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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.166, #2)

Kinetics of Ergothioneine Inhibition of Mushroom Tyrosinase by Wayne C. Liao; Wen Hong Wu; Pei-Chuan Tsai; Hui-Feng Wang; Yi-Hsin Liu; Chin-Feng Chan (pp. 259-267).

The native amino acid ergothioneine, a thiourea derivative of histidine, inhibits mushroom tyrosinase activity in a dose-dependent manner, with an IC₅₀ value of 1.025 mg/ml (4.47 mM). By contrast, histidine exhibited no inhibitory effect on mushroom tyrosinase activity. We characterized ergothioneine as a noncompetitive tyrosinase inhibitor using a Lineweaver–Burk plot of experimental kinetic data. The IC₅₀ value for ergothioneine scavenging of 2,2-diphenyl-1-picrylhydrazyl was 6.110 ± 0.305 mg/ml, much higher than the IC₅₀ for inhibition of tyrosinase activity which indicating ergothioneine on tyrosinase shows a weak correlation to its antioxidative activity. The results demonstrated that ergothioneine has a potent inhibition effect on tyrosinase enzyme activity, resulting from the presence of the sulfur substituted imidazole ring in ergothioneine.

Keywords: Ergothioneine; Histidine; Tyrosinase; DPPH

Chemiluminescent Detection of Carbohydrates in the Tumoral Breast Diseases by Vanessa Passos Brustein; Carmelita Lima Bezerra Cavalcanti; Mario Ribeiro de Melo-Junior; Maria Tereza Santos Correia; Eduardo Isidoro Carneiro Beltrão; Luiz Bezerra Carvalho Jr. (pp. 268-275).

Nowadays, there is an increase of investigations into the fibroadenoma, mainly because some studies have shown that the occurrence of fibroadenoma is linked to an increased risk of developing breast carcinoma. Currently, the chemiluminescence biomarkers are applied for validation methods and screening. Here, a lectin chemiluminescence is proposed as new histochemistry method to identify carbohydrates in mammary tumoral tissues. The lectins concanavalin A (Con A) and peanut agglutinin (PNA) conjugated to acridinium ester were used to characterize the glycocode of breast tissues: normal, fibroadenoma, and invasive duct carcinoma (IDC). The lectin chemiluminescence expressed in relative light units (RLU) was higher in fibroadenoma and IDC than in normal tissue for both lectins tested. The relationship RLU emission versus tissue area described a linear and hyperbolic curve for IDC and fibroadenoma, respectively, using Con A whereas hyperbolic curves for both transformed tissues using PNA. RLU was abolished by inhibiting the interaction between tissues and lectins using their specific carbohydrates: methyl-α-d-mannoside (Con A) and galactose (PNA). The intrinsic fluorescence emission did not change with combination of the lectins (Con A/PNA) to the acridinium ester for hydrophobic residues. These results represent the lectin chemiluminescence as an alternative of histochemistry method for tumoral diagnosis in the breast.

Keywords: Chemiluminescence; Concanavalin A (Con A); Peanut agglutinin (PNA); Human mammary tissues; Histochemistry

Trifluoroethanol-Induced Changes in Activity and Conformation of Manganese-Containing Superoxide Dismutase by Shang-Jun Yin; Zhi-Rong Lü; Daeui Park; Hae Young Chung; Jun-Mo Yang; Hai-Meng Zhou; Guo-Ying Qian; Yong-Doo Park (pp. 276-288).

Superoxide dismutase (SOD, EC 1.15.1.1) plays an important role in antioxidant defense in organisms exposed to oxygen. However, there is a lack of research into the regulation of SOD activity and structural changes during folding, especially for SOD originating from extremophiles. We studied the inhibitory effects of trifluoroethanol (TFE) on the activity and conformation of manganese-containing SOD (Mn-SOD) from Thermus thermophilus. TFE decreased the degree of secondary structure of Mn-SOD, which directly resulted in enzyme inactivation and disrupted the tertiary structure of Mn-SOD. The kinetic studies showed that TFE-induced inactivation of Mn-SOD is a first-order reaction and that the regional Mn-contained active site is very stable compared to the overall structure. We further simulated the docking between Mn-SOD and TFE (binding energy for Dock 6.3, −9.68 kcal/mol) and predicted that the LEU9, TYR13, and HIS29 residues outside of the active site interact with TFE. Our results provide insight into the inactivation of Mn-SOD during unfolding in the presence of TFE and allow us to describe ligand binding via inhibition kinetics combined with computational predictions.

Keywords: Active site; Docking simulation; Folding; Inactivation kinetics; Manganese; Superoxide dismutase; Trifluoroethanol

Decarboxylation of Ferulic Acid to 4-Vinyl Guaiacol by Streptomyces setonii by Belén Max; Julia Carballo; Sandra Cortés; Jose M. Domínguez (pp. 289-299).

4-Vinyl guaiacol (3-methoxy 4-hydroxystyrene) can be obtained by decarboxylation of ferulic acid by the strain Streptomyces setonii ATCC 39116. The formation of this metabolite was favoured by microaerobic conditions and the culture medium employed, increasing progressively the product concentration from 543.3 up to 885.1 mg/l when aeration level was diminished, reaching a highest volumetric productivity of 70.4 mg/l h and a product yield of 1.11 mol/mol. The identity of the metabolite was confirmed by gas chromatography–mass spectrometry. A metabolic study of ferulic acid and the main degradation products (ferulic acid, 4-vinyl guaiacol, protocatechuic acid, vanillyl alcohol, vanillic acid and vanillin) suggested that ferulic acid was the only substrate capable to be transformed into 4-vinyl guaiacol by this strain of S. setonii.

Keywords: 4-Vinyl guaiacol; Ferulic acid; Decarboxylation; Deacetylation; Streptomyces setonii

Selection of a New Whole Cell Biocatalyst for the Synthesis of 2-Deoxyribose 5-Phosphate by Ana L. Valino; Martín A. Palazzolo; Adolfo M. Iribarren; Elizabeth Lewkowicz (pp. 300-308).

2-Deoxyribose 5-phosphate (DR5P) is a key intermediate in the biocatalyzed preparation of deoxyribonucleosides. Therefore, DR5P production by means of simpler, cleaner, and economic pathways becomes highly interesting. One strategy involves the use of bacterial whole cells containing DR5P aldolase as biocatalyst for the aldol addition between acetaldehyde and d-glyceraldehyde 3-phosphate or glycolytic intermediates that in situ generate the acceptor substrate. In this work, diverse microorganisms capable of synthesizing DR5P were selected by screening several bacteria genera. In particular, Erwinia carotovora ATCC 33260 was identified as a new biocatalyst that afforded 14.1-mM DR5P starting from a cheap raw material like glucose.

Keywords: 2-Deoxyribose 5-phosphate; Aldolases; 2-Deoxyribose 5-phosphate aldolase; Nucleosides; Erwinia carotovora

Molecular Cloning and Characterization of a Putative cDNA Encoding Endoglucanase IV from Trichoderma Viride and its Expression in Bombyx Mori by Xing-hua Li; Peng Zhang; Shuang Liang; Fang Zhou; Mei-xian Wang; Roy Bhaskar; Firdose Ahmad Malik; Yan-shan Niu; Yun-gen Miao (pp. 309-320).

The development of cellulase production technology has greatly contributed to the successful use of cellulosic materials as renewable carbon sources. In this study, a putative endoglucanase IV (EG IV) complementary DNA was cloned from the mycelium of a strain of the filamentous fungus Trichoderma viride using a PCR-based exon-splicing method and expressed in both a silkworm BmN cell line and in silkworm larvae. Western blot analysis detected a band of 42 kDa in BmN cells after infection with a recombinant mBacmid/BmNPV/EG IV baculovirus. Sequence alignment analysis of the T. viride EG IV gene showed two domains that were highly conserved with glycosyl hydrolases and a funga-type cellulose-binding domain. Analysis of variance showed that silkworms infected with recombinant baculoviruses exhibited significantly higher enzyme activity that was 48.84% higher than silkworms infected with blank baculoviruses and 46.61% higher than normal silkworms. The expressed bioactive EG IV was also stable at the pH range from 5.0 to 10.0. The availability of large quantities of bioactive EG IV in silkworm provided a possibility to produce cellulase transgenic silkworm, which express bioactive cellulase specially in its digestive tract and improve its metabolism efficiency of mulberry leaves. Its application in the sericulture industry may be very promising.

Keywords: Trichoderma viride ; Cellulase; Bombyx mori ; Exon splicing; BmNPV baculovirus expression system

Production, Partial Characterization, and Use of a Red Biochrome Produced by Serratia sakuensis subsp. nov Strain KRED for Dyeing Natural Fibers by J. Vaidyanathan; Z. Bhathena-Langdana; R. V. Adivarekar; M. Nerurkar (pp. 321-335).

We have described a novel red biochrome, 514 Da in size, produced by solid-state cultivation of a bacterial isolate obtained from garden soil. The growth requirements of the isolate, the chemical characteristics of the biochrome produced, and the application of the biochrome in dying of silk, wool, and cotton fabrics have been studied. The biochrome obtained after 52 h of incubation and having a λ _max of 535 nm was used for dyeing the fabrics. We found that silk, wool, and cotton fabrics dyed with this new natural red compound have high color strength values and dye uptake along with good color fastness as well as antibacterial activity.

Keywords: Natural dyes; Bacteria; Speckled colonies; Tripyrrol compound; Dyeing; Natural fibers; Antibacterial activity

Xylanase and β-Xylosidase Production by Aspergillus ochraceus: New Perspectives for the Application of Wheat Straw Autohydrolysis Liquor by Michele Michelin; Maria de Lourdes T. M. Polizeli; Denise S. Ruzene; Daniel P. Silva; António A. Vicente; João A. Jorge; Héctor F. Terenzi; José A. Teixeira (pp. 336-347).

The xylanase biosynthesis is induced by its substrate—xylan. The high xylan content in some wastes such as wheat residues (wheat bran and wheat straw) makes them accessible and cheap sources of inducers to be mainly applied in great volumes of fermentation, such as those of industrial bioreactors. Thus, in this work, the main proposal was incorporated in the nutrient medium wheat straw particles decomposed to soluble compounds (liquor) through treatment of lignocellulosic materials in autohydrolysis process, as a strategy to increase and undervalue xylanase production by Aspergillus ochraceus. The wheat straw autohydrolysis liquor produced in several conditions was used as a sole carbon source or with wheat bran. The best conditions for xylanase and β-xylosidase production were observed when A. ochraceus was cultivated with 1% wheat bran added of 10% wheat straw liquor (produced after 15 min of hydrothermal treatment) as carbon source. This substrate was more favorable when compared with xylan, wheat bran, and wheat straw autohydrolysis liquor used separately. The application of this substrate mixture in a stirred tank bioreactor indicated the possibility of scaling up the process to commercial production.

Keywords: Xylanase; β-xylosidase; Wheat bran; Wheat straw autohydrolysis liquor; Bioreactor

Adding Value to the Oil Cake as a Waste from Oil Processing Industry: Production of Lipase and Protease by Candida utilis in Solid State Fermentation by Omar Ali Saied Moftah; Sanja Grbavčić; Milena Žuža; Nevena Luković; Dejan Bezbradica; Zorica Knežević-Jugović (pp. 348-364).

Olive oil cake is a by-product from the olive oil processing industry and can be used for the lipase and protease production by Candida utilis in solid state fermentation. Different carbon and nitrogen sources were evaluated, and the results showed that the supplementation of the substrate with maltose and starch as carbon sources and yeast extract as a nitrogen source significantly increased the lipase production. The best results were obtained with maltose, whereas rather low lipase and protease activities were found with glucose and oleic acid. Response surface methodology and a five-level–three-factor central composite rotatable design were used to evaluate the effects of the initial moisture content, inoculum size and fermentation time on both lipase and protease activity levels. A lipase activity value of ≈25 U g^-1 and a protease activity value of 110 U g^-1 were obtained under the optimized fermentation conditions. An alkaline treatment of the substrate appeared to be efficient, leading to increases of 39% and 133% in the lipase and protease production, respectively. The results showed that the olive cake could be a good source for enzyme production by solid state fermentation.

Keywords: Olive oil cake; Candida utilis ; Lipase; Protease; Optimization; Solid state fermentation

In Vitro Azadirachtin Production by Hairy Root Cultivation of Azadirachta indica in Nutrient Mist Bioreactor by Smita Srivastava; A. K. Srivastava (pp. 365-378).

Azadirachtin, a well-known biopesticide is a secondary metabolite conventionally extracted from the seeds of Azadirachta indica. The present study involved in vitro azadirachtin production by developing hairy roots of A. indica via Agrobacterium rhizogenes-mediated transformation of A. indica explants. Liquid culture of hairy roots was established in shake flask to study the kinetics of growth and azadirachtin production. A biomass production of 13.3 g/L dry weight (specific growth rate of 0.7 day⁻¹) was obtained after 25 days of cultivation period with an azadirachtin yield of 3.3 mg/g root biomass. To overcome the mass transfer limitation in conventionally used liquid-phase reactors, batch cultivation of hairy roots was carried out in gas-phase reactors (nutrient spray and nutrient mist bioreactor) to investigate the possible scale-up of A. indica hairy root culture. The nano-size nutrient mist particles generated from the nozzle of the nutrient mist bioreactor could penetrate till the inner core of the inoculated root matrix, facilitating uniform growth during high-density cultivation of hairy roots. A biomass production of 9.8 g/L dry weight with azadirachtin accumulation of 2.8 mg/g biomass (27.4 mg/L) could be achieved in 25 days of batch cultivation period, which was equivalent to a volumetric productivity of 1.09 mg/L per day of azadirachtin.

Keywords: Azadirachtin; Hairy roots; Azadirachta indica ; Gas-phase reactors; Nutrient mist bioreactor; Nutrient spray bioreactor

Pathophysiology of Microwave Radiation: Effect on Rat Brain by Kavindra Kumar Kesari; Sanjay Kumar; Jitendra Behari (pp. 379-388).

The study aims to investigate the effect of 2.45 GHz microwave radiation on Wistar rats. Rats of 35 days old with 130 ± 10 g body weight were selected for this study. Animals were divided into two groups: sham exposed and experimental (six animals each). Animals were exposed for 2 h a day for 45 days at 2.45 GHz frequency (power density, 0.21 mW/cm²). The whole body specific absorption rate was estimated to be 0.14 W/kg. Exposure took place in a ventilated plexiglas cage and kept in an anechoic chamber under a horn antenna. After completion of the exposure period, rats were killed, and pineal gland and whole brain tissues were isolated for the estimation of melatonin, creatine kinase, caspase 3, and calcium ion concentration. Experiments were performed in a blind manner and repeated. A significant decrease (P < 0.05) was recorded in the level of pineal melatonin of exposed group as compared with sham exposed. A significant increase (P < 0.05) in creatine kinase, caspase 3, and calcium ion concentration was observed in whole brain of exposed group of animals as compared to sham exposed. One-way analysis of variance method was adopted for statistical analysis. The study concludes that a reduction in melatonin or an increase in caspase-3, creatine kinase, and calcium ion may cause significant damage in brain due to chronic exposure of these radiations. These biomarkers clearly indicate possible health implications of such exposures.

Keywords: Melatonin; Caspase-3; Creatine kinase; Calcium ion concentration; Microwave

Additional Paper Waste in Pulping Sludge for Biohydrogen Production by Heat-Shocked Sludge by Prapaipid Chairattanamanokorn; Supachok Tapananont; Siriporn Detjaroen; Juthatip Sangkhatim; Patana Anurakpongsatorn; Pramote Sirirote (pp. 389-401).

Dark anaerobic fermentation is an interesting alternative method for producing biohydrogen (H₂) as a renewable fuel because of its low cost and various usable organic substrates. Pulping sludge from wastewater treatment containing plentiful cellulosic substrate could be feasibly utilized for H₂ production by dark fermentation. The objective of this study was to investigate the optimal proportion of pulping sludge to paper waste, the optimal initial pH, and the optimal ratio of carbon and nitrogen (C/N) for H₂ production by anaerobic seed sludge pretreated with heat. The pulping sludge was pretreated with NaOH solution at high temperature and further hydrolyzed with crude cellulase. Pretreatment of the pulping sludge with 3% NaOH solution under autoclave at 121 °C for 2 h, hydrolysis with 5 FPU crude cellulase at 50 °C, and pH 4.8 for 24 h provided the highest reducing sugar production yield (229.68 ± 2.09 mg/g_TVS). An initial pH of 6 and a C/N ratio of 40 were optimal conditions for H₂ production. Moreover, the supplement of paper waste in the pulping sludge enhanced the cumulative H₂ production yield. The continuous hydrogen production was further conducted in a glass reactor with nylon pieces as supporting media and the maximum hydrogen production yield was 151.70 ml/g_TVS.

Keywords: Biohydrogen production; Paper waste; Pretreatment of substrate; Pulping sludge

Development of Saccharomyces cerevisiae Producing Higher Levels of Sulfur Dioxide and Glutathione to Improve Beer Flavor Stability by Yefu Chen; Xu Yang; Shijie Zhang; Xiaoqiong Wang; Changhui Guo; Xuewu Guo; Dongguang Xiao (pp. 402-413).

Sulfur compounds, such as sulfite (SO₂), hydrogen sulfide (H₂S), and glutathione (GSH), play different roles in beer flavor stability. SO₂ and GSH have antiaging effects which are helpful to improve the flavor stability of beer, whereas H₂S is undesirable to beer flavor because of its unpleasant aroma. Here, we report the development of Saccharomyces cerevisiae which produces higher levels of SO₂ and GSH but lower level of H₂S to improve beer flavor stability by nongenetic engineering approaches. After two rounds of UV mutagenesis coupled with specific plate screening methods, one promising mutant named MV16 was obtained. Compared with the original strain, the SO₂ and GSH production of MV16 in fermenting liquor increased by 31% and 30.2%, respectively, while H₂S content decreased by 74.9%, and the DPPH radical clearance and the resistance staling value of beer fermented by MV16 increased by 24.6% and 33.0%, respectively. The antioxidizability of the mutant was improved significantly. The strategy adopted in our study could be used to obtain S. cerevisiae of improved antiaging properties, and the mutant would be safe for public use.

Keywords: Saccharomyces cerevisiae ; Flavor stability; Sulfur dioxide (SO₂); Hydrogen sulfide (H₂S); Glutathione (GSH)

Genome Shuffling Enhanced ε-Poly-l-Lysine Production by Improving Glucose Tolerance of Streptomyces graminearus by Shu Li; Feng Li; Xu-Sheng Chen; Liang Wang; Jian Xu; Lei Tang; Zhong-Gui Mao (pp. 414-423).

The productivity of ε-poly-l-lysine (ε-PL) in currently reported wild-type strains is low. Here we improved glucose tolerance of a Streptomyces graminearus strain LS-B1 by genome shuffling while simultaneously enhancing the ε-PL productivity. The starting population was generated by ultraviolet irradiation and nitrosoguanidine mutagenesis and then subjected for recursive protoplast fusion. The positive colonies from library, created by fusing the inactivated protoplasts were screened on agar plates containing different concentrations of glucose. Characterization of all recombinants and wild-type strain in shake-flask fermentation indicated the compatibility of two phenotypes of glucose tolerance and ε-PL yield enhancement. The best performing recombinant, F3-4, was isolated after three rounds of genome shuffling, whose ε-PL production was about 88% higher than that of the parent strain. In batch fermentation test, the ε-PL concentration was obtained as 2.4 g/L by F3-4 compared with 1.6 g/L of wild type. Fed-batch fermentation by F3-4 was carried out and the ε-PL production accumulated to 13.5 g/L when initial glucose concentration was improved from 50 to 85 g/L. Enzyme activities of hexokinase, pyruvate kinase, and citrate synthase revealed that the glycolytic pathway and tricarboxylic acid circle way in F3-4 were more active than those in wild type, which was a possible reason for enhanced ε-PL production.

Keywords: ε-Poly-l-lysine; Genome shuffling; Streptomyces graminearus ; Fermentation

Purification and Characterization of a Mannose Recognition Lectin from Oreochromis niloticus (Tilapia Fish): Cytokine Production in Mice Splenocytes by Cynarha Daysy Cardoso da Silva; Marília Cavalcanti Coriolano; Mércia Andréa da Silva Lino; Cristiane Moutinho Lagos de Melo; Ranilson de Souza Bezerra; Elba Verônica Matoso Maciel de Carvalho; Athiê Jorge Guerra dos Santos; Valéria Rêgo Alves Pereira; Luana Cassandra Breitenbach Barroso Coelho (pp. 424-435).

The aim of this work was to purify and partially characterize a mannose recognition lectin from Nile tilapia (Oreochromis niloticus) serum, named OniL. OniL was isolated through precipitation with ammonium sulfate and affinity chromatography (Concanavalin A–Sepharose 4B). In addition, we evaluated carbohydrate specificity, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) profiles, and in vitro immunomodulatory activity on mice splenocyte experimental cultures through cytotoxic assays and cytokine production. The ammonium sulfate fraction F2 showed the highest specific hemagglutinating activity (331) and was applied to affinity matrix. Adsorbed proteins (OniL) were eluted with methyl-α-d-mannopyranoside. OniL, a 17-kDa protein by SDS–PAGE constituted by subunits of 11 and 6.6 kDa, showed highest affinity for methyl-α-d-mannopyranoside and d-mannose. Immunological assays, in vitro, showed that OniL did not show cytotoxicity against splenocytes, induced higher IFN-γ production and lower IL-10 as well as nitrite release. In conclusion, OniL lectin was successfully purified and showed a preferential Th1 response in mice splenocytes.

Keywords: Oreochromis niloticus ; Tilapia; Lectin purification; Immunomodulatory activity

Determination of Dimethyl Phthalate in Environment Water Samples by a Highly Sensitive Indirect Competitive ELISA by Mingcui Zhang; Shaohui Liu; Huisheng Zhuang; Yurong Hu (pp. 436-445).

Recent controversy over the discovery of clouding agents containing the banned chemical di(2-ethylhexyl) phthalate in beverages in 2011 in Taiwan has caused public concerns. For the detection of dimethyl phthalate (DMP) in environment water samples, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed in this paper. Dimethyl 4-aminophthalate (4-DMAP) was covalently attached to bovine serum albumin as immunogen by a diazotization method. The conjugation of DMAP and ovalbumin as coating antigen was obtained in the same way. Polyclonal antibody was obtained from New Zealand white rabbits. Under the optimized conditions, DMP was detected in the concentration range of 0.02–419 ng/mL with a detection limit of 0.01 ng/mL. The proposed method has been applied to the analysis of river water, lake water, and rain water samples. Satisfactory recoveries were obtained ranging from 90.6% to 105.5%. The cross-reactivities of the anti-DMP antibody to seven structurally related phthalate esters were below 10%. The data demonstrated that the ic-ELISA method described in our study is a simple, sensitive, and specific method and showed that this assay is a reliable tool to detect DMP in water samples.

Keywords: ELISA; Dimethyl phthalate; Polyclonal antibody; Water samples; Phthalate esters

An Optimum Fermentation Model Established by Genetic Algorithm for Biotransformation from Crude Polydatin to Resveratrol by Yang Chong; Aixia Yan; Xiaoying Yang; Yangliu Cai; Jinchun Chen (pp. 446-457).

Natural resveratrol is widely used in medicine, food, and cosmetic because of its pharmacological properties. Due to its low content in plants, this study was conducted to increase the yield of resveratrol by microorganism transformation. Fungi Aspergillus niger AN-2436 was employed in biotransformation to produce resveratrol from polydatin, and genetic algorithm (GA) was used to optimize the fermentation conditions. A transformation ratio of higher than 95% was achieved in the following conditions: culture temperature of 30.3 °C, inoculum size of 20% (v/v), rotating speed of 147 rpm, and cultivation time of 36 h. Compared with the polydatin absorbance under the experimental conditions obtained by single-factor, orthogonal experiments and average absorbance, the GA provides the optimum experimental conditions, under which the largest transformation rate was achieved. The final transformation product obtained was identified as resveratrol, and it was proved by high-performance liquid chromatography, infrared, mass spectrometry, and nuclear magnetic resonance.

Keywords: Microorganism transformation; Polydatin; Resveratrol; Fungi Aspergillus niger ; Genetic algorithm (GA); Fermentation optimization

Pretreatment of Corn Stover with Twin-Screw Extrusion Followed by Enzymatic Saccharification by Shujing Zhang; Yixiang Xu; Milford A. Hanna (pp. 458-469).

Pretreatment of biomass before subjecting it to enzyme saccharification is crucial with regards to facilitating access of enzyme to biomass. Extrusion, as a continuous and cost-effective pretreatment method, combines heating with high shear and mixing opening cell walls at the microscopic scale, thus largely increasing the specific surface area (SSA) of biomass for enzyme adsorption. The objective of this study was to examine the effect of extrusion as a pretreatment method and the underlying factors ruling the improvement of sugar yields. The optimum glucose, xylose, and combined sugar recoveries were 48.79%, 24.98%, and 40.07%, respectively, at 27.5% moisture content and 80 rpm screw speed. These yields were 2.2, 6.6, and 2.6 times higher than those for untreated corn stover. X-ray diffraction analysis showed that the crystallinity index was not a good indicator of sugar yield. However, scanning electron microscopy showed that the cellulose network was exposed due to the destruction of the lignin sheath. The Langmuir adsorption model was shown to be an effective tool for the estimation of the SSA of corn stover. The SSA of pretreated samples was significantly amplified over the control, revealing that extrusion can open the cell wall at the microscopic scale, which was especially favorable on sugar yields.

Keywords: Corn stover; Pretreatment; Twin-screw extrusion; Enzymatic hydrolysis; Sugar yields

Fermentation of Reactive-Membrane-Extracted and Ammonium-Hydroxide-Conditioned Dilute-Acid-Pretreated Corn Stover by David L. Grzenia; S. Ranil Wickramasinghe; Daniel J. Schell (pp. 470-478).

Acid-pretreated biomass contains various compounds (acetic acid, etc.) that are inhibitory to fermentative microorganisms. Removing or deactivating these compounds using detoxification methods such as overliming or ammonium hydroxide conditioning (AHC) improves sugar-to-ethanol yields. In this study, we treated the liquor fraction of dilute-acid-pretreated corn stover using AHC and a new reactive membrane extraction technique, both separately and in combination, and then the sugars in the treated liquors were fermented to ethanol with the glucose–xylose-fermenting bacterium, Zymomonas mobilis 8b. We performed reactive extraction with mixtures of octanol/Alamine 336 or oleyl alcohol/Alamine 336. The best ethanol yields and rates were achieved for oleyl alcohol-extracted hydrolysates followed by AHC hydrolysates, while octanol-extracted hydrolysates were unfermentable because highly toxic octanol was found in the hydrolysate. Adding olive oil significantly improved yields for octanol-extracted hydrolysate. Additional work is underway to determine if this technology is a cost-effective alternative to traditional hydrolysate conditioning processes.

Keywords: Pretreatment; Bioethanol; Membrane; Extraction; Fermentation

Isolation and Characterization of a Novel Plasma Membrane Intrinsic Protein Gene, LcPIP1, in Leymus chinensis that Enhances Salt Stress Tolerance in Saccharomyces cerevisiae by Pengda Ma; Jingying Liu (pp. 479-485).

A novel plasma membrane intrinsic, LcPIP1, was isolated from Leymus chinensis using RACE method. The LcPIP1 has 288 amino acids with an estimated molecular mass of 30.6 kDa. Semi RT-PCR analysis indicated that the expression level of LcPIP1 was obviously higher in leaf than root. The LcPIP1 was also found to be induced by salt stress. In addition, transformed with the LcPIP1, Saccharomyces cerevisiae could increase tolerance to salt stress. These results indicate that the LcPIP1 gene seems to play a role in resistance against salt stress.

Keywords: Aquaporin; Plasma membrane intrinsic protein; Salt stress; Leymus chinensis

Overview of Fungal Lipase: A Review by Abhishek Kumar Singh; Mausumi Mukhopadhyay (pp. 486-520).

Lipases (triacylglycerolacyl hydrolases, EC3.1.1.3) are class of enzymes which catalyze the hydrolysis of long-chain triglycerides. In this review paper, an overview regarding the fungal lipase production, purification, and application is discussed. The review describes various industrial applications of lipase in pulp and paper, food, detergent, and textile industries. Some important lipase-producing fungal genera include Aspergillus, Penicillium, Rhizopus, Candida, etc. Current fermentation process techniques such as batch, fed-batch, and continuous mode of lipase production in submerged and solid-state fermentations are discussed in details. The purification of lipase by hydrophobic interaction chromatography is also discussed. The development of mathematical models applied to lipase production is discussed with special emphasis on lipase engineering.

Keywords: Lipase; Enzyme; Modeling; Production; Purification

Erratum to: Proteomic Analysis of Differential Protein Expression of Achilles Tendon in a Rabbit Model by Two-Dimensional Polyacrylamide Gel Electrophoresis at 21 Days Postoperation by Jiasharete Jielile; Ainuer Jialili; Gulnur Sabirhazi; Nuerai Shawutali; Darebai Redati; Jiangtao Chen; Bin Tang; Jingping Bai; Kayrat Aldyarhan (pp. 521-521).

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.166, #2)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.166, #2)