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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.169, #2)

Improved Thermostability of Lipase B from Candida antarctica by Directed Evolution and Display on Yeast Surface by Xiang-Qian Peng (pp. 351-358).

To expand the functionality of lipase B from Candida antarctica (CALB), we used the multi-site saturation mutagenesis methods to create CALB mutants with improved thermal stability. The times of residual activity of the mutants mCALB168 and mCALB7 were 14 and 6 times higher than that of the displayed WT-CALB at 60 °C. Amino acid substitutions at positions 218, 219, 220, and 221 in mCALB7 introduced two additional hydrogen bonds and altered part of the surface domain from 217 to 224. In mCALB168, we introduced mutations T57A/R168K, which formed three additional hydrogen bonds. The mutants displayed on yeast significantly increased the thermostability in an aqueous system at 60 °C. These results indicate that yeast surface display technology could dramatically improve the stability of CALB.

Keywords: Candida antarctica lipase B; Multi-site saturation mutagenesis; Thermostability; Yeast cell surface display

Formation Kinetics of Potential Fermentation Inhibitors in a Steam Explosion Process of Corn Straw by Yuzhen Zhang; Lan Wang; Hongzhang Chen (pp. 359-367).

The weak acids, furan derivatives, and phenolic compounds formed during lignocellulose pretreatment are potential inhibitors of subsequent enzymatic and microbial processes. In this work, the effects of the steam explosion process on the formation of weak acids, furan derivatives, and phenolic compounds were explored. The correlations of different steam explosion conditions and formation kinetics of degradation products showed that the formation of weak acids and furan derivatives was in the first-order reactions, which are expressed as $$ A imes {e^{{{{{-{E_a}}} left/ {RT } ight.}}}}cdot t $$ . The formation of weak acids and furan derivatives increases with pretreatment temperature and time. On the other hand, the formation of phenolic compounds showed typical characteristics of continuous reaction, expressed as $$ {{{{A_1} imes {e^{{{{{-{E_{a1 }}}} left/ {RT } ight.}}}}}} left/ {{{A_2} imes {e^{{{{{-{E_{a2 }}}} left/ {RT } ight.}}}}cdot t}} ight.} $$ . The formation was affected by the active energies in two stages, temperature and time, and thus existed at extreme value. This work revealed the formation rules of weak acids, furan derivatives, and phenolic compounds in a steam explosion process and provided theoretical guidelines for improving the process and limiting the production of certain inhibitors.

Keywords: Detoxification; Kinetics; Biomass; Bioconversion; Corn straw

DNA Degradation of Genetically Modified Cottonseed Meal During Feed Processing by Qingfeng Guan; Xiumin Wang; Da Teng; Yalin Yang; Jianhua Wang (pp. 368-379).

The effects of dry heating, wet heating, and extrusion on the degradation of DNA in cottonseed meal (CSM) were studied using polymerase chain reaction (PCR) and real-time PCR approach. Both the sad1 DNA, ranging between 128 and 883 bp in size, and the cry1Ab/c gene, ranging between 183 and 652 bp in size, were detectable in all dry-heated CSM and cottonseed. During wet heating, the sad1 gene (≥883 bp) and the cry1Ab/c (≥952 bp) gene were thoroughly degraded at 105 and 120 °C, respectively. Sizes from 128 to 530 bp for the sad1 gene and sizes from 183 to 652 bp for the cry1Ab/c gene were detected during extrusion at temperatures ranging from 75 to 135 °C. Fragments ≤883 bp for the sad1 gene and ≤952 bp for the cry1Ab/c gene were detected in all of the extruded samples with water content varying between 26 and 34 %. The copy number ratio of cry1Ab/c to sad1 in samples of Bt cottonseed meal decreased rapidly when the temperature increased during the heating process. In conclusion, feed processing markedly degrades the larger DNA fragments of sad1 and cry1Ab/c, with high temperature and water content being the main factors for that degradation.

Keywords: Cottonseed meal; Extrusion; DNA; Degradation; Feed processing

The Metabolic Advantage of Choline Lactate in Growth Media: An Experimental Analysis with Staphylococcus lentus by Sudharshan Sekar; Surianarayanan Mahadevan; Perinkulam Ravi Deepa; Bhuvanesh Kumar Shanmugam; B. V. N. Phani Kumar; Asit Baran Mandal (pp. 380-392).

The metabolic effectiveness of choline lactate in the growth media was investigated relative to conventional carbon source for growing Staphylococcus lentus, a bacterial strain commonly used in bioremediation of industrial effluents and xenobiotic detoxification. Bacterial growth thermodynamics was determined by biocalorimetry. ¹³C NMR and FTIR spectroscopic analyses traced the consumption of choline lactate at specific time intervals of bacterial growth. Under aerobic conditions, it is apparent that S. lentus initially metabolized lactate for its energy needs, while the choline cation of the ionic salt seemed to provide its C and N for biosynthetic intermediates for cell structure/function, in the growing bacterial colony. Urea accumulation after 40 h of bacterial growth was recorded. Possible metabolic trajectory of choline lactate consumed during S. lentus growth is suggested here. The theoretical estimation of heats of reaction for the proposed metabolic pathway (455 kJ/mol) was comparable with the experimentally obtained reaction enthalpy (435 kJ/mol), which further validated the proposed metabolic pathway. The biomass and energy profile of bacteria growth in choline media was found to be more favorable than in glucose media. The ionic liquid, choline lactate, offers a metabolically and energetically efficient carbon (and nitrogen) source for growing S. lentus.

Keywords: Choline lactate; Ionic liquid; Growth media; Metabolic pathway; ¹³C NMR; Biocalorimetry; S. lentus

Proteome-Based Profiling of Hypercellulase-Producing Strains Developed Through Interspecific Protoplast Fusion Between Aspergillus nidulans and Aspergillus tubingensis by Baljit Kaur; Manju Sharma; Rohit Soni; H. S. Oberoi; B. S. Chadha (pp. 393-407).

Thirty heterokaryons, formed by protoplast fusion of Aspergillus nidulans and Aspergillus tubingensis, were selected on the basis of their ability to grow on 2-deoxyglucose (0.2 %, w/v) and intermediate spore color. These heterokaryons were studied for cellulase production using shake flask and solid substrate cultures at 40 °C. Fusants 51 and 28 exhibited appreciably higher levels of endoglucanase, cellobiohydrolase, β-glucosidase, and FPase activities when compared with parental strains. Employing proteomic-based approaches, the differential expression of proteins in secretome of fusants and parental strains were analyzed using two-dimensional electrophoresis. The expression of some of the proteins in the fusants was found to be up/downregulated. The upregulated proteins in the fusant 51 were identified by liquid chromatography–mass spectroscopy as endoxylanase, endochitinase, β-glucosidase, as well as hypothetical proteins. The cellulases produced by fusants 28 and 51 showed improved saccharification of alkali treated rice straw when compared with the parental strains.

Keywords: Interspecific; Protoplast fusion; Heterokaryons; Cellulase hyper producers; Proteomics; Saccharification

Molecular and Biochemical Characterization in Rauvolfia tetraphylla Plantlets Grown from Synthetic Seeds Following In Vitro Cold Storage by Mohammad Faisal; Abdularhaman A. Alatar; Ahmad K. Hegazy (pp. 408-417).

Synseed technology is one of the most important applications of plant biotechnology for in vitro conservation and regeneration of medicinal and aromatic plants. In the present investigation, synseeds of Rauvolfia tetraphylla were produced using in vitro-proliferated shoots upon complexation of 3 % sodium alginate and 100 mM CaCl₂. The encapsulated buds were stored at 4, 8, 12, and 16 °C and high conversion was observed in synseeds stored at 4 °C for 4 weeks. The effect of different medium strength on in vitro conversion response of synseed was evaluated and the maximum conversion (80.6 %) into plantlets was recorded on half-strength woody plant medium supplemented with 7.5 μM 6-benzyladenine and 2.5 μM α-naphthalene acetic acid after 8 weeks of culture. Plantlets with well-developed shoot and roots were hardened and successfully transplanted in field condition. After 4 weeks of transfer to ex vitro conditions, the performance of synseed-derived plantlets was evaluated on the basis of some physiological and biochemical parameters and compared with the in vivo-grown plants. Short-term storage of synthetic seeds at low temperature had no negative impact on physiological and biochemical profile of the plants that survived the storage process. Furthermore, clonal fidelity of synseed-derived plantlets was also assessed and compared with mother plant using rapid amplified polymorphic DNA and inter-simple sequence repeats analysis. No changes in molecular profiles were found among the regenerated plantlets and comparable to mother plant, which confirm the genetic stability among clones. This synseed protocol could be useful for in vitro clonal multiplication, conservation, and short-term storage and exchange of germplasm of this antihypertensive drug-producing plant.

Keywords: Cold storage; Genetic stability; Germplasm storage; Plant regeneration; Synthetic seed

Bioconversion of Sodium Dodecyl Sulphate to Rhamnolipid by Pseudomonas aeruginosa: A Novel and Cost-Effective Production Strategy by Sharrel Rebello; Aju K. Asok; Sunil V. Joseph; Biljo V. Joseph; Leny Jose; Sathish Mundayoor; Jisha M.S. (pp. 418-430).

The utility of rhamnolipids in industry is currently limited due to the high constraints in its economic production. In this scenario, the novel utility of sodium dodecyl sulphate (SDS) as carbon source could serve as promising cost-effective strategy. Screening of effective SDS biodegraders led to the isolation of Pseudomonas aeruginosa S15 capable of concomitant SDS degradation and biosurfactant synthesis. SDS-based rhamnolipid production was proved on SDS minimal agar plates using cetyl trimethylammonium bromide–methylene blue method and optimised in SDS-based minimal salt (SBS) medium. SDS proved to be an ideal carbon source for rhamnolipid synthesis with a high substrate to product conversion rate yielding 6.9 g/l of rhamnolipids from 1 g/l SDS in 5 days. Fast atom bombardment mass spectroscopy analysis of the purified biosurfactant proved the presence of mono- and di-rhamnolipids, viz., Rha-C₁₀-C₁₀, Rha-C₁₀-C₁₂ and Rha-Rha-C₁₀-C₁₀ with surface active properties. The secreted rhamnolipids were not utilised by S15 as a carbon source, but it caused a dispersion of bacterial biofilms in SBS medium. To the best of our knowledge, this is the first report on bioconversion of synthetic detergent to biodetergent. This SDS-based novel methodology presents a more economised mode of rhamnolipid synthesis utilising SDS as sole carbon source.

Keywords: Biosurfactant; Bioremediation; Sodium dodecyl sulphate; Detergent; Biofilm

Recombinant OmpL1 Protein as a Diagnostic Antigen for the Detection of Canine Leptospirosis by M. Subathra; T M A Senthilkumar; P. Ramadass (pp. 431-437).

Microscopic agglutination test (MAT) is the standard method for the diagnosis of leptospirosis, which is laborious and the interpretation of the results is subjective. The present work describes the use of recombinant-based IgG ELISA for the serodiagnosis of leptospirosis. We used recombinant outer membrane protein OmpL1 as an antigen for conducting IgG enzyme-linked immunosorbent assay (ELISA). A total of 475 canine serum samples were subjected to IgG ELISA; 294 sera were positive to ELISA, while 283 were positive to MAT. All samples that were positive to MAT were positive to ELISA also, however, few samples which were negative to MAT were positive to ELISA, which suggested that recombinant-based IgG ELISA showed 100 % sensitivity when compared to MAT. Thus, this present study showed that recombinant OmpL1-based IgG ELISA appears to be a better alternative to MAT for the diagnosis of leptospirosis and rOmpL1 protein could be used as a potential diagnostic antigen in different assay formats for leptospirosis.

Keywords: Canine leptospirosis; Enzyme-linked immunosorbent assay; Microscopic agglutination test; Recombinant OmpL1

Identifying the Regulative Role of NF-κB Binding Sites Within Promoter Region of Human Matrix Metalloproteinase 9 (mmp-9) by TNF-α Induction by Hsi Tien Wu; Syu Sheng Sie; Tang Ching Kuan; Chih Sheng Lin (pp. 438-449).

Matrix metalloproteinase 9 (MMP-9), a member of MMP family, is involved in many physiological processes, including cardiovascular disease (CVD). Tumor necrosis factor-α (TNF-α) is considered a cytokine with pleiotropic biological capabilities and leads to the process of CVD when TNF-α is abnormally released and stimulates MMP-9 expression and activation. In this study, we investigated the molecular mechanism of TNF-α-regulated MMP-9 expression. The experimental results confirm that TNF-α could upregulate MMP-9 expression in heart myoblast H9c2 cells of rat. To evaluate the MMP-9 regulation at transcriptional level, a DNA fragment of 2.2 kb (−2168/+18) of human mmp-9 promoter region was cloned and constructed in a vector of luciferase reporter gene. The 2.2-kb sequences were identified as having three candidate nuclear factor-κ B (NF-κB) binding sites: NF-κB I (−1418/−1409), NF-κB II (−626/−617), and NF-κB III (−353/−345). A series of reporter vectors with the mutated NF-κB sites of mmp-9 promoter sequences were constructed and transfected into H9c2 cells. The results show that the NF-κB II binding site (−626/−617) within the promoter region of mmp-9 plays a key role in upregulation of mmp-9 expression by TNF-α induction. In addition, we also first identified that the NF-κB I, similar to c-Rel, might be one of the NF-κB families to regulate mmp-9 expression.

Keywords: Matrix metalloproteinases 9 (MMP-9); Nuclear factor-κ B (NF-κB); Tumor necrosis factor-α (TNF-α); Transcription factor; Cell line H9c2

MC8 Peptide-Mediated Her-2 Receptor Targeting Based on PEI-β-CyD as Gene Delivery Vector by Wei Deng; Hefang Xiao; Xiangfu Zeng; Yiping Hu (pp. 450-461).

A novel vector with high gene delivery efficiency and special cell targeting ability was developed using a good strategy that utilized low molecular weight polyethylenimine (PEI; molecular weight, 600 KDa [PEI600]) cross-linked to β-cyclodextrin (β-CyD) via a facile synthetic route. Human epidermal growth factor receptor 2 (Her-2) are highly expressed in a variety of human cancer cells and are potential targets for cancer therapy. MC8 peptides, which have been proven to combine especially with Her-2 on cell membranes were coupled to PEI-β-CyD using N-succinimidyl-3-(2-pyridyldithio) propionate as a linker. The ratios of PEI600, β-CyD, and peptide were calculated based on proton integral values obtained from the ¹H-NMR spectra of the resulting products. Electron microscope observations showed that MC8-PEI-β-CyD can efficiently condense plasmid DNA (pDNA) into nanoparticles of about 200 nm, and MTT assays suggested the decreased toxicity of the polymer. Experiments on gene delivery efficiency in vitro showed that MC8-PEI-β-CyD/pDNA polyplexes had significantly greater transgene activities than PEI-β-CyD/pDNA in the Skov3 and A549 cells, which positively expressed Her-2, whereas, no such effect was observed in the MCF-7 cells, which negatively expressed Her-2. Our current research indicated that the synthesized nonviral vector shows improved gene delivery efficiency and targeting specificity in Her-2 positive cells.

Keywords: Her-2; Nonviral vector; Gene delivery; Gene therapy

Enhancement of Long-Chain Fatty Acid Production in Escherichia coli by Coexpressing Genes, Including fabF, Involved in the Elongation Cycle of Fatty Acid Biosynthesis by Seunghan Lee; Sunhee Lee; Yeo Joon Yoon; Jinwon Lee (pp. 462-476).

The goal of this study was to increase the production of long-chain fatty acids and to change the composition of fatty acids through the overexpression of genes involved in the fatty acid synthase (FAS) pathway and utilizing characteristics of a specific gene, namely, fabF. The four genes, fabB, fabG, fabZ, and fabI, are Escherichia coli homologues and function in the elongation cycle of fatty acid biosynthesis. FabB (fabB), an activator of FAS, is a β-oxoacyl-ACP synthase, which catalyzes the addition of acyl-ACP to malonyl-ACP to generate β-oxoacyl-ACP. FabF (fabF) participates at the same step as FabB in the elongation cycle and is structurally and functionally similar to FabB. Hence, we attempted to see if FabF was an activator of FAS, like FabB, with the rationale that these two enzymes have striking similarities. FabF exhibits thermal regulation in that enzyme activity increases at lower temperatures. To confirm its role as an activator of FAS, fabF was overexpressed solely or with other genes in the elongation cycle through biochemical engineering. The fabF recombinants were cultured at different temperatures, resulting in increased total and unsaturated fatty acid accumulation in all the recombinants, compared to wild type, at lower temperatures.

Keywords: E. coli MG1655; Fatty acid biosynthesis; Long-chain fatty acid; Elongation cycle; fabF ; Thermal regulation; 3-Oxoacyl-[acyl-carrier-protein] synthase II

Computing and Interpreting Specific Production Rates in a Chemostat in Steady State According to the Luedeking-Piret model by Jacques Thierie (pp. 477-492).

The Luedeking–Piret model is an empirical relationship which is very widely used in cell cultures to evaluate specific production rates of some products (metabolites or others). It constitutes a very common method of calculation as much in fundamental as in applied research and especially for designing and optimizing industrial processes in very varied fields. However, this model appears to be frequently deficient and has to be greatly adapted, practically, one might say, for each individual case. Obviously, this is a very great drawback, requiring a great deal of time spent on it and one that greatly lessens the ‘universality’ of the model. This work reveals that it is possible to give the initial Luedeking–Piret model a much more general scope. The used method revealed metabolic switches that have never been suspected until now. Confirmation of the method would certainly give a precious general tool both to optimize production processes and to increase understanding of some physiological states of cells in chemostat.

Keywords: Metabolite production; Chemostat; Continuous cultures; Bioreactor design; Bioreactor optimisation

Wine Aroma Improvement Using a β-Glucosidase Preparation from Aureobasidium pullulans by Milla A. Baffi; Thaise Tobal; João Henrique Ghilardi Lago; Maurício Boscolo; Eleni Gomes; Roberto Da-Silva (pp. 493-501).

Microbial β-glucosidases have been used for the enhancement of wine aroma. Nevertheless, few enzymes are active in the conditions of winemaking. In this work, the production of a β-glucosidase by an Aureobasidium pullulans strain (Ap-β-gl) isolated from grape ecosystems was evaluated. The maximum enzymatic synthesis using submerged fermentation was after 96 h of growth in complex media containing 20 g/L of cellobiose as the sole carbon source. The crude enzyme (Ap-β-gl) showed optimal pH at 5.5 and two peaks of optimum temperature (at 45 and 70 °C). It showed a wide range of pH stability, stability at low temperatures, and tolerance to ethanol, showing suitable characteristics for winemaking conditions. The hydrolysis of glycosidic terpenes by Ap-β-gl was studied, and its ability to efficiently release free terpenols was demonstrated by gas chromatography/mass spectrometry. The enzymatic treatment notably increased the amount of monoterpenes, showing good prospects for its potential application for the development of aroma in wines.

Keywords: Yeast; Aureobasidium pullulans ; β-Glucosidase; Wine; Aroma

Il17A Autoantibody Induced by Recombinant Protein Ag85A-Il17A by Rong Jin; Sheng Guo; Liangxia Wu; Xiaoyong Fan; Hui Ma; Douglas B. Lowrie; Jianhua Zhang (pp. 502-510).

IL17A is a widely studied cytokine which plays an important role in autoimmune diseases as well as asthma. In this study, murine IL17a gene fragment was inserted into pET28a-Ag85A and it was expressed as a fusion protein named Ag85A-IL17A. Recombinant protein Ag85A-IL17A was expressed as an open reading frame of 1,287 bp, encoding a polypeptide of 429 amino acid residues with a predicted molecular mass of 50 kDa. It was expressed in Escherichia coli. BL21 and purified by metal affinity chromatography using a nickel–nitrilotriacetic acid column. Bioinformatics revealed that Ag85A-IL17A contained B cell epitopes of IL17A. Purified proteins Ag85A-IL17A and Ag85A were injected with adjuvants into mice then antibody responses in sera were measured by enzyme-linked immunosorbent assay and western blot assay. Experiments showed that purified protein Ag85-IL17A could bind to a standard IL17A antibody and could induce IL17A autoantibody in mice.

Keywords: IL17A; Ag85A; Cloning; Expression; Autoantibody; B cell epitope

Potential Probiotic Lactic Acid Bacteria of Human Origin Induce Antiproliferation of Colon Cancer Cells via Synergic Actions in Adhesion to Cancer Cells and Short-Chain Fatty Acid Bioproduction by Mongkol Thirabunyanon; Penrat Hongwittayakorn (pp. 511-525).

The activities and modes of probiotic action of lactic acid bacteria isolated from infant feces were investigated for alternative application in the prevention and biotherapy of colon cancer. From a total of 81 isolates of Gram-positive rod and cocci bacteria obtained from healthy infants, only 15 isolates had the probiotic criteria which included growth inhibition against eight food-borne pathogens, no blood hemolysis, and tolerance to gastrointestinal tract properties such as pH 2.5 and 0.3 % bile salt. Four probiotic bacteria showed antiproliferation of colon cancer cells with the use of MTT and Trypan blue exclusion assay at the rates of 17–35 %. Through comparison of probiotic 16S rRNA sequences, they were identified as Pediococcus pentosaceus FP3, Lactobacillus salivarius FP25, L. salivarius FP35, and Enterococcus faecium FP51. Finding the mechanism of proliferative inhibition of colon cancer cells in this study indicated synergic induction by probiotic bacteria directly adhered to these cancer cells and triggered the bioproduction of short-chain fatty acids, mainly butyric and propionic acids. This study suggested that the use of these probiotics may be suitable as an alternative bioprophylactic and biotherapeutic strategy for colon cancer.

Keywords: Caco-2; Colon cancer; Food-borne pathogens; Lactic acid bacteria; Probiotic; Short-chain fatty acids

Extraction and Characterization of Extracellular Polymeric Substances in Biofilm and Sludge via Completely Autotrophic Nitrogen Removal Over Nitrite System by You-Peng Chen; Chun Li; Jin-Song Guo; Fang Fang; Xu Gao; Peng Zhang; Shan Li (pp. 526-538).

Extracellular polymeric substances (EPS) were extracted from sludge and biofilm via the completely autotrophic nitrogen removal over nitrite (CANON) system. Tightly bound (TB)-EPS were extracted using four physical methods, namely, cationic exchange resin (CER), sonication, heating, and steaming. CER was the most effective and most suitable method for extraction among the four methods. Moreover, the ultraviolet–vis spectra of TB-EPS indicated that few cells were destroyed by the CER method. The major component contents of total EPS, proteins, carbohydrates, humic substances, and DNA in sludge were 60.77, 49.84, 21.63, and 9.01 mg/g volatile suspended solids (VSS) and 90.03, 29.01, 15.96, and 10.04 mg/g VSS in biofilm, respectively. The Fourier transform infrared (FT-IR) spectra results indicated differences in the EPS functional groups between biofilm and sludge. The results of the batch experiments showed that the biofilm activity was significantly higher than that of the sludge in the CANON system. Furthermore, biomass activity was probably influenced by the EPS composition and distribution in the sludge and biofilm.

Keywords: EPS; CANON; FT-IR; Biomass activity; Biofilm; Sludge

Biodegradation of Bisphenol A and Decolorization of Synthetic Dyes by Laccase from White-Rot Fungus, Trametes polyzona by Thanunchanok Chairin; Thitinard Nitheranont; Akira Watanabe; Yasuhiko Asada; Chartchai Khanongnuch; Saisamorn Lumyong (pp. 539-545).

Purified laccase from Trametes polyzona WR710-1 was used as biocatalyst for bisphenol A biodegradation and decolorization of synthetic dyes. Degradation of bisphenol A by laccase with or without redox mediator, 1-hydroxybenzotriazole (HBT) was studied. The quantitative analysis by HPLC showed that bisphenol A rapidly oxidized by laccase with HBT. Bisphenol A was completely removed within 3 h and 4-isopropenylphenol was found as the oxidative degradation product from bisphenol A when identified by GC-MS. All synthetic dyes used in this experiment, Bromophenol Blue, Remazol Brilliant Blue R, Methyl Orange, Relative Black 5, Congo Red, and Acridine Orange were decolorized by Trametes laccase and the percentage of decolorization increased when 2 mM HBT was added in the reaction mixture. This is the first report showing that laccase from T. polyzona is an affective enzyme having high potential for environmental detoxification, bisphenol A degradation and synthetic dye decolorization.

Keywords: Laccase; Tramete polyzona ; Bisphenol A degradation; Synthesis dyes decolorization

Hypolipidaemic Effects of Methanol Extract of Holoptelea integrifolia (Roxb.) Planchon Bark in Diet-Induced Obese Rats by Arun Koorappally Subash; Anu Augustine (pp. 546-553).

The leaf and bark paste of Holoptelea integrifolia is traditionally used for the treatment of obesity in Asian countries. However, no scientific studies have been undertaken to reveal the actual mechanism of action. The present study aimed to investigate the hypolipidaemic effect of H. integrifolia and its mechanism in diet-induced obese rat model. After 4 weeks of oral administration, blood samples were collected for the estimation of serum lipids, lecithin: cholesterolacyltransferase (LCAT) apolipoproteins (apo) and liver for HMG-CoA reductase (HMGR) assay. The faecal samples were also collected to estimate the faecal fat content. The H. integrifolia treatment markedly lowered body weight, serum lipids and apo B and increase high-density lipoprotein-cholesterol and apo A1 concentrations. In this study, HMGR activity was enormously reduced, which helps to reduce cholesterol biosynthesis and an increase in LCAT activity was also observed. The detailed faecal analysis showed a remarkable increase in faecal lipids, which indicates the ability to inhibit intestinal fat absorption. The methanol fraction of H. integrifolia on LC-MS and tandem mass spectrometry analysis shows the presence of a compound, 3-(7-ethoxy-4-methyl-2-oxo-2H-chromen-3-yl)propanoate (C1). The result showed that the significant hypolipidaemic effect of H. integrifolia may be linked to its ability to inhibit HMGR activity and block intestinal fat absorption.

Keywords: Holoptelea integrifolia ; Diet-induced obesity; HMG-CoA reductase; LCAT; Apolipoproteins

Xylitol Production by Genetically Engineered Trichoderma reesei Strains Using Barley Straw as Feedstock by Mehdi Dashtban; Greg Kepka; Bernhard Seiboth; Wensheng Qin (pp. 554-569).

Xylitol, a naturally occurring five-carbon sugar alcohol derived from d-xylose, is currently in high demand by industries. Trichoderma reesei, a prolific industrial cellulase and hemicellulase producing fungus, is able to selectively use d-xylose from hemicelluloses for xylitol production. The xylitol production by T. reesei can be enhanced by genetic engineering of blocking further xylitol metabolism in the d-xylose pathway. We have used two different T. reesei strains which are impaired in the further metabolism of xylitol including a single mutant in which the xylitol dehydrogenase gene was deleted (∆xdh1) and a double mutant where additionally l-arabinitol-4-dehydrogenase, an enzyme which can partially compensate for xylitol dehydrogenase function, was deleted (∆lad1∆xdh1). Barely straw was first pretreated using NaOH and Organosolv pretreatment methods. The highest xylitol production of 6.1 and 13.22 g/L was obtained using medium supplemented with 2 % Organosolv-pretreated barley straw and 2 % d-xylose by the ∆xdh1 and ∆lad1∆xdh1 strains, respectively.

Keywords: Agricultural residues; Trichoderma reesei ; Xylitol production

Comparative Study of Chromium Biosorption by Mesorhizobium amorphae Strain CCNWGS0123 in Single and Binary Mixtures by Pin Xie; Xiuli Hao; Osama Abdalla Mohamad; Jianqiang Liang; Gehong Wei (pp. 570-587).

The appearance of chromium in the aqueous effluent is a major concern for the modern industry. In this work, Mesorhizobium amorphae strain CCNWGS0123 was investigated as a biosorbent to remove chromium from aqueous solutions. The optimum pH for Cr(III) and Cr(VI) biosorption were 4 and 2, respectively. This isolate showed an experimental maximum Cr(III) adsorption capacity of 53.52 mg L⁻¹, while the result was 47.67 mg L⁻¹ for Cr(VI), with an initial 100 mg L⁻¹ Cr ions and 1.0 g L⁻¹ biomass. In terms of time equilibrium, Cr(III) ion was more readily adsorbed than Cr(VI) by this isolate. The biosorption data of both ions fit the Langmuir isotherm better than that of Freundlich model. Meanwhile, this organism exhibited a good capability to release Cr ions, with desorption efficiency of 70 % for Cr(III) and 76 % for Cr(VI). Fourier transform infrared spectroscopy analysis showed that –OH, –COO, –NH, amide I, and C=O were involved in Cr(III) and Cr(VI) binding. The biosorbent was further characterized by scanning electron microscopy and energy-dispersive X-ray spectrometry, which indicated an accumulation of chromium on the cellular level. In the binary mixtures, the removal ratio of total Cr and Cr(III) increased from pH 2 to 4. The highest removal ratio of the total Cr was observed in the 25/25 mg L⁻¹ mixture at pH 4. In addition, the removal efficiency of Cr(VI) was closely influenced by Cr(III) in the mixture, decreasing to 23.57 mg g⁻¹ in the 100/100 mg L⁻¹ mixture system, due to the competition of Cr(III). The potential usage of the chromium-resistant rhizobium for the remediation of chromium-contaminated effluents has been demonstrated based on the above results.

Keywords: Biosorption; Cr(VI); Cr(III); M. amorphae CCNWGS0123; FTIR; SEM; EDX

Establishment and Quality Assessment of Tissue Cultures in Glycyrrhiza uralensis Fisch. by Juan Wang; Wenyuan Gao; Liming Zhang; Luqi Huang (pp. 588-594).

In this study, seedling, callus, cell, and adventitious root of Glycyrrhiza uralensis Fisch. have been established. In order to find the best one for producing G. uralensis active constituents, triterpenoid saponins and flavonoids in native root and tissue cultures were determined, and the contents in different G. uralensis materials were analyzed using cluster analysis. The contents of triterpenoid saponins and glycyrrhizic acid in tissue cultures were much lower than that in native G. uralensis. The total flavonoids content we determined in adventitious root was 6.31 mg g⁻¹, which was close to that of native root (9.82 mg g⁻¹). Based on the cluster analysis, we found that G. uralensis cultures were not suitable for production of glycyrrhizic acid, while adventitious root had a greater capability of flavonoids production comparing to seedling, callus, and cell.

Keywords: Glycyrrhiza uralensis Fisch.; Tissue culture; Glycyrrhizic acid; Flavonoids; Cluster analysis

Pythium irregulare Fermentation to Produce Arachidonic Acid (ARA) and Eicosapentaenoic Acid (EPA) Using Soybean Processing Co-products as Substrates by JunYi Lio; Tong Wang (pp. 595-611).

Arachidonic acid (ARA) and eicosapentaenoic acid (EPA) were produced by Pythium irregulare fungus using soybean cotyledon fiber and soy skim, two co-products from soybean aqueous processing, as substrates in different fermentation systems. Parameters such as moisture content, substrate glucose addition, incubation time, and vegetable oil supplementation were found to be important in solid-state fermentation (SSF) of soybean fiber, which is to be used as animal feed with enriched long-chain polyunsaturated fatty acids (PUFA). Soybean fiber with 8 % (dwb) glucose supplementation for a 7-day SSF produced 1.3 mg of ARA and 1.6 mg of EPA in 1 g of dried substrate. When soy skim was used as substrate for submerged fermentation, total ARA yield of 125.7 mg/L and EPA yield of 92.4 mg/L were achieved with the supplementation of 7 % (w/v) soybean oil. This study demonstrates that the values of soybean fiber and soy skim co-products could be enhanced through the long-chain PUFA production by fermentation.

Keywords: Arachidonic acid; Eicosapentaenoic acid; Polyunsaturated fatty acids; Solid-state fermentation; Soybean cotyledon fiber; Soy skim; Submerged fermentation

Production, Characterization, and Application of an Organic Solvent-Tolerant Lipase Present in Active Inclusion Bodies by Suxia Li; Kang Lin; Huaiyu Pang; Yixin Wu; Jianhe Xu (pp. 612-623).

An organic solvent-tolerant lipase from Serratia marcescens ECU1010 (rSML) was overproduced in Escherichia coli in an insoluble form. High concentrations of both biomass (50 g cell wet weight/L culture broth) and inclusion bodies (10.5 g/L) were obtained by applying a high-cell-density cultivation procedure. Activity assays indicated that the enzymatic activity of rSML reached 600 U/L. After treatment with isopropyl ether for 12 h, the maximum lipase activity reached 6,000 U/L. Scanning electron microscopy and Fourier transform infrared microspectroscopy revealed the activation mechanism of rSML in the presence of organic solvents. rSML was stable in broad ranges of temperatures and pH values, as well as in a series of organic solvents. Besides, rSML showed the best enantioselectivity for the kinetic resolution of (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester. These features render the S. marcescens ECU1010 lipase attractive for biotechnological applications in the field of organic synthesis and pharmaceutical industry.

Keywords: Lipase; Active inclusion body; Organic solvent tolerance; (±)-MPGM; Serratia marcescens ECU1010

Effects of Phytohormones and Jasmonic Acid on Glucosinolate Content in Hairy Root Cultures of Sinapis alba and Brassica rapa by Anja Kastell; Iryna Smetanska; Christian Ulrichs; Zhenzhen Cai; Inga Mewis (pp. 624-635).

Although some study have established hairy root cultures from brassicaceous plants with glucosinolates (GS) as characteristic secondary metabolite, studies are missing which compare hairy roots with the corresponding mother plants. Therefore, two different plant species—Sinapis alba and Brassica rapa subsp. rapa pygmeae teltoviensis—were transformed with the Agrobacterium rhizogenes strain A4. Aliphatic and indolyl GS were present in B. rapa, exhibiting larger quantities in leaves than in roots. Aromatic p-hydroxybenzyl GS were found particularly in the leaves of S. alba. However, the proportion of indolyl GS increased suddenly in transformed hairy roots of S. alba and B. rapa. Cultivation with the phytohormone kinetin (0.5 mg L⁻¹) enhanced GS accumulation in B. rapa hairy roots, however not in S. alba, but 2,4-D (0.4 mg L⁻¹) induced de-differentiation of roots in both species and reduced GS levels. GS levels especially of 1-methoxyindol-3ylmethyl GS increased in hairy roots in response to JA, but root growth was inhibited. While 2 weeks of cultivation in 100 to 200 μM JA were determined at optimum for maximum GS yield in S. alba hairy root cultures, 4 weeks of cultivation in 50 to 100 μM JA was the optimum for B. rapa.

Keywords: Hairy root culture; Glucosinolates; Brassica rapa ; Sinapis alba ; Jasmonic acid; Kinetin; 2,4-D

Expression of Cholera Toxin B–Lumbrokinase Fusion Protein in Pichia pastoris—The Use of Transmucosal Carriers in the Delivery of Therapeutic Proteins to Protect Rats Against Thrombosis by Guan Chunfeng; Li Xiaozhou; Wang Gang; Ji Jing; Jin Chao; Tchouopou Lontchi Josine (pp. 636-650).

Cholera toxin B-subunit (CTB) has been widely used to facilitate antigen delivery by serving as an effective mucosal carrier molecule for the induction of oral tolerance. However, whether CTB can be used as a transmucosal carrier in the delivery of not only vaccines but also therapeutic proteins has not been widely studied. Thus, we investigate here the concept of receptor-mediated oral delivery of lumbrokinase (LK) proteins which is an important fibrinolytic enzyme derived from earthworm. CTB and LK, separated by a furin cleavage site, was expressed via Pichia pastoris. The activity and proper folding of recombinant protein in yeast were confirmed by Western blot analysis, fibrin plate assays, and G_M1-ganglioside ELISA. Following oral administration of recombinant protein, the thrombosis model of rats and mice revealed that the oral treatment of rCTB–LK has a more significant anti-thrombotic effect on animals compared with rLK. It is possible to conclude that CTB can successfully enhance its fusion protein LK to be absorbed. The use of CTB as a transmucosal carrier in the delivery of not only vaccines but also therapeutic proteins was supported.

Keywords: Lumbrokinase; Cholera toxin B-subunit; Transmucosal carriers; Thrombosis

Facile Production and Rapid Purification of Functional Recombinant Qβ Replicase Heterotetramer Complex by Karthikeyan Gunasekaran; Peter L. Bergquist; Anwar Sunna (pp. 651-659).

We describe an improved method for the production of recombinant Qβ replicase heterotetramer. The successful expression of the soluble Qβ RNA polymerase complex depends on the EF-Ts and EF-Tu subunits being co-expressed prior to β-subunit expression. Efficient co-expression requires two different inducible operons to co-ordinate the expression of the heterotrimer. The complete heterotetramer enzyme complex is achieved by production of the recombinant S1-subunit of Qβ replicase in a separate host. This approach represents a facile way for producing and purifying large amounts of soluble and active recombinant Qβ replicase tetramer without the necessity of a His-tag for purification.

Keywords: Qβ replicase heterotetramer; RNA-dependent RNA polymerase; RNA amplification; Isothermal amplification; Replicase purification

Decolorization of Synthetic Dyes by Crude and Purified Laccases from Coprinus comatus Grown Under Different Cultures: The Role of Major Isoenzyme in Dyes Decolorization by Man Jiang; Zhen Ten; Shaojun Ding (pp. 660-672).

Coprinus comatus laccase isoenzyme induction and its effect on decolorization were investigated. The C/N ratio, together with aromatic compounds and copper, significantly influenced laccase isoenzyme profile and enzyme activity. This fungus produced six laccase isoenzymes in high-nitrogen low-carbon cultures but much less in low-nitrogen high-carbon (LNHC) cultures. The highest laccase level (3.25 IU/ml), equivalent to a 12.6-fold increase compared with unsupplemented controls (0.257 IU/ml), was recorded after 13 days in LNHC cultures supplemented with 2.0 mM 2-toluidine. Decolorization of twelve synthetic dyes belonging to anthraquinone, azo, and triphenylmethane dyes, by crude laccases with different proportion of isoenzymes produced under selected culture conditions, illustrated that the LacA is the key isoenzyme contributed to dyes decolorization especially in the presence of 1-hydroxybenzotriazol, which was further confirmed by dyes decolorization with purified LacA in the same condition. The crude laccase only was able to decolorize over 90 % of Reactive Brilliant Blue K-3R, Reactive Dark Blue KR, and Malachite Green, and higher decolorization for broader spectrum of synthetic dyes was obtained in presence of redox mediator, suggesting that C. comatus had high potential to decolorize various synthetic dyes as well as the recalcitrant azo dyes.

Keywords: Decolorization; Coprinus comatus ; Crude laccase; Isoenzyme; Synthetic dye

Use of Different Extracts of Coffee Pulp for the Production of Bioethanol by Evandro Galvão Tavares Menezes; Juliana Ribeiro do Carmo; Aline Galvão Tavares Menezes; José Guilherme Lembi Ferreira Alves; Carlos José Pimenta; Fabiana Queiroz (pp. 673-687).

Coffee is one of the most important agricultural products in Brazil. More than 50 % of the coffee fruit is not used for the production of commercial green coffee and is therefore discarded, usually ending up in the environment. The goal of this work was to select an efficient process for obtaining coffee pulp extract and to evaluate the use of this extract in bioethanol production. The effects of heat treatment and trituration on the yield and composition of the extract were investigated by measuring the amounts of reducing sugars, starch, pectin, and phenolic compounds. The extraction process was most efficient at room temperature using grinding followed by pressing. Five different fermentation media were tested: sugarcane juice or molasses diluted with water or with coffee pulp extract and a medium with only coffee pulp extract. Batch fermentations were carried out at 30 °C for 24 h, and samples were taken to obtain measurements of the total reducing sugars, cell count, and ethanol concentration. The addition of coffee pulp extract did not influence the fermentation or yeast viability, and it can thus be mixed with sugarcane juice or molasses for the production of bioethanol, with a yield of approximately 70 g/L.

Keywords: Pulp coffee; Extraction; Fermentable sugars; Ethanolic fermentation; Saccharomyces cerevisiae

Short Hairpin RNA-Induced Myostatin Gene Silencing in Caprine Myoblast Cells In Vitro by Ajai K. Tripathi; Umed V. Ramani; Amrutlal K. Patel; Dharamshibhai N. Rank; Chaitanya G. Joshi (pp. 688-694).

Myostatin (MSTN) belongs to the transforming growth factor (TGF)-β superfamily and is a potent negative regulator of skeletal muscle development and growth. Dysfunction of MSTN gene either by natural mutation or induced through genetic manipulation (knockout or knockdown) has been reported to increase the remarkable muscle mass in mammalian species. RNA interference (RNAi) is the most promising method for inhibition of gene expression that can be utilized for MSTN gene knockdown by developing short hairpin RNA (shRNA) construct against it. We utilized three antisense RNA expressing vectors with six constructs to knockdown MSTN gene in in vitro caprine myoblast cell culture system. We observed that all six shRNA constructs were successful in MSTN silencing with efficiency ranging from 7 to 46 % by quantitative real-time PCR and up to 19 % by western blotting. The significant upregulation of interferon response gene OAS1 (5- to 11-fold) in cells transfected with shRNA constructs were indicative of induction of interferon response. This RNAi-based method of increasing muscle mass could provide an alternative strategy to gene knockout methods for improving the production traits and economic properties of livestock.

Keywords: Myostatin; RNAi; Caprine; Silencing; Myoblast

An Extremely Simple and Effective Colony PCR Procedure for Bacteria, Yeasts, and Microalgae by Heiko Packeiser; Chanyuen Lim; Balaji Balagurunathan; Jinchuan Wu; Hua Zhao (pp. 695-700).

An extremely simple and effective colony PCR procedure is established for both gram-negative and gram-positive bacteria, yeasts, and microalgae. Among the four lysis buffers examined, Y-PER is observed to be more effective than Tris/EDTA, 0.2 % SDS, and 10 mM EDTA in the extraction of PCR-quality genomic DNA from those microorganisms. Vortexing or pipetting agitation of the cells in Y-PER for 5–10 s was sufficient to release genomic DNA for all the test bacteria and yeasts, and most microalgae. Additional incubation at 98 °C for 5 min for further cell disruption was essential only for Chlorella vulgaris due to its notoriously rigid cell wall.

Keywords: Colony PCR; Bacteria; Yeasts; Microalgae

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.169, #2)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.169, #2)