Applied Biochemistry and Biotechnology (v.175, #8)

In Vitro Antifungal Activity and Probable Fungicidal Mechanism of Aqueous Extract of Barleria Grandiflora by Suman Kumari; Preeti Jain; Bhawana Sharma; Preeti Kadyan; Rajesh Dabur (3571-3584).
Barleria grandiflora Dalz. (Acanthaceae) is being used in India to treat different types of disorders including skin infections. Therefore, there are good possibilities to find antifungal compounds in its extracts with novel mechanism of action. The main objectives of the present study were to evaluate the antifungal activity of plant extracts and to study its effects on metabolic pathways of A. fumigatus. The microbroth dilution assay was used to explore antifungal activity and MIC of various extracts. Metabolic profiles of control and treated cultures were collected from Q-TOF-MS interfaced with HPLC. Affected metabolic pathways of A. fumigatus after the treatment were analyzed by discrimination analysis of mass data. Antifungal activities were observed in hot and cold water extracts of the plant. Hot water extract of B. grandiflora showed significant activity against tested fungi in the range 0.625–1.25 mg/mL. Partial least discrimination analysis revealed that the hot water plant extract downregulated amino acid, glyoxylate pathway, and methylcitrate pathways at the same time due to the synergistic effects of secondary metabolites. Hot water extract also downregulated several other metabolic pathways unique to fungi indicating its specific activity toward fungi. B. grandiflora showed promising antifungal activity which can further be exploited by identification of active compounds, to inhibit the specific fungal pathways and development of novel therapeutic antifungal drugs.
Keywords: Aspergillus fumigatus ; Invasive aspergillosis; Barleria grandiflora Dalz; HPLC-Q-TOF-MS; Aminoadipate pathway; Glyoxylate pathway; Methylcitrate pathway

Genome-Wide Analysis and Differential Expression of Chitinases in Banana Against Root Lesion Nematode (Pratylenchus coffeae) and Eumusa Leaf Spot (Mycosphaerella eumusae) Pathogens by S. Backiyarani; S. Uma; S. Nithya; A. Chandrasekar; M. S. Saraswathi; R. Thangavelu; M. Mayilvaganan; P. Sundararaju; N. K. Singh (3585-3598).
Knowledge on structure and conserved domain of Musa chitinase isoforms and their responses to various biotic stresses will give a lead to select the suitable chitinase isoform for developing biotic stress-resistant genotypes. Hence, in this study, chitinase sequences available in the Musa genome hub were analyzed for their gene structure, conserved domain, as well as intron and exon regions. To identify the Musa chitinase isoforms involved in Pratylenchus coffeae (root lesion nematode) and Mycosphaerella eumusae (eumusa leaf spot) resistant mechanisms, differential gene expression analysis was carried out in P. coffeae- and M. eumusae-challenged resistant and susceptible banana genotypes. This study revealed that more number of chitinase isoforms (CIs) were responses upon eumusa leaf spot stress than nematode stress. The nematode challenge studies revealed that class II chitinase (GSMUA_Achr9G16770_001) was significantly overexpressed with 6.75-fold (with high fragments per kilobase of exon per million fragments mapped (FPKM)) in resistant genotype (Karthobiumtham-ABB) than susceptible (Nendran-AAB) genotype, whereas when M. eumusae was challenge inoculated, two class III CIs (GSMUA_Achr9G25580_001 and GSMUA_Achr8G27880_001) were overexpressed in resistant genotype (Manoranjitham-AAA) than the susceptible genotype (Grand Naine-AAA). However, none of the CIs were found to be commonly overexpressed under both stress conditions. This study reiterated that the chitinase genes are responding differently to different biotic stresses in their respective resistant genotypes.
Keywords: Banana; Chitinase isoforms; Nematode; Eumusa leaf spot; Digital gene expression; Illumina

Comparison and Evaluation of Two Diagnostic Methods for Detection of npt II and GUS Genes in Nicotiana tabacum by Mohammad Amin Almasi; Mehdi Aghapour-ojaghkandi; Khadijeh Bagheri; Mohammadreza Ghazvini; Seyed Mohammad Hosseyni-dehabadi (3599-3616).
To diminish the time required for some diagnostic assays including polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP) and also a visual detection protocol on the basis of npt II and GUS genes in transgenic tobacco plants were used. Agrobacterium tumefaciens-mediated transformation of Nicotiana tabacum leaf discs was performed with plant transformation vector of pBI 121. From kanamycin-resistant plants selected by their antibiotic resistance, four plants were selected for DNA isolation. Presence of the transgene was confirmed in the transformants by PCR and LAMP. In this regard, all LAMP and PCR primers were designed on the basis of the gene sequences of npt II and GUS. The LAMP assay was applied for direct detection of gene marker from plant samples without DNA extraction steps (direct LAMP assay). Also, a novel colorimetric LAMP assay for rapid and easy detection of npt II and GUS genes was developed here, its potential compared with PCR assay. The LAMP method, on the whole, had the following advantages over the PCR method: easy detection, high sensitivity, high efficiency, simple manipulation, safety, low cost, and user friendly.
Keywords: β-Glucuronidase (GUS); LAMP assay; Neomycin phosphotransferase II (npt II); Nicotiana tabacum ; PCR assay

Effects of C-Terminal Domain Truncation on Enzyme Properties of Serratia marcescens Chitinase C by Fu-Pang Lin; Chun-Yi Wu; Hung-Nien Chen; Hui-Ju Lin (3617-3627).
A chitinase gene (SmChiC) and its two C-terminal truncated mutants, SmChiCG426 and SmChiCG330 of Serratia marcescens, were constructed and cloned by employing specific polymerase chain reaction (PCR) techniques. SmChiCG426 is derived from SmChiC molecule without its C-terminal chitin-binding domain (ChBD) while SmChiCG330 is truncated from SmChiC by its C-terminal deletion of both ChBD and fibronectin type III domain (FnIII). To study the role of the C-terminal domains of SmChiC on the enzyme properties, SmChiC, SmChiCG426, and SmChiCG330 were expressed in Escherichia coli by using the pET-20b(+) expression system. The His-tag affinity-purified SmChiC, SmChiCG426, and SmChiCG330 enzymes had a calculated molecular mass of 51, 46, and 36 kDa, respectively. Certain biochemical characterizations indicated that the enzymes had similar physicochemical properties, such as the optimum pH (5), temperature (37 °C), thermostability (50 °C), and identical hydrolyzing product (chitobiose) from both the soluble and insoluble chitin substrates. The overall catalytic efficiency k cat /K M was higher for both truncated enzymes toward the insoluble α-chitin, whereas the binding abilities toward the insoluble α-chitin substrate were reduced moderately. The fluorescence and circular dichroism (CD) spectroscopy data suggested that both mutants retained a similar folding conformation as that of the full-length SmChiC enzyme. However, a CD-monitored melting study showed that the SmChiCG330 had no obvious transition temperature, unlike the SmChiC and SmChiCG426.
Keywords: Serratia marcescens ; Chitinase C; C-terminal truncation; Circular dichroism

Xylitol Bioproduction in Hemicellulosic Hydrolysate Obtained from Sorghum Forage Biomass by Danielle Camargo; Luciane Sene; Daniela Inês Loreto Saraiva Variz; Maria das Graças de Almeida Felipe (3628-3642).
This study evaluated the biotechnological production of xylitol from sorghum forage biomass. The yeast Candida guilliermondii was cultivated in hemicellulosic hydrolysates obtained from biomass of three sorghum varieties (A, B, and C). First, the biomass was chemically characterized and subjected to dilute acid hydrolysis to obtain the hemicellulosic hydrolysates which were vacuum-concentrated and detoxified with activated charcoal. The hemicellulosic hydrolysates (initial pH 5.5) were supplemented with nutrients, and fermentations were conducted in 125-mL Erlenmeyer flasks containing 50 mL medium, under 200 rpm, at 30 °C for 96 h. Fermentations were evaluated by determining the parameters xylitol yield (Y P/S ) and productivity (QP), as well as the activities of the enzymes xylose reductase (XR) and xylitol dehydrogenase (XDH). There was no significant difference among the three varieties with respect to the contents of cellulose, hemicellulose, and lignin, although differences were found in the hydrolysate fermentability. Maximum xylitol yield and productivity values for variety A were 0.35 g/g and 0.16 g/L.h−1, respectively. It was coincident with XR (0.25 U/mg prot) and XDH (0.17 U/mg prot) maximum activities. Lower values were obtained for varieties B and C, which were 0.25 and 0.17 g/g for yield and 0.12 and 0.063 g/L.h-1 for productivity.
Keywords: Sorghum biomass; Hemicellulosic hydrolysate; Candida guilliermondii ; Xylitol

Proteomic Analysis of Mature Lagenaria siceraria Seed by Neha Kumari; Md. Tajmul; Savita Yadav (3643-3656).
Lagenaria siceraria (bottle gourd) class belongs to Magnoliopsida family curcurbitaceae that is a traditionally used medicinal plant. Fruit of this plant are widely used as a therapeutic vegetable in various diseases, all over the Asia and Africa. Various parts of this plant like fruit, seed, leaf and root are used as alternative medicine. In the present study, primarily, we have focused on proteomic analysis of L. siceraria seed using phenol extraction method for protein isolation. Twenty-four colloidal coomassie blue stained protein spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) after resolving on two-dimensional gel electrophoresis. Out of 24 identified protein spots, four were grouped as unidentified proteins which clearly suggest that less work has been done in the direction of plant seed proteomics. These proteins have been found to implicate in various functions such as biosynthesis of plant cell wall polysaccharides and glycoproteins, serine/threonine kinase activity, plant disease resistance and transferase activity against insects by means of insecticidal and larval growth inhibitory, anti-HIV, antihelmintic and antimicrobial properties. By Blast2GO annotation analysis, amongst the identified proteins of L. siceraria, molecular function for majority of proteins has indispensable role in catalytic activity, few in binding activity and antioxidant activity; it is mostly distributed in cell, organelle, membrane and macromolecular complex. Most of them involved in biological process such as metabolic process, cellular process, response to stimulus, single organism process, signalling, biological recognition, cellular component organization or biogenesis and localization.
Keywords: Lagenaria siceraria ; Proteomics; MALDI-TOF/MS; Blast2go; Annotation

Effect of Carbonyl Inhibitors and Their H2O2 Detoxification on Lactic Acid Fermentation by Jing Li; Caiqing Zhu; Maobing Tu; Pingping Han; Yonnie Wu (3657-3672).
Biomass degradation compounds significantly inhibit biochemical conversion of biomass prehydrolysates to biofuels and chemicals, such as lactic acid. To characterize the structure-activity relationship of carbonyl inhibition on lactic acid fermentation, we examined effects of eight carbonyl compounds (furfural, 5-hydroxymethyl furfural, vanillin, syringaldehyde, 4-hydroxybenzaldehyde, phthalaldehyde, benzoic acid, and pyrogallol aldehyde) and creosol on lactic acid production by Lactobacillus delbrueckii. Pyrogallol aldehyde reduced the cell growth rate by 35 % at 1.0 mM and inhibited lactic acid production completely at 2.0 mM. By correlating the molecular descriptors to the inhibition constants in lactic acid fermentation, we found a good relationship between the hydrophobicity (Log P) of aldehydes and their inhibition constants in fermentation. The inhibitory effect of carbonyl inhibitors appeared to correlate with their thiol reactivity as well. In addition, we found that H2O2 detoxified pyrogallol aldehyde and phthalaldehyde inhibitory activity. H2O2 detoxification was applied to real biomass prehydrolysates in lactic acid fermentation.
Keywords: Lactic acid fermentation; H2O2 detoxification; Carbonyl compounds; Inhibition; Lactobacillus delbrueckii

Increased Vulnerability to Physical Stress by Inactivation of NdgR in Streptomyces coelicolor by Bo-Rahm Lee; Da-Hye Yi; Eunjung Song; Shashi Kant Bhatia; Ju Hee Lee; Yun-Gon Kim; Sung-Hee Park; Yoo Kyung Lee; Byung-Gee Kim; Yung-Hun Yang (3673-3682).
The antibiotic production and spore formation process in Streptomyces coelicolor need complex decision making processes by several regulatory units. These regulatory units are involved in both primary and secondary metabolism. As a result, most regulators have several functions, and those are worthwhile themes to study about different functions of a known regulator. In this study, a deletion mutant of ndgR, which encodes the nitrogen-dependent growth regulator, was examined by the cell viability test, TEM, and growth in N-acetylglucosamine/asparagine (GlcNAc/Asn) liquid medium. The results of the study show that NdgR is also involved in the structure of the cell membrane affecting survival under physical shocks. Deletion of ndgR leads to abnormal cell membrane resulting in the vulnerable cells to physical stress caused by shaking with beads in liquid culture condition. This empirical observation is the first meaningful explanation to why ndgR mutant could not grow well in a liquid minimal medium due to the defect of N-acetylglucosamine (GlcNAc) utilization and phospholipid synthesis.
Keywords: Streptomyces coelicolor ; ndgR ; Physical stress; N-acetylglucosamine; Cell membrane

The increase in drug resistance to current antifungal drugs brings enormous challenges to the management of Candida infection. Therefore, there is a continuous need for the discovery of new antimicrobial agents that are effective against Candida infections especially from natural source especially from medical plants. The present investigation describes the synergistic anticandidal activity of two asarones (∞ and β) purified from Acorus calamus in combination with three clinically used antifungal drugs (fluconazole, clotrimazole, and amphotericin B). The synergistic anticandidal activities of asarones and drugs were assessed using the checkerboard microdilution and time-kill assays. The results of the present study showed that the combined effects of asarones and drugs principally recorded substantial synergistic activity (fractional inhibitory concentration index (FICI) <0.5). Time-kill study by combination of the minimal inhibitory concentration (MIC) of asarones and drugs (1:1) recorded that the growth of the Candida species was significantly arrested between 0 and 2 h and almost completely attenuated between 2 and 6 h of treatment. These findings have potential implications in adjourning the development of resistance as the anticandidal activity is achieved with lower concentrations of asarones and drugs. The combination of asarones and drugs also significantly inhibit the biofilm formation by Candida species, and this would also help to fight against drug resistance because biofilms formed by Candida species are ubiquitous in nature and are characterized by their recalcitrance toward antimicrobial treatment. The in vitro synergistic activity of asarones and drugs against pathogenic Candida species is reported here for the first time.
Keywords: Asarones; Azoles; Candida species; Polyene; Synergistic

Actinobacteria is a prolific producer of complex natural products; we isolated a potential marine Streptomyces sp. PM49 strain from Bay of Bengal coastal area of India. The strain PM49 exhibited highly efficient antibacterial properties on multidrug-resistant pathogens with a zone of inhibition of 14–17 mm. SSF was adopted for the production of the secondary metabolites from PM49 with ISP2; utilizing agricultural wastes for compound extraction was also attempted. Bioactive fraction of Rf value 0.69 resolved using chloroform and ethyl acetate (1:1, v/v) was obtained and subjected to further analysis. Based on UV, IR, ESI-MS, and 1H and 13C NMR spectral analysis, it was revealed that the compound is closely similar to cyslabdan with a molecular mass of 467.66 corresponding to the molecular formula C25H41NO5S. ESBL and MBL production was screened in the hospital test isolates of Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, and Staphylococcus aureus. PCR amplification in the phenotypically positive strains was positive for bla IMP, bla SHV, bla CTX-M, and mec genes. The β-lactamase enzyme from tested strains had cephalosporinase activity with a 31-kDa protein and isolated compound from the strain possessing β-lactamase inhibitory potential. MIC of the active fraction was 16–32 μg/ml on ATCC strains; the ceftazidime and meropenem sensitive and resistant test strains showed MIC of 64–256 μg/ml. The Streptomyces sp. PM49 aerial mycelium was rectiflexibile; the 16S rRNA showed 99 % identity with Streptomyces rochei and submitted at Genbank with accession no JX904061.1.
Keywords: Streptomyces sp; Multidrug-resistant pathogens; β-Lactamase inhibition; Cyslabdan-like compound

Enhanced Biological Straw Saccharification Through Coculturing of Lignocellulose-Degrading Microorganisms by Mohamed Taha; Esmaeil Shahsavari; Khalid Al-Hothaly; Aidyn Mouradov; Andrew T. Smith; Andrew S. Ball; Eric M. Adetutu (3709-3728).
Lignocellulosic waste (LCW) is an abundant, low-cost, and inedible substrate for the induction of lignocellulolytic enzymes for cellulosic bioethanol production using an efficient, environmentally friendly, and economical biological approach. In this study, 30 different lignocellulose-degrading bacterial and 18 fungal isolates were quantitatively screened individually for the saccharification of four different ball-milled straw substrates: wheat, rice, sugarcane, and pea straw. Rice and sugarcane straws which had similar Fourier transform-infrared spectroscopy profiles were more degradable, and resulted in more hydrolytic enzyme production than wheat and pea straws. Crude enzyme produced on native straws performed better than those on artificial substrates (such as cellulose and xylan). Four fungal and five bacterial isolates were selected (based on their high strawase activities) for constructing dual and triple microbial combinations to investigate microbial synergistic effects on saccharification. Combinations such as FUNG16-FUNG17 (Neosartorya fischeri–Myceliophthora thermophila) and RMIT10-RMIT11 (Aeromonas hydrophilaPseudomonas poae) enhanced saccharification (3- and 6.6-folds, respectively) compared with their monocultures indicating the beneficial effects of synergism between those isolates. Dual isolate combinations were more efficient at straw saccharification than triple combinations in both bacterial and fungal assays. Overall, co-culturing can result in significant increases in saccharification which may offer significant commercial potential for the use of microbial consortia.
Keywords: Lignocellulosic straws; Microbial consortia; Saccharification; Cellulolytic–xylanolytic enzymes

We constructed and applied a recombinant, permeabilized Escherichia coli strain for the multistep synthesis of UDP-glucose. Sucrose phosphorylase (E.C. of Leuconostoc mesenteroides was over expressed and the pgm gene encoding for phosphoglucomutase (E.C. was deleted in E. coli to yield the E. coli JW 0675-1 SP strain. The cells were permeabilized with the detergent Triton X-100 at 0.05 % v/v. The synthesis of UDP-glucose with permeabilized cells was then optimized with regard to pH, cell density during the synthesis and growth phase during cell harvest, metal cofactor, other media components, and temperature. In one configuration sucrose, phosphate, UMP, and ATP were used as substrates. At pH 7.8, 27 mg/ml cell dry weight, cell harvest during the early stationary phase of growth and Mn2+ as cofactor a yield of 37 % with respect to UMP was achieved at 33 °C. In a second step, ATP was regenerated by feeding glucose and using only catalytic amounts of ATP and NAD+. A UDP-glucose yield of 60 % with respect to UMP was obtained using this setup. With the same setup but without addition of external ATP, the yield was 54 %.
Keywords: Permeabilization; Permeabilized cells; E. coli ; UDP-glucose; Sucrose phosphorylase; Multi-enzyme catalyst; Triton X-100; Manganese; ATP regeneration

Among all VEGF-A isoforms, VEGF-111 is particularly important in molecular biology research owing to its potent angiogenic properties and its remarkable resistance to proteolysis. These features make it a potential candidate for therapeutic use in ischemic diseases. VEGF-111 is not expressed in normal cells, but expression is induced by UV-B irradiation and exposure to genotoxic agents. Here, to increase expression at the transcriptional and translational levels, we synthesized and cloned recombinant VEGF-111 cDNA. Two fragments encoding exons 1–4 and intron 4/5 plus exon 8a were amplified and cloned into the pBud.CE4.1 vector using a class IIs restriction enzyme-based method. The expression of VEGF-111 in CHO-dhfr − and HEK 293 cell lines was evaluated with real-time PCR, dot blotting, and ELISA. VEGF expression was increased about 10- and 18-fold in transfected CHO-dhfr − and HEK 293 cells, respectively. Dot blotting and ELISA confirmed successful production of VEGF-111 in both cell lines.
Keywords: VEGF-111; Real-time PCR; Dot blotting; ELISA; Intron 4/5

Isolation and Purification of Trypsin Inhibitors from the Seeds of Abelmoschus moschatus L. by Muni Kumar Dokka; Lavanya Seva; Siva Prasad Davuluri (3750-3762).
Four trypsin inhibitors, AMTI-I, AMTI-II, AMTI-III, and AMTI-IV, have been isolated and purified to homogeneity from the seeds of Abelmoschus moschatus following ammonium sulphate fractionation, DEAE-cellulose ion exchange chromatography and gel permeation on Sephadex G-100, and their molecular weights were determined to be 22.4, 21.2, 20.8 and 20.2 kDa respectively by SDS-PAGE. While all the four inhibitors were very active against bovine trypsin, two of them (AMTI-III and AMTI-IV) showed moderate activity towards bovine chymotrypsin. AMTI-I and AMTI-II were found to be glycoproteins with neutral sugar content of 2.8 and 4 %, respectively, and all the four inhibitors were devoid of free sulphhydryl groups. The inhibitors were quite stable up to 80 °C for 10 min and were not affected at alkaline as well as acidic conditions tested. Treating them with 8 M urea and 1 % SDS for 24 h at room temperature did not result in any loss of their antitryptic activities. However, they lost considerable antitryptic activity when treated with 6 M guanidine hydrochloride. Activities of the inhibitors were unaffected even after their reduction with DTT suggesting that disulphide bonds are not needed for their inhibitory activities.
Keywords: Abelmoschus moschatus ; Antitryptic; Trypsin inhibitors

Biological removal of hydrogen sulfide in biogas is an increasingly adopted alternative to the conventional physicochemical processes, because of its economic and environmental benefits. In this study, a microaerobic biofiltration system packed with polypropylene carrier was used to investigate the removal of high concentrations of H2S contained in biogas from an anaerobic digester. The results show that H2S in biogas was removed completely under different inlet concentrations of H2S from 2065 ± 234 to 7818 ± 131 ppmv, and the elimination capacity of H2S in the filter achieved about 122 g H2S/m3/h. It was observed that the content of CH4 in biogas increased after the biogas biodesulfurization process, which was beneficial for the further utilization of biogas. The elemental sulfur and sulfate were the main sulfur species of H2S degradation, and elemental sulfur was dominant (about 80 %) under high inlet H2S concentration. The results of terminal restriction fragment length polymorphism (T-RFLP) and fluorescence in situ hybridization (FISH) show that the population of sulfide-oxidizing bacteria (SOB) species in the filter changed with different concentrations of H2S. The microaerobic biofiltration system allows the potential use of biogas and the recovery of elemental sulfur resource simultaneously.
Keywords: Hydrogen sulfide; Biofiltration; Microaerobic; Biogas; Desulfurization

We report a fully automated DNA purification platform with a micropored membrane in the channel utilizing centrifugal microfluidics on a lab-on-a-disc (LOD). The microfluidic flow in the LOD, into which the reagents are injected for DNA purification, is controlled by a single motor and laser burst valve. The sample and reagents pass successively through the micropored membrane in the channel when each laser burst valve is opened. The Coriolis effect is used by rotating the LOD bi-directionally to increase the purity of the DNA, thereby preventing the mixing of the waste and elution solutions. The total process from the lysed sample injection into the LOD to obtaining the purified DNA was finished within 7 min with only one manual step. The experimental result for Salmonella shows that the proposed microfluidic platform is comparable to the existing devices in terms of the purity and yield of DNA.
Keywords: Lab-on-a-disc; DNA purification; Coriolis effect; Centrifugal microfluidics

Saccharification of β-Chitin From Squid Pen by a Fermentation Method Using Recombinant Chitinase-Secreting Escherichia coli by Takamitsu Minamoto; Narumi Takahashi; Sayaka Kitahara; Yuko Shinozaki; Takako Hirano; Wataru Hakamata; Toshiyuki Nishio (3788-3799).
Two strains [BL21(DE3) and HMS174(DE3)] of Escherichia coli harboring the recombinant chitinase expression plasmid pVP-Chi, which contains Vibrio parahaemolyticus chitinase gene with an attached signal sequence, were prepared. These E. coli transformants produced a large amount of recombinant chitinase, which hydrolyzes chitin to yield di-N-acetylchitobiose (GlcNAc)2, under the presence of isopropyl-1-thio-β-D-galactopyranoside (IPTG), and secreted the enzyme into their culture fluid with the aid of the signal peptide. Cultivation of these E. coli transformants in Luria-Bertani medium containing squid pen β-chitin and IPTG gave rise to the decomposition of this polysaccharide and the accumulation of (GlcNAc)2 in the culture fluid. Through these experiments, we confirmed that the use of strain HMS174(DE3) was preferable for the stable accumulation of (GlcNAc)2 in the culture fluid during cultivation owing to lower (GlcNAc)2 assimilation compared to BL21(DE3). Next, using E. coli HMS174(DE3) transformants, we conducted saccharification of different forms (fluffy fiber, flake, and powder) of β-chitin samples prepared from squid pens in Bacterion-N-KS(B)K medium containing 2 % of each sample under the presence of IPTG. In these experiments, (GlcNAc)2 was isolated with a more than 20 % stoichiometric yield from each culture supernatant through charcoal column chromatography followed by recrystallization.
Keywords: Squid pen β-chitin; Saccharification; Recombinant chitinase; Escherichia coli ; Fermentation method

Manganese Peroxidases from Ganoderma applanatum Degrade β-Carotene Under Alkaline Conditions by Isabel Lanfermann; Diana Linke; Manfred Nimtz; Ralf G. Berger (3800-3812).
A β-carotene-degrading enzyme activity was observed in liquid cultures of the basidiomycete Ganoderma applanatum. Supplementing the cultures with β-carotene induced the bleaching activity. Purification via hydrophobic interaction, ion exchange and size exclusion chromatography followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) resulted in a single protein band. LC-ion-trap-MS analyses and gene amplification identified two manganese peroxidase isoenzymes with 97.8 % identity on the amino acid level. These showed an estimated molecular mass of 48 kDa and an isoelectric point of 2.6. Properties not yet described for other manganese peroxidases were hydrogen-peroxide-independent catalysis and two maxima of the bleaching activity, a distinct one at pH 5 and a lower one at pH 8. During simulated washing studies, the applicability of the isoenzymes for the brightening of carotenoids under alkaline conditions was proven. The new enzymes may replace common bleaching agents to produce environmentally more compatible detergent formulations.
Keywords: Biocatalysis; Enzyme activity; Carotene degradation; Manganese peroxidase; Purification; Filamentous fungi; Ganoderma applanatum ; Washing; Alkaline activity

Engineering Tobacco to Remove Mercury from Polluted Soil by S. Chang; F. Wei; Y. Yang; A. Wang; Z. Jin; J. Li; Y. He; H. Shu (3813-3827).
Tobacco is an ideal plant for modification to remove mercury from soil. Although several transgenic tobacco strains have been developed, they either release elemental mercury directly into the air or are only capable of accumulating small quantities of mercury. In this study, we constructed two transgenic tobacco lines: Ntk-7 (a tobacco plant transformed with merT-merP-merB1-merB2-ppk) and Ntp-36 (tobacco transformed with merT-merP-merB1-merB2-pcs1). The genes merT, merP, merB1, and merB2 were obtained from the well-known mercury-resistant bacterium Pseudomonas K-62. Ppk is a gene that encodes polyphosphate kinase, a key enzyme for synthesizing polyphosphate in Enterobacter aerogenes. Pcs1 is a tobacco gene that encodes phytochelatin synthase, which is the key enzyme for phytochelatin synthesis. The genes were linked with LP4/2A, a sequence that encodes a well-known linker peptide. The results demonstrate that all foreign genes can be abundantly expressed. The mercury resistance of Ntk-7 and Ntp-36 was much higher than that of the wild type whether tested with organic mercury or with mercuric ions. The transformed plants can accumulate significantly more mercury than the wild type, and Ntp-36 can accumulate more mercury from soil than Ntk-7. In mercury-polluted soil, the mercury content in Ntp-36’s root can reach up to 251 μg/g. This is the first report to indicate that engineered tobacco can not only accumulate mercury from soil but also retain this mercury within the plant. Ntp-36 has good prospects for application in bioremediation for mercury pollution.
Keywords: Phytoremediation; Mercury; Vacuole; Cytoplasm; Chelatin

Nephelium lappaceum L., commonly known as “rambutan,” is a typical tropical tree and is well known for its juicy and sweet fruit which has an exotic flavor. Chemical studies on rambutan have led to the identification of various components such as monoterpene lactones and volatile compounds. Here, a 22.5-kDa trypsin inhibitor (N . lappaceum trypsin inhibitor (NLTI)) was isolated from fresh rambutan seeds using liquid chromatographical techniques. NLTI reduced the proteolytic activities of both trypsin and α-chymotrypsin. Dithiothreitol reduced the trypsin inhibitory activity of NLTI at a concentration of 1 mM, indicating that an intact disulfide bond is essential to the activity. NLTI inhibited HIV-1 reverse transcriptase with an IC50 of 0.73 μM. In addition, NLTI manifested a time- and dose-dependent inhibitory effect on growth in many tumor cells. NLTI is one of the few trypsin inhibitors with nitric oxide-inducing activity and may find application in tumor therapy.
Keywords: Rambutan; Nephelium lappaceum L.; Nitric oxide; Protease inhibitor; Liquid chromatography; Antitumor

Evaluation of MutS and Mut+ Pichia pastoris Strains for Membrane-Bound Catechol-O-Methyltransferase Biosynthesis by A. Q. Pedro; D. Oppolzer; M. J. Bonifácio; C. J. Maia; J. A. Queiroz; L. A. Passarinha (3840-3855).
Catechol-O-methyltransferase (COMT, EC is an enzyme that catalyzes the methylation of catechol substrates, and while structural and functional studies of its membrane-bound isoform (MBCOMT) are still hampered by low recombinant production, Pichia pastoris has been described as an attractive host for the production of correctly folded and inserted membrane proteins. Hence, in this work, MBCOMT biosynthesis was developed using P. pastoris X33 and KM71H cells in shake flasks containing a semidefined medium with different methanol concentrations. Moreover, after P. pastoris glass beads lysis, biologically and immunologically active hMBCOMT was found mainly in the solubilized membrane fraction whose kinetic parameters were identical to its correspondent native enzyme. In addition, mixed feeds of methanol and glycerol or sorbitol were also employed, and its levels quantified using liquid chromatography coupled to refractive index detection. Overall, for the first time, two P. pastoris strains with opposite phenotypes were applied for MBCOMT biosynthesis under the control of the strongly methanol-inducible alcohol oxidase (AOX) promoter. Moreover, this eukaryotic system seems to be a promising approach to deliver MBCOMT in high quantities from fermentor cultures with a lower cost-benefit due to the cheaper cultivation media coupled with the higher titers tipically achieved in biorreactors, when compared with previously reported mammallian cell cultures.
Keywords: COMT; Pichia pastoris ; Membrane protein; Methanol feeding; Alcohol oxidase promoter; Fermentation

Development of PCR-Based Technique for Detection of Purity of Pashmina Fiber from Textile Materials by Rajiv Kumar; D. B. Shakyawar; P.K. Pareek; A. S. M. Raja; L. L. L. Prince; Satish Kumar; S. M. K. Naqvi (3856-3862).
Pashmina fiber is one of major specialty animal fiber in India. The quality of Pashmina obtained from Changthangi and Chegu goats in India is very good. Due to restricted availability and high prices, adulteration of natural prized fibers is becoming a common practice by the manufacturers. Sheep wool is a cheap substitute, which is usually used for adulteration and false declaration of Pashmina-based products. Presently, there is lack of cost-effective and readily available methodology to identify the adulteration of Pashmina products from other similar looking substitutes like sheep wool. Polymerase chain reaction (PCR)-based detection method can be used to identify origin of animal fiber. Extraction of quality DNA from dyed and processed animal fiber and textile materials is a limiting factor in the development of such detection methods. In the present study, quality DNA was extracted from textile materials, and PCR-based technique using mitochondrial gene (12S rRNA) specific primers was developed for detection of the Pashmina in textile blends. This technique has been used for detection of the adulteration of the Pashmina products with sheep wool. The technique can detect adulteration level up to 10 % of sheep/goat fibers in textile blends.
Keywords: Pashmina; Cottage-based industry; Fiber identification; PCR; Textile materials

Heavy Metal Removal from Multicomponent System by the Cyanobacterium Nostoc muscorum: Kinetics and Interaction Study by Arindam Sinha Roy; Jayeeta Hazarika; N. Arul Manikandan; Kannan Pakshirajan; Mayashree B. Syiem (3863-3874).
In this study, Nostoc muscorum, a native cyanobacterial species isolated from a coal mining site, was employed to remove Cu(II), Zn(II), Pb(II) and Cd(II) from aqueous solution containing these metals in the mixture. In this multicomponent study, carried out as per the statistically valid Plackett-Burman design of experiments, the results revealed a maximum removal of both Pb(II) (96.3 %) and Cu(II) (96.42 %) followed by Cd(II) (80.04 %) and Zn(II) (71.3 %) at the end of the 60-h culture period. Further, the removal of these metals was attributed to both passive biosorption and accumulation by the actively growing N. muscorum biomass. Besides, the specific removal rate of these metals by N. muscorum was negatively correlated to its specific growth rate. For a better understanding of the effect of these metals on each other’s removal by the cyanobacteria, the results were statistically analyzed in the form of analysis of variance (ANOVA) and Student’s t test. ANOVA of the metal bioremoval revealed that the main (individual) effect due to the metals was highly significant (P value <0.05) on each other’s removal. Student’s t test results revealed that both Zn(II) and Pb(II) strongly inhibited both Cu(II) removal (P value <0.01) and Cd(II) removal (P value <0.02). All these results not only demonstrated a very good potential of the cyanobacteria in the bioremoval of these metals but also the effect of individual metals on each other’s removal in the multicomponent system.
Keywords: Nostoc muscorum ; Multicomponent system; Heavy metals; Bioremoval; Design of experiments

Investigation, Expression, and Molecular Modeling of ORF2, a Metagenomic Lipolytic Enzyme by Rosmeriana Afnis Marioto Garcia; Mariana Rangel Pereira; Thaís Carvalho Maester; Eliana Gertrudes de Macedo Lemos (3875-3887).
One clone exhibiting lipolytic activity was selected among 30 positives from a metagenomic library of a microbe consortium specialized in petroleum hydrocarbon degradation. From this clone, a sublibrary was constructed and a metagenome contig was assembled and analyzed using the ORF Finder; thus, it was possible to identify a potential ORF that encodes a lipolytic enzyme, denoted ORF2. This ORF is composed of 1035-bp 345 amino acids and displayed 98 % identity with an alpha/beta hydrolase from Pseudomonas nitroreducens (accession number WP024765380.1). When analyzed against a metagenome database, ORF2 also showed 76 % of sequence identity with a hypothetical protein from a marine metagenome (accession number ECT55726.1). The ProtParam analyses indicated that the recombinant protein ORF2 has a molecular mass approximately 39 kDa, as expected from its amino acid sequence, and based on phylogenetic analysis and molecular modeling, it was possible to suggest that ORF2 is a new member from family V. This enzyme exhibits the catalytic triad and conserved motifs typical from this family, wherein the serine residue is located in the central position of the conserved motif GASMGG. The orf2 gene was cloned in the expression vector pET28a, and the recombinant protein was superexpressed in Escherichia coli BL21(DE3) cells. The lipolytic activity of protein bands presented in a SDS-PAGE gel was confirmed by zymogram analyses, indicating ORF2 activity. These discoveries raise the possibility of employing this protein in biotechnological applications, such as bioremediation.
Keywords: Esterase; Lipase; Metagenome; Tributyrin; Protein modeling

Cell Surface Display of Yarrowia lipolytica Lipase Lip2p Using the Cell Wall Protein YlPir1p, Its Characterization, and Application as a Whole-Cell Biocatalyst by Evgeniya Y. Yuzbasheva; Tigran V. Yuzbashev; Natalia I. Perkovskaya; Elizaveta B. Mostova; Tatiana V. Vybornaya; Aleksei V. Sukhozhenko; Ilya Y. Toropygin; Sergey P. Sineoky (3888-3900).
The Yarrowia lipolytica lipase Lip2p was displayed on the yeast cell surface via N-terminal fusion variant using cell wall protein YlPir1p. The hydrolytic activity of the lipase displayed on Y. lipolytica cells reached 11,900 U/g of dry weight. However, leakage of enzyme from the cell wall was observed. The calculated number of recombinant enzyme displayed on the cell surface corresponds to approximately 6 × 105 molecules per cell, which is close to the theoretical maximum (2 × 106 molecules/cell). Furthermore, the leaking enzyme was presented as three N-glycosylated proteins, one of which corresponds to the whole hybrid protein. Thus, we attribute the enzyme leakage to the limited space available on the cell surface. Nevertheless, the surface-displayed lipase exhibited greater stability to short-term and long-term temperature treatment than the native enzyme. Cell-bound lipase retained 74 % of its original activity at 60 °C for 5 min of incubation, and 83 % of original activity after incubation at 50 °C during 5 h. Cell-bound lipase had also higher stability in organic solvents and detergents. The developed whole-cell biocatalyst was used for recycling biodiesel synthesis. Two repeated cycles of methanolysis yielded 84.1 and 71.0 % methyl esters after 33- and 45-h reactions, respectively.
Keywords: Yarrowia lipolytica ; Cell surface display; Lipase Lip2p; Cell wall protein YlPir1p; Whole-cell biocatalyst; Biodiesel

Biogas by Semi-Continuous Anaerobic Digestion of Food Waste by Cunsheng Zhang; Haijia Su; Zhenbin Wang; Tianwei Tan; Peiyong Qin (3901-3914).
The semi-continuous anaerobic digestion of food waste was investigated in 1-L and 20-L continuously stirred tank reactors (CSTRs), to identify the optimum operation condition and the methane production of the semi-continuous anaerobic process. Results from a 1-L digester indicated that the optimum organic loading rate (OLR) for semi-continuous digestion is 8 g VS/L/day. The corresponding methane yield and chemical oxygen demand (COD) reduction were 385 mL/g VS and 80.2 %, respectively. Anaerobic digestion was inhibited at high OLRs (12 and 16 g VS/L/day), due to volatile fatty acid (VFA) accumulation. Results from a 20-L digester indicated that a higher methane yield of 423 mL/g VS was obtained at this larger scale. The analysis showed that the methane production at the optimum OLR fitted well with the determined kinetics equation. An obvious decrease on the methane content was observed at the initial of digestion. The increased metabolization of microbes and the activity decrease of methanogen caused by VFA accumulation explained the lower methane content at the initial of digestion.
Keywords: Semi-continuous; Anaerobic digestion; Food waste; Methane; Biogas; Kinetic equation

Cloning and Expression of the xynA1 Gene Encoding a Xylanase of the GH10 Group in Caulobacter crescentus by Luciana Graciano; Juliana Moço Corrêa; Fabíola Giovanna Nesello Vieira; Adilson Bosetto; Eduardo Alexandre Loth; Marina Kimiko Kadowaki; Rinaldo Ferreira Gandra; Rita de Cássia Garcia Simão (3915-3929).
Caulobacter crescentus (NA1000 strain) are aquatic bacteria that can live in environments of low nutritional quality and present numerous genes that encode enzymes involved in plant cell wall deconstruction, including five genes for β-xylosidases (xynB1xynB5) and three genes for xylanases (xynA1xynA3). The overall activity of xylanases in the presence of different agro-industrial residues was evaluated, and it was found that the residues from the processing of corn were the most efficient in inducing bacterial xylanases. The xynA1 gene (CCNA_02894) encoding a predicted xylanase of group 10 of glyco-hydrolases (GH10) that was efficiently overexpressed in Escherichia coli LMG194 using 0.02 % arabinose, after cloning into the vector pJet1.2blunt and subcloning into the expression vector pBAD/gIII, provided a fusion protein that contained carboxy-terminal His-tags, named XynA1. The characterization of pure XynA1 showed an enzymatic activity of 18.26 U mL−1 and a specific activity of 2.22 U mg−1 in the presence of xylan from beechwood as a substrate. XynA1 activity was inhibited by EDTA and metal ions such as Cu2+ and Mg2+. By contrast, β-mercaptoethanol, dithiothreitol (DTT), and Ca2+ induced recombinant enzyme activity. Kinetic data for XynA1 revealed K M and V max values of 3.77 mg mL−1 and 10.20 μM min−1, respectively. Finally, the enzyme presented an optimum pH of 6 and an optimum temperature of 50 °C. In addition, 80 % of the activity of XynA1 was maintained at 50 °C for 4 h of incubation, suggesting a thermal stability for the biotechnological processes. This work is the first study concerning the cloning, overexpression, and enzymatic characterization of C. crescentus xylanase.
Keywords: Xylanase; Caulobacter crescentus ; Agro-industrial residues; Cloning; Characterization