accentuated cell sensitivity to common culture procedures (centrifugation, trypsinization), initial cell concentration, time given at each step of serum reduction, and, most importantly, medium supplements used to support adaptation. Indeed, only one of the five supplement combinations assessed (rhinsulin, ammonium metavanadate, nickel chloride, and stannous chloride) succeeded for the Chinese hamster ovary-K1 cell line used. This work also revealed that the chemically defined EX-CELL medium benefits mAb production in comparison with the general purpose Dulbecco’s Modified Eagle’s Medium, but the complete removal of serum attenuates these positive effects.
Keywords: Chinese hamster ovary cells; Monoclonal antibody; Production; Serum-free medium; Trace elements; Cell adaptation

Indole-3-Acetic Acid Biosynthesis in Fusarium delphinoides Strain GPK, a Causal Agent of Wilt in Chickpea by Guruprasad B. Kulkarni; S Sanjeevkumar; B. Kirankumar; M. Santoshkumar; T. B. Karegoudar (1292-1305).
Fusarium delphinoides (Ascomycota; Nectriaceae) is an indole-3-acetic acid (IAA) producing plant pathogen and a causal agent of wilt in chickpea. The IAA biosynthetic pathway in F. delphinoides strain GPK (FDG) was examined by analyzing metabolic intermediates and by feeding experiments. Gas chromatograph (GC) analysis of FDG culture filtrates showed the presence of metabolic intermediates of indole-3-pyruvic acid (IPyA), indole-3-acetamide (IAM), and tryptamine (TRA) pathways. The different IAA biosynthetic pathways were further confirmed by identifying the presence of different enzymes of these pathways. Substrate specificity study of aromatic amino acid aminotransferase revealed that the enzyme is highly specific for tryptophan (Trp) and α-ketoglutarate (α-kg) as amino group donor and acceptor, respectively. Furthermore, the concentration-dependent effect of exogenous IAA on fungal growth was established. Low concentration of exogenous IAA increases the fungal growth and at high concentration it decreases the growth of FDG.
Keywords: Indole-3-acetic acid; Fusarium delphinoides ; Phytopathogen; IAA biosynthetic pathway; Aminotransferase

A trypsin inhibitor with a molecular mass around 17 kDa was purified from the seeds of Phaseolus vulgaris cv. ‘brown kidney bean’. The purification protocol involved, in sequence, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on Q-Sepharose and Mono Q, and gel filtration on Superdex 75. The molecular size and N-terminal amino acid sequence of the trypsin inhibitor resembled leguminous Bowman–Birk protease inhibitors (BBIs), signifying that brown kidney bean trypsin inhibitor is a BBI. Brown kidney bean trypsin inhibitor exhibited pronounced thermostability and pH stability. Its trypsin inhibitory activity was retained at all pH values (0–14) and up to 90 °C. The trypsin inhibitor also inhibited the proliferation of human breast cancer MCF7 cells with an IC50 of 71.52 μM. On the other hand, it only slightly inhibited proliferation of hepatoma HepG2 and embryonic liver WRL68 cells at a concentration over 110 μM.
Keywords: Leguminous; Trypsin inhibitor; Bowman–Birk; Anti-proliferative

The aim of this study is to compare the effect of different homogenization treatments on the physicochemical properties and the hydrolysis rate of a pure bleached cellulose. Results obtained show that homogenization treatments improve the enzymatic hydrolysis rate of the cellulose fibers by 25 to 100 %, depending of the homogenization treatment applied. Characterization of the samples showed also that homogenization had an impact on some physicochemical properties of the cellulose. For moderate treatment intensities (pressure below 500 b and degree of homogenization below 25), an increase of water retention values (WRV) that correlated to the increase of the hydrolysis rate was highlighted. Result also showed that the overall crystallinity of the cellulose properties appeared not to be impacted by the homogenization treatment. For higher treatment intensities, homogenized cellulose samples developed a stable tridimentional network that contributes to decrease cellulase mobility and slowdown the hydrolysis process.
Keywords: Homogenization; Microcrystalline cellulose; Enzymatic hydrolysis; Crystallinity; Water retention

The square pyramidal copper(II) complexes of N, O- donor ligand and ciprofloxacin have been synthesized. Synthesized complexes were characterized by physicochemical parameters like elemental analysis, electronic, FT–IR and LC–MS spectra. The complexes were screened for their antimicrobial activity against Gram(+Ve), i.e. Staphylococcus aureus, Bacillus subtilis, and Gram(−Ve), i.e. Serratia marcescens, Pseudomonas aeruginosa and Escherichia coli, microorganisms in terms of minimum inhibitory concentration and colony-forming unit. To determine the binding mode of complexes with Herring Sperm DNA, absorption titration and viscosity measurement were employed. DNA cleavage activity was carried out by gel electrophoresis experiment using supercoiled form of pUC19 DNA. The complexes were tested for their superoxide dismutase mimic activity in terms of IC50 value. Synthesized complexes were also screened for their cytotoxicity using brine shrimp lethality assay method.
Keywords: Antimicrobial activity; DNA binding; DNA cleavage; SOD mimic; Cytotoxicity

Tetramethylpyrazine (TTMP) is a widely used flavoring additive with a nutty and roasted taste. Solid-state fermentation (SSF) of wheat bran for producing TTMP was studied with Bacillus subtilis CCTCC M208157, which was an exogenous precursor-independent TTMP-producing strain. Factors influencing endogenous precursor supply and TTMP formation in this strain were investigated. According to the findings, glucose and diammonium phosphate contributed to TTMP production but excess salts caused an inhibition on cell growth and TTMP formation. Then a two-step supply strategy was applied: 10 % glucose was added at the beginning of the process to allow acetoin formation, which was the precursor of TTMP, while 3 % diammonium phosphate was added only after acetoin accumulation reached its maximum. By applying this strategy, acetoin increased from 5.44 to 13.2 g/kg dry substrate (kgds), and consequently the yield of TTMP increased by 6.8 folds from 0.44 to 3.01 g/kgds. This was the first report of using a two-step supply strategy for TTMP production by SSF, which proved to be conducive to TTMP production in this strain.
Keywords: Tetramethylpyrazine; Solid-state fermentation; Bacillus subtilis ; Two-step supply strategy

Biotechnological and Molecular Approaches for Vanillin Production: a Review by Baljinder Kaur; Debkumar Chakraborty (1353-1372).
Vanillin is one of the most widely used flavoring agents in the world. As the annual world market demand of vanillin could not be met by natural extraction, chemical synthesis, or tissue culture technology, thus biotechnological approaches may be replacement routes to make production of bio-vanillin economically viable. This review’s main focus is to highlight significant aspects of biotechnology with emphasis on the production of vanillin from eugenol, isoeugenol, lignin, ferulic acid, sugars, phenolic stilbenes, vanillic acid, aromatic amino acids, and waste residues by applying fungi, bacteria, and plant cells. Production of biovanillin using GRAS lactic acid bacteria and metabolically engineered microorganisms, genetic organization of vanillin biosynthesis operons/gene cassettes and finally the stability of biovanillin generated through various biotechnological procedures are also critically reviewed in the later sections of the review.
Keywords: Vanillin; Ferulic acid; Eugenol; Isoeugenol; Lactic acid bacteria; Genetic organization; Metabolic engineering; Biotransformation

Production and Partial Characterization of Cellulases and Xylanases from Trichoderma atroviride 676 Using Lignocellulosic Residual Biomass by André Luiz Grigorevski-Lima; Mariana Menezes Quadros de Oliveira; Rodrigo Pires do Nascimento; Elba Pinto da Silva Bon; Rosalie Reed Rodrigues Coelho (1373-1385).
Trichoderma atroviride 676 was studied to evaluate its efficiency in the production of some lignocellulolytic enzymes, using lignocellulosic residual biomass. Best results were obtained when 3.0 % (w/v) untreated sugarcane bagasse was used (61.3 U mL−1 for xylanase, 1.9 U mL−1 for endoglucanase, 0.25 U mL−1 for FPase, and 0.17 U mL−1 for β-glucosidase) after 3–4 days fermentation. The maximal enzymatic activity for endoglucanase, FPase, and xylanase were observed at 50–60 °C and pH 4.0–5.0, whereas thermal stability at 50 °C (CMCase and FPase) or 40 °C (xylanase) was obtained after 8 h. Zymograms have shown two bands of 104 and 200 kDa for endoglucanases and three bands for xylanase (23, 36, and 55.7 kDa). The results obtained with T. atroviride strain 676 were comparable to those obtained with the cellulolytic strain Trichoderma reesei RUT-C30, indicating, in the studied conditions, its great potential for biotechnological application, especially lignocellulose biomass hydrolysis.
Keywords: Trichoderma atroviride ; Sugarcane bagasse; Cellulose; Xylanase; Corn steep liquor

Hatching enzyme (HE) is of importance to degrade egg membrane to let the larvae be free. HE was purified and characterized from starfish blastula. The specific activity and the purification ratio of the purified HE with 110.9 kDa of molecular weight were 449.62 U/mg and 7.42-fold, respectively. Its optimal pH and temperature for activity were pH 8.0 and 30 °C, respectively. This enzyme was relatively stable in the range of pH 4.0–6.0 and 30–40 °C. This enzyme was inhibited by ethylene diamine tetraacetic acid (EDTA) and ethylene glycol tetraacetic acid, and also done moderately by Leupeptin, tosyl-lysine chloromethyl ketone, tosyl-phenylalanine chloromethyl ketone, and phenyl-methanesulfonyl fluoride. Zn2+ ion activated HE activity strongly and recovered the EDTA-pretreated activity more than did Ca2+, Mg2+, and Cu2+. Based on the results above, the starfish HE was classified as a zinc metallo- and trypsin-like serine protease. The values of Km, Vmax, and Kcat of the starfish HE on dimethyl casein were 0.31 mg/ml, 0.17 U/ml, and 122.70 s−1, respectively, whereas 1.09 mg/ml, 0.12 U/ml, and 771.98 s−1 on type I collagen. Therefore, the starfish HE could be a potential cosmeceutical because of its strong cleavage specificity on type I collagen.
Keywords: Hatching enzyme; Starfish; Asterias amurensis ; Purification; Characterization; Cleavage specificity

Enzyme “hexachlorocyclohexane (HCH) dehydrochlorinase LinA” mediates first step of aerobic microbial degradation of a chlorinated insecticide γ-HCH. The archetypal LinA-type1 consists of 156 amino acids that include a directly repeated hexapeptide motif GIHFAP at positions 141–146 and 148–153. Analysis of a series of LinA mutants, containing none, one, two, or three units of this repeated motif revealed that two units, as present in wild-type LinA, are required for its optimal activity and stability. Moreover, the presence of a bend in its secondary structure due to a proline residue that precedes the distal repeated unit contributes to enhanced LinA activity.
Keywords: LinA; Hexachlorocyclohexane; Dehydrochlorinase; Repeated motif

Analysis of Dormant Bud (Banjhi) Specific Transcriptome of Tea (Camellia sinensis (L.) O. Kuntze) from cDNA Library Revealed Dormancy-Related Genes by Krishnaraj Thirugnanasambantham; Gajjeraman Prabu; Senthilkumar Palanisamy; Suresh Ramraj Subhas Chandrabose; Abul Kalam Azad Mandal (1405-1417).
Bud dormancy is of ecological and economical interest due to its impact on tea (Camellia sinensis (L.) O. Kuntze) plant growth and yield. Growth regulation associated with dormancy is an essential element in plant’s life cycle that leads to changes in expression of large number of genes. In order to identify and provide a picture of the transcriptome profile, cDNA library was constructed from dormant bud (banjhi) of tea. Sequence and gene ontology analysis of 3,500 clones, in many cases, enabled their functional categorization concerning the bud growth. Based on the cDNA library data, the putative role of identified genes from tea is discussed in relation to growth and dormancy, which includes morphogenesis, cellular differentiation, tropism, cell cycle, signaling, and various metabolic pathways. There was a higher representation of unknown processes such as unknown molecular functions (65.80 %), unknown biological processes (62.46 %), and unknown cellular components (67.42 %). However, these unknown transcripts represented a novel component of transcripts in tea plant bud growth and/or dormancy development. The identified transcripts and expressed sequence tags provides a valuable public resource and preliminary insights into the molecular mechanisms of bud dormancy regulation. Further, the findings will be the target of future expression experiments, particularly for further identification of dormancy-related genes in this species.
Keywords: Banjhi; Bud; cDNA; Dormancy; Library; Tea; Transcriptome

Operational Stability to Changes in Composition of Herbicide Mixtures Fed to a Laboratory-Scale Biobarrier by O. Ramos-Monroy; N. Ruiz-Ordaz; J. Galíndez-Mayer; C. Juárez-Ramirez; I. Nava-Arenas; Y. Ordaz-Guillén (1418-1430).
The main objective of this work was to evaluate the operational stability of a laboratory-scale aerobic biobarrier designed for the treatment of water contaminated by mixtures of three herbicides frequently found in agricultural runoffs, atrazine, simazine and 2,4-dichlorophenoxyacetic acid (2,4-D). The microbial consortium used to degrade the herbicides was composed by six cultivable bacterial strains, identified as members of the genera Variovorax, Sphingopyxis, Hydrocarboniphaga, Methylobacterium, Pseudomonas and Acinetobacter. The effect caused by a seventh member of the microbial consortium, a ciliated protozoa of the genus Colpoda, on the herbicides biodegradation kinetics, was also evaluated. The biodegradation of five combinations of the herbicides 2,4-D, atrazine and simazine was studied in the biobarrier, operated in steady state continuous culture at different volumetric loading rates. In all cases, removal efficiencies determined by chemical oxygen demand (COD) and HPLC were nearly 100 %. These results, joined to the null accumulation of aromatic byproducts of atrazine and simazine catabolism, show that after 495 days of operation, in the presence of the protozoa, the adaptability of the microbial consortium to changing environmental conditions allowed the complete removal of the mixture of herbicides.
Keywords: Protozoan grazing; Colpoda ; Triazine herbicides; Biodegradation; Biofilm reactor; Biobarrier

Liquid Formulations for Long-Term Storage of Monoclonal IgGs by Monika Mueller; Maybelle Q. T. Loh; Doris H. Y. Tee; Yuansheng Yang; Alois Jungbauer (1431-1448).
Proteins like immunoglobulin (IgGs) are prone to degradation by a variety of pathways. In this study, a stabilizing formulation for long-term storage of a panel of seven monoclonal IgGs was found using differential scanning calorimetry (DSC). In the chosen formulations, the IgGs were subjected to stress, accelerated and real-time storage, and analyzed by size exclusion chromatography to determine fragment and aggregate content, and fluorescence-activated cell sorting to measure immunoreactivity. All IgGs showed the greatest conformational stability near their isoelectric point which was enhanced by adding sorbitol, sucrose, glycine, and sodium chloride. Optimized formulations, found by DSC, containing 20 % sorbitol and 1 M glycine prevented IgG aggregation and fragmentation and conserved immunoreactivity against shear stress, multiple freeze–thaw cycles, accelerated storage at 37 °C, and 12 months storage at 4 and −20 °C. Relatively poor thermal stability of the antigen-binding fragment domain was shown to limit storage stability of IgGs. This study confirms the predictive power of DSC to find storage formulations which protect IgGs during stress and long-term storage from aggregation and degradation. Liquid formulations found in this study may have a broad utility for other IgGs.
Keywords: IgG; Stability; DSC; Formulation; Long-term storage

Biological N Removal from Wastes Generated from Amine-Based CO2 Capture: Case Monoethanolamine by Ingrid Hauser; Ana B. Colaço; Julie A. Skjæran; Aslak Einbu; Kjetill Østgaard; Hallvard F. Svendsen; Francisco J. Cervantes (1449-1458).
Large-scale amine-based CO2 capture will generate waste containing large amounts of ammonia, in addition to contaminants such as the actual amine as well as degradation products thereof. Monoethanolamine (MEA) has been a dominant amine applied so far in this context. This study reveals how biological N removal can be achieved even in systems heavily contaminated by MEA in post- as well as pre-denitrification treatment systems, elucidating the rate-limiting factors of nitrification as well as aerobic and denitrifying biodegradation of MEA. The hydrolysis of MEA to ammonia readily occurred both in post- and pre-denitrification treatment systems with a hydraulic retention time of 7 h. MEA removal was ≥99 ± 1 % and total nitrogen removal 77 ± 10 % in both treatment systems. This study clearly demonstrates the advantage of pre-denitrification over post-denitrification for achieving biological nitrogen removal from MEA-contaminated effluents. Besides the removal of MEA, the removal efficiency of total nitrogen as well as organic matter was high without additional carbon source supplied.
Keywords: Biodegradation; Carbon capture and storage; Denitrification; Monoethanolamine; Nitrification

Effective Solubilization Procedure for Analysis of Silkworm Hemolymph Proteins by Two-Dimensional Gel Electrophoresis by Mallikarjun Gadwala; Neetha Kari; Narayan Moger; Shyam Kumar Vootla (1459-1466).
The outstanding capability of two-dimensional gel electrophoresis in separating all types of proteins basically depends on the efficiency of sample preparation. Sample preparation is one of the most critical steps in two-dimensional gel electrophoresis. Unfortunately, due to severe solubility, resolution of protein on gel is usually hampered, and thus, analysis remains a difficult task. However, technically several problems are generally encountered during protein extraction and isoelectric focusing. In the present investigation, we emphasized on evaluation and comparison of six different protein solubilization methods intended for resolving and analyzing silkworm hemolymph proteins by two-dimensional gel electrophoresis. Our findings revealed that the buffer composition of 8 M urea, 4 % 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 40 mM Tris base, 65 mM dithiothreitol, and 0.2 % ampholyte can effectively solubilize and yields maximum protein spots.
Keywords: Silkworm hemolymph; Lysis buffer; Two-dimensional gel electrophoresis; Solubilization buffer