Applied Biochemistry and Biotechnology (v.171, #6)

β-Galactosidase-Catalyzed Synthesis of Galactosyl Chlorphenesin and Its Characterization by Sang-Eun Lee; Tae-Min Jo; Hyang-Yeol Lee; Jongsung Lee; Kyung-Hwan Jung (1299-1312).
We synthesized galactosyl chlorphenesin (CPN-G) using β-gal-containing Escherichia coli (E. coli) cells in which the conversion yield of chlorphenesin (CPN) to CPN-G reached about 64 % during 12 h. CPN-G was identified and characterized using high-performance liquid chromatography, liquid chromatography-mass spectrometry, Fourier transform-infrared spectrometry, and nuclear magnetic resonance analysis (1H and 13C). We verified that a galactose was covalently bound to a CPN alcohol group during CPN-G synthesis throughout these analyses. In particular, by the hydrolysis of CPN-G using β-gal, it was confirmed that a galactose was bound to CPN. The minimal inhibitory concentration (MIC) results showed that the CPN-G MICs were fairly similar to those of CPN. HACAT cell viability was significantly higher in CPN-G-treated cells than in CPN-treated cells at concentrations of 0.0–20.0 mM. Finally, we accomplished the synthesis of less toxic CPN-G, compared with CPN, using β-gal-containing E. coli cells as whole cells without changes in the MICs against microorganisms.
Keywords: Chlorphenesin; Galactosylation; β-Galactosidase; Escherichia coli ; HACAT cell

Sorption of Copper(II) and Silver(I) by Four Bacterial Exopolysaccharides by M. Deschatre; F. Ghillebaert; J. Guezennec; C. Simon Colin (1313-1327).
Metal remediation was studied by the sorption of analytical grade copper Cu(II) and silver Ag(I) by four exopolysaccharides (EPS) produced by marine bacteria. Colorimetric analysis showed that these EPS were composed of neutral sugars, uronic acids (>20 %), acetate, and sulfate (29 %). Metal sorption experiments were conducted in batch process. Results showed that the maximum sorption capacities calculated according to Langmuir model were 400 mg g−1 EPS (6.29 mmol g−1) and 333 mg g−1 EPS (3.09 mmol g−1) for Cu(II) and Ag(I), respectively. Optimum pH values of Ag(I) sorption were determined as 5.7. Experiment results also demonstrated the influence of initial silver concentration and EPS concentrations. Microanalyzing coupled with scanning electron microscopy demonstrated the presence of metal and morphological changes of the EPS by the sorption of metallic cations. The Fourier transform infrared spectroscopy analysis indicated possible functional groups (e.g., carboxyl, hydroxyl, and sulfate) of EPS involved in the metal sorption processes. These results showed that EPS from marine bacteria are very promising for copper and silver remediation. Further development in dynamic and continuous process at the industrial scale will be established next.
Keywords: Sorption; Bacterial exopolysaccharide; Copper and silver removal; Bioremediation

Strategies for Enhancing the Production of Penicillin G Acylase from Bacillus badius: Influence of Phenyl Acetic Acid Dosage by Karthikeyan Rajendran; Surianarayanan Mahadevan; Rajendhran Jeyaprakash; Gunasekaran Paramasamy; Asit Baran Mandal (1328-1338).
Bacillus badius isolated from soil has been identified as potential producer of penicillin G acylase (PGA). In the present study, batch experiments performed at optimized inoculum size, temperature, pH, and agitation yielded a maximum PGA of 9.5 U/ml in shake flask. The experiments conducted in bioreactor with different oxygen flow rates revealed that 0.66 vvm oxygen flow rate could be sufficient for the maximum PGA activity of 12.7 U/ml. From a detailed investigation on the strategies of the addition of phenyl acetic acid (PAA) for increasing the production of PGA, it was found that the controlled addition of 10 ml of 0.1 % (w/v) PAA once in every 2 h from 6th hour of growth showed the maximum PGA activity of 32 U/ml. Thus, our studies for the first time showed that at concentration above 0.1 % (w/v) PAA, the PGA production decreased. This selective condition paves the way for less costly bioprocess for the production of PGA.
Keywords: Bacillus badius (B. badius); Penicillin G Acylase (PGA); Phenyl acetic acid (PAA); Bioreactor; Optimization

Four kinds of worts with different nitrogen compositions were used to examine their effects on fermentation performance of brewer's yeast. The absorption pattern of peptides with different molecular weights (Mw) in yeast cells during wort fermentation was also investigated. Results showed that both the nitrogen composition and level had significant impacts on the yeast biomass accumulation, ethanol production, and free amino nitrogen and sugars consumption rates. Worts supplemented with wheat gluten hydrolysates increased 11.5% of the biomass, 5.9% of fermentability, and 0.6% of ethanol content and decreased 25.6% of residual sugar content during wort fermentation. Moreover, yeast cells assimilated peptides with various Mw differently during fermentation. Peptides with Mw below 1 kDa decreased quickly, and the rate of assimilation was more than 50% at the end of fermentation, while those with Mw above 10 kDa almost could not be assimilated by yeast. All these results further indicated that the level and composition of wort nitrogen had significant impacts on the growth and fermentation performances of brewer's yeast, and peptides with Mw below 1 kDa were one of preferred nitrogen sources for brewer's yeast.
Keywords: Nitrogen composition; Wheat gluten hydrolysate; Brewer's yeast; Fermentation performance; Absorption pattern

Batch cultivation of Azadirachta indica hairy roots was carried out in different liquid-phase bioreactor configurations (stirred-tank, bubble column, bubble column with polypropylene basket, and polyurethane foam disc as root supports) to investigate possible scale-up of the A. indica hairy root culture for in vitro production of the biopesticide azadirachtin. The hairy roots failed to grow in the conventional bioreactor designs (stirred tank and bubble column). However, modified bubble column reactor (with polyurethane foam as root support) configuration facilitated high-density culture of A. indica hairy roots with a biomass production of 9.2 g l−1dry weight and azadirachtin yield of 3.2 mg g−1 leading to a volumetric productivity of azadirachtin as 1.14 mg l−1 day−1. The antifeedant activity in the hairy roots was also evaluated by no choice feeding tests with known concentrations of the hairy root powder and its solvent extract separately on the desert locust Schistocerca gregaria. The hairy root powder and its solvent extract demonstrated a high level of antifeedant activity (with an antifeedant index of 97 % at a concentration of 2 % w/v and 83 % at a concentration of 0.05 % (w/v), respectively, in ethanol).
Keywords: Azadirachtin; Hairy roots; Azadirachta indica ; Bubble column reactor; Polyurethane foam; Antifeedant index

Application of Lipopeptide Biosurfactant Isolated from a Halophile: Bacillus tequilensis CH for Inhibition of Biofilm by Arun Kumar Pradhan; Nilotpala Pradhan; Gangotri Mall; Himadri Tanaya Panda; Lala Behari Sukla; Prasanna Kumar Panda; Barada Kanta Mishra (1362-1375).
Biosurfactants are amphiphilic molecules having hydrophobic and hydrophilic moieties produced by various microorganisms. These molecules trigger the reduction of surface tension or interfacial tension in liquids. A biosurfactant-producing halophile was isolated from Lake Chilika, a brackish water lake of Odisha, India (19°41′39″N, 85°18′24″E). The halophile was identified as Bacillus tequilensis CH by biochemical tests and 16S rRNA gene sequencing and assigned accession no. KC851857 by GenBank. The biosurfactant produced by B. tequilensis CH was partially characterized as a lipopeptide using thin-layer chromatography, Fourier transform infrared spectroscopy, and nuclear magnetic resonance techniques. The minimum effective concentration of a biosurfactant for inhibition of pathogenic biofilm (Escherichia coli and Streptococcus mutans) on hydrophilic and hydrophobic surfaces was found to be 50 μg ml−1. This finding has potential for a variety of applications.
Keywords: Halophiles; Bacillus tequilensis CH; Lipopeptides; Biosurfactants; Biofilm

Improvement of Strain Penicillium sp. EZ-ZH190 for Tannase Production by Induced Mutation by E. Zakipour-Molkabadi; Z. Hamidi-Esfahani; M. A. Sahari; M. H. Azizi (1376-1389).
In the search for an efficient producer of tannase, Penicillium sp. EZ-ZH190 was subjected to mutagenesis using heat treatment and strain EZ-ZH290 was isolated. The maximum tannase in this mutant strain was 4.32 U/mL with an incubation period of 84 h as compared to wild strain EZ-ZH190 where the incubation period was 96 h with a maximum enzyme activity of 4.33 U/mL. Also, the Penicillium sp. EZ-ZH290 tannase had a maximum activity at 40 °C and pH 5.5. Then, the spores of strain EZ-ZH290 were subjected to γ irradiation mutagenesis and strain EZ-ZH390 was isolated. Strain EZ-ZH390 exhibited higher tannase activity (7.66 U/mL) than the parent strain EZ-ZH290. It was also found that Penicillium sp. EZ-ZH390 tannase had an optimum activity at 35 °C and a broad pH profile with an optimum at pH 5.5. The tannase pH stability of Penicillium sp. EZ-ZH390 and its maximum production of tannase followed the same trend for five generations confirming the occurrence of stable mutant. This paper is shown that γ irradiation can mutate the Penicillium sp. leading to increase the tannase production.
Keywords: Penicillium sp. EZ-ZH190; Tannase; Mutagenesis; Heat treatment; γ irradiation

Recombinant human platelet derived growth factor BB (rhPDGF-BB) is clinically approved for treating diabetic neuropathic ulcers. Plant-based expression systems offer less expensive ways of producing recombinant drugs, which do not require purification for clinical use. From this perspective, rhPDGF-BB is an ideal candidate for expression in plants as it can be applied topically. Here, we report a proof of concept study, in which rhPDGF-BB was expressed in tobacco plants, and its biological activity was tested in vitro. The mature human platelet derived growth factor BB (hPDGF-BB) gene was codon-optimized for tobacco and fused with ER targeting and retention signals, 5′ and 3′ UTRs of arc5-1 gene along with CaMV 35S promoter, and then, transferred by Agrobacterium-mediated transformation. Gene and protein expression of hPDGF-BB were confirmed by PCR and immunoblot studies. Bioactivity of hPDGF-BB expressed protein was determined by in vitro assays such as proliferation and migration in NIH3T3 cells. Our data reveals that total soluble proteins containing hPDGF-BB from transgenic plants showed a 4.5-fold increase in fibroblast proliferation compared to non-transgenic plants. Furthermore, plant-made rhPDGF-BB induced chemotaxis of treated cells and promoted wound healing in vitro. These results clearly demonstrate that functionally active rhPDGF-BB protein can be produced in plants and might have therapeutic benefits.
Keywords: rhPDGF-BB; Diabetic foot ulcer; Tobacco; NIH3T3 cells; Chemotaxis

An ultrasensitive fluorimetric biosensor for the detection of chemical warfare agent sulfur mustard (SM) was developed using its monofunctional analogue. SM is a vesicant and a potent chemical threat owing to its direct toxic effects on eyes, lungs, skin and DNA. This work investigates the quenching action of the analyte on chlorophyll fluorescence as elucidated by nuclear magnetic resonance, Fourier transform infrared spectroscopy and mass spectrometry studies suggesting the electrophilic attack of carbonium ion on nitrogens of the porphyrin moiety of chlorophyll. The properties of immobilisation matrix were optimised and scanning electron microscope observations confirmed improvement in pore size of sol–gels by addition of 32 % (v/v) glycerol, a feature enabling enhanced sensitivity towards the analyte. Chlorophyll embedded sol–gel was treated with increasing concentrations of monofunctional SM and the corresponding drop in maximum fluorescence intensity as measured by emission at 673 nm was observed, which varied linearly and had a detection limit of 7.68 × 10−16 M. The biosensor was found to be 6 orders of magnitude more sensitive than the glass microfibre-based disc biosensor previously reported by us.
Keywords: Biosensor; Fluorescence; Sulfur mustard; Sol–gel; Ultrasensitive

Enhanced Hydrolysis and Acidification of Waste Activated Sludge by Biosurfactant Rhamnolipid by Xin Yi; Kun Luo; Qi Yang; Xiao-Ming Li; Wei-Guang Deng; Hong-Bo Cheng; Zi-long Wang; Guang-Ming Zeng (1416-1428).
The effect of biosurfactant rhamnolipid (RL) on hydrolysis and acidification of waste activated sludge (WAS) was investigated. The results indicated that RL could greatly reduce the surface tension of sludge, resulting in stimulating the hydrolysis rate of WAS and enhancing the production of short-chain fatty acids (SCFAs). With the increase of RL dosage from 0.2 to 0.5 g/g DS, the maximum soluble chemical oxygen demand (SCOD), protein and carbohydrate concentration increased correspondingly. After 6 h of hydrolysis, SCOD, protein and carbohydrate concentration increased from 371.9, 93.3 and 9.0 mg/l to 3,994.5, 800.0 and 401.4 mg/l at RL 0.3 g/g DS, respectively. Furthermore, the release of NH4 +-N, PO4 3−-P and the accumulation of SCFAs also improved in the presence of RL. The maximum SCFAs was 1,829.9 mg COD/l at RL 0.3 g/g DS, while it was only 377.7 mg COD/l for the blank test. The propionic acid and acetic acid were the mainly SCFAs produced, accounting for 50–60% of total SCFAs.
Keywords: Anaerobic processes; Batch processing; Fermentation; Rhamnolipid; Surface tension; Waste treatment

Studies on lipase production and characterization were carried out with a bacterial strain Staphylococcus arlettae JPBW-1 isolated from rock salt mine, Darang, HP, India. Higher lipase activity has been obtained using 10 % inoculum with 5 % of soybean oil as carbon source utilizing a pH 8.0 in 3 h at 35 °C and 100 rpm through submerged fermentation. Partially purified S. arlettae lipase has been found to be active over a broad range of temperature (30–90 °C), pH (7.0–12.0) and NaCl concentration (0–20 %). It has shown extreme stability with solvents such as benzene, xylene, n-hexane, methanol, ethanol and toluene up to 30 % (v/v). The lipase activity has been found to be inhibited by metal ions of K+, Co2+ and Fe 2+ and stimulated by Mn2+, Ca2+ and Hg2+. Lipase activity has been diminished with denaturants, but enhanced effect has been observed with surfactants, such as Tween 80, Tween 40 and chelator EDTA. The K m and V max values were found to be 7.05 mM and 2.67 mmol/min, respectively. Thus, the lipase from S. arlettae may have considerable potential for industrial application from the perspectives of its tolerance towards industrial extreme conditions of pH, temperature, salt and solvent.
Keywords: Production; Characterization; Staphylococcus arlettae lipase; Submerged fermentation

A Novel Polysaccharide from Lentinus edodes Mycelia Exhibits Potential Antitumor Activity on Laryngeal Squamous Cancer Cell Line Hep-2 by Xiangyu Cao; Ruihua Liu; Jianli Liu; Yapeng Huo; Wei Yang; Meng Zeng; Chao Yang (1444-1453).
A novel polysaccharide [Lentinus edodes mycelia polysaccharide 2 (LMP2)] with a molecular weight of 2.27 × 104 Da was isolated from the Lentinus edodes mycelia and purified by Sephadex G-200 and diethylaminoethyl-32 column chromatography. HPLC results indicated that LMP2 contained mannose, arabinose, galactose, xylose, and rhamnose with a relative molar ratio of 1:0.74:3.23:1.18:10.98. Its antitumor activity was evaluated in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation assay, and transwell assay. LMP2 showed a remarkable inhibitory effect on the proliferation of Hep-2 cells, and at the concentration of 200 mg/ml, the inhibition ratio was 37.2 % after 72 h. Furthermore, colony formation of Hep-2 cells was reduced significantly after treatment with LMP2. Invasion of Hep-2 cells was inhibited significantly by LMP2. These results suggested that LMP2 could be explored as a potential antitumor material for laryngeal carcinoma.
Keywords: Lentinus edodes mycelia; Polysaccharide; Purification; Antitumor; Laryngeal carcinoma

Human non-pancreatic secretory phospholipase A2 (hnpsPLA2) catalyzes the sn-2 acyl hydrolysis of phospholipids. It was reported that hnpsPLA2 is involved in various diseases like inflammation, cancer, and so on. This enzyme also exhibits anti-bacterial and anti-virus activities. It is active over a broad pH range, with higher activity at alkaline conditions. In order to make it suitable as a potential bactericide, high activity at neutral pH is preferable. We have tried to tailor the pH dependence of hnpsPLA2 activity by replacing its surface charged residues. Three surface charge replacements, Arg42Glu, Arg100Glu, and Glu89Lys, showed increased activities at neutral pH, which are 2.3, 2.8, and 2.3 times that of the wild-type enzyme at pH 7. Both the positive-to-negative and negative-to-positive mutations lowered the optimum enzymatic reaction pH of hnpsPLA2, indicating that the enzyme pH profile depends on a delicate balance of charged residues. The activity changes are in good agreement with the recently proposed calcium-coordinated catalytic triad mechanism. This study also provides a general means of enhancing hnpsPLA2 activity at low pH.
Keywords: Phospholipase A2 ; Protein engineering; Enzyme reaction optimum pH; Catalytic mechanism; The catalytic triad mechanism; The calcium-coordinated oxyanion mechanism

Effect of Phosphate on Glucosamine Production by Ethanolic Fungus Mucor indicus by Marzieh Mohammadi; Akram Zamani; Keikhosro Karimi (1465-1472).
In this study, the effect of phosphorous compound concentration on the production of glucosamine by Mucor indicus was investigated. Changes in the yield of ethanol, the major metabolite of the fungus, were also followed besides. The alkali insoluble material of the biomass of the fungus mainly contained phosphates and polymers of glucosamine and N-acetyl glucosamine, i.e., chitin and chitosan. Yields of glucosamine (78–113 g/kg dry fungal biomass) and ethanol (200–370 g/kg glucose) were significantly affected by the phosphorous concentration. The results showed that lower concentrations of phosphorous favored the production of glucosamine while higher ethanol as well as biomass yields was obtained at higher concentrations. The best concentration was 0.5 g/l where glucosamine yield was 0.37 g/l (11 % of the biomass). At this phosphate concentration, ethanol and biomass yields were 360 and 76 g/kg glucose, respectively. On average, proteins comprised 51.5 % of the biomass. Glycerol was the second important metabolite during the fermentation by the fungus which appeared at lower yields (20–34 g/kg glucose).
Keywords: Mucor indicus ; Glucosamine (GlcN); Ethanol; Phosphorous concentration

The present study explores the potential of extracellular fungal organophosphate (OP) hydrolase for the degradation of monocrotophos. Extracellular OP hydrolases were isolated and purified from five different fungal isolates viz. Aspergillus niger (M1), Aspergillus flavus (M2), Penicillium aculeatum (M3), Fusarium pallidoroseum (M4), and Macrophomina sp. (M5) by AmSO4 precipitation, dialysis, and G-100 chromatography. M3 showed highest percentage yield of 68.81 followed by 55.41 % for M1. Each of the purified enzyme fraction constituted of two different subunits of 33- and 67-kDa molecular weight. Optimum enzyme fraction (150 μg ml−1) rapidly degraded monocrotophos within 120 h in phosphorus-free liquid culture medium (CZM) with K deg of 0.0368, 0.0138, 0.048, 0.016, 0.0138, and 0.048 day−1 and half-life of 0.79, 2.11, 0.6, 1.8, and 2.11 days for M1, M2, M3, M4, and M5, respectively. The results were further confirmed by high performance thin layer chromatography and Fourier transform infrared which indicate the disappearance of monocrotophos by hydrolytic cleavage of vinyl phosphate bond. The overall order of enzymatic degradation was found to be P. aculeatum > A. niger > F. pallidoroseum > A. flavus = Macrophomina sp. Hence, the study concludes that extracellular OP hydrolases efficiently degraded monocrotophos and could be used as a potential candidate for the detoxification of this neurotoxin pesticide.
Keywords: Degradation; Fungi; Monocrotophos; OP hydrolases

Using a methanotrophic consortium (that includes Methylosinus sporium NCIMB 11126, Methylosinus trichosporium OB3b, and Methylococcus capsulatus Bath) isolated from a landfill site, the potential for partial oxidation of methane into methanol through selective inhibition of methanol dehydrogenase (MDH) over soluble methane monooxygenase (sMMO) with some selected MDH inhibitors at varied concentration range, was evaluated in batch serum bottle and bioreactor experiments. Our result suggests that MDH activity could effectively be inhibited either at 40 mM of phosphate, 100 mM of NaCl, 40 mM of NH4Cl or 50 μM of EDTA with conversion ratios (moles of CH3OH produced per mole CH4 consumed) of 58, 80, 80, and 43 %, respectively. The difference between extent of inhibition in MDH activity and sMMO activity was significantly correlated (n = 6, p < 0.05) with resultant methane to methanol conversion ratio. In bioreactor study with 100 mM of NaCl, a maximum specific methanol production rate of 9 μmol/mg h was detected. A further insight with qPCR analysis of MDH and sMMO coding genes revealed that the gene copy number continued to increase along with biomass during reactor operation irrespective of presence or absence of inhibitor, and differential inhibition among two enzymes was rather the key for methanol production.
Keywords: Methane oxidation; Methanol synthesis; Methanol dehydrogenase (MDH); Soluble methane monooxygenase (sMMO); Microbial consortium

Green Synthesis and Characterization of Hybrid Collagen–Cellulose–Albumin Biofibers from Skin Waste by Manickam Amsaveni; Ayyappan Anumary; Meiyazhagan Ashokkumar; Bangaru Chandrasekaran; Palanisamy Thanikaivelan (1500-1512).
Collagen (C) and cellulose are prominent biopolymers from the animal and plant kingdom and widely used in bioengineering. Albumin, on the other hand, is the most abundant plasma protein present in mammalian blood. In this work, collagen extracted from animal skin waste was blended with hydroxyethyl cellulose (HEC) and bovine serum albumin (A) and wet-spun to form hybrid biodegradable C/HEC/A fibers. They were further cross-linked with glutaraldehyde vapors and analyzed. X-ray diffraction and infra-red spectroscopic studies of the hybrid fibers display peaks corresponding to collagen, cellulose, and albumin. Incorporation of cellulose into the biopolymeric matrix leads to a reasonable improvement in mechanical, swelling, and thermal properties of hybrid fibers. Addition of albumin improves the regularity of fiber surface without altering the porosity as observed under a microscope. Hence, the formed hybrid biofibers can be potentially used as a suture material as well as for different biomedical applications due to their improved properties.
Keywords: Biowaste; Plant waste; Blood; Fiber; Morphology; Trimmings

Response of Human Mesenchymal Stem Cells to Patterned and Randomly Oriented Poly(Vinyl Alcohol) Nano-fibrous Scaffolds Surface-Modified with Arg-Gly-Asp (RGD) Ligand by Yasaman Zamani; Mohammad Rabiee; Mohammad Ali Shokrgozar; Shahin Bonakdar; Mohammadreza Tahriri (1513-1524).
The aim of this study was to obtain a better insight of how nano-fibrous scaffolds can affect human mesenchymal stem cells responses. Therefore, in this study, using electrospinning technique, poly(vinyl alcohol) (PVA) nano-fibers with two different patterns were prepared. In the first structure, PVA nano-fibers were oriented randomly and in the second structure, nano-fibers were electrospun in such a way that a special pattern was obtained. In order to enhance their stability, scaffolds were cross-linked using glutaraldehyde vapor. RGD immobilization was used to improve cell adhesion properties of the scaffolds. SEM micrographs demonstrated that the cell adhesion was effectively enhanced after RGD immobilization and higher cell densities were observed on RGD-modified scaffolds. Randomly oriented nano-fibers showed better cell adhesion compared to patterned structure. Patterned structure also revealed slightly lower cell viability compared to random nano-fibers. Finally, it was assumed that randomly oriented nano-fibers provide a more favorable surface for cells.
Keywords: PVA; Electrospun nano-fibers; Surface; Pattern; RGD ligand; hMSCs response

Enhanced Lignin Biodegradation by a Laccase-Overexpressed White-Rot Fungus Polyporus brumalis in the Pretreatment of Wood Chips by Sun-Hwa Ryu; Myung-Kil Cho; Myungkil Kim; Sang-Min Jung; Jin-Ho Seo (1525-1534).
The laccase gene of Polyporus brumalis was genetically transformed to overexpress its laccase. The transformants exhibited increased laccase activity and effective decolorization of the dye Remazol Brilliant Blue R than the wild type. When the transformants were pretreated with wood chips from a red pine (softwood) and a tulip tree (hardwood) for 15 and 45 days, they showed higher lignin-degradation activity as well as higher wood-chip weight loss than the wild type. When the wood chips treated with the transformant were enzymatically saccharified, the highest sugar yields were found to be 32.5 % for the red pine wood and 29.5 % for the tulip tree wood, on the basis of the dried wood weights, which were 1.6-folds higher than those for the wild type. These results suggested that overexpression of the laccase gene from P. brumalis significantly contributed to the pretreatment of lignocellulose for increasing sugar yields.
Keywords: Lignin biodegradation; Laccase; Polyporus brumalis ; Pretreatment; White-rot fungi

Expression of Biologically Active Human Recombinant Interferon Alpha 2b in Human Breast Cancer Cell Line Bcap-37 by Hui Li; Xiangping Li; Qingyou Liu; Zhendan Shi; Deshun Shi (1535-1544).
Human interferon alpha 2b (IFNα-2b) is a pleiotropic cytokine used to treat various viral diseases and cancers. Conventionally, recombinant human IFNα-2b used in clinics was produced by prokaryotic expression system, which always lack of enough biological activity due to limitations on proper folding and post-translational modifications, so the eukaryotic expression system are becoming prevailing method for the production of recombinant proteins. In this study, human breast cancer cell Bcap-37 was firstly used as host for the expression of human IFNα-2b, with the expression vector pIRES2-IFN-EGFP, in which IFNα-2b gene is under the control of CMV promoter. The expression of recombinant IFNα-2b was detected by Western blot and ELISA. Results showed that the concentration of the secreted recombinant IFNα-2b in culture medium was 435.7 pg/mL/24 h. Biological activity of the recombinant IFNα-2b was assayed by detecting the expression of IFN-inducible genes, including MxA, OAS, PKR, and Caspase1 through QRT-PCR. Results demonstrated that recombinant IFNα-2b possess the biological activities. Compared to non-transgenic cells, the expression levels of the aforementioned four IFN-inducible genes were increased by 18.098-, 1.843-, 2.21-, and 3.066-folds, respectively. We got to a conclusion that the human breast cancer cell Bcap-37 could express bioactive recombinant IFNα-2b.
Keywords: Bcap-37; Biological active; IFN-inducible genes; Interferon alpha 2b