Applied Biochemistry and Biotechnology (v.162, #1)

Gene Cloning, Overexpression, and Characterization of a Xylanase from Penicillium sp. CGMCC 1669 by Wanli Liu; Pengjun Shi; Qiang Chen; Peilong Yang; Guozeng Wang; Yaru Wang; Huiying Luo; Bin Yao (1-12).
A xylanase-encoding gene, xyn11F63, was isolated from Penicillium sp. F63 CGMCC1669 using degenerated polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR techniques. The full-length chromosomal gene consists of 724 bp, including a 73-bp intron, and encodes a 217 amino acid polypeptide. The deduced amino acid sequence of xyn11F63 shows the highest identity of 70% to the xylanase from Penicillium sp. strain 40, which belongs to glycosyl hydrolases family 11. The gene was overexpressed in Pichia pastoris, and its activity in the culture medium reached 516 U ml−1. After purification to electrophoretic homogeneity, the enzyme showed maximal activity at pH 4.5 and 40°C, was stable at acidic buffers of pH 4.5–9.0, and was resistant to proteases (proteinase K, trypsin, subtilisin A, and α-chymotrypsin). The specific activity, K m, and V max for oat spelt xylan substrate was 7,988 U mg−1, 22.2 mg ml−1, and 15,105.7 μmol min−1 mg−1, respectively. These properties make XYN11F63 a potential economical candidate for use in feed and food industrial applications.
Keywords: Xylanase; Penicillium sp. F63 CGMCC 1669; Pichia pastoris ; Overexpression

New Ether-Functionalized Ionic Liquids for Lipase-Catalyzed Synthesis of Biodiesel by Hua Zhao; Zhiyan Song; Olarongbe Olubajo; Janet V. Cowins (13-23).
Ionic liquids (ILs) are being explored as solvents for the enzymatic methanolysis of triglycerides. However, most available ILs (especially hydrophobic ones) have poor capability in dissolving lipids, while hydrophilic ILs tend to cause enzyme inactivation. Recently, we synthesized a new type of ether-functionalized ionic liquids (ILs) carrying anions of acetate or formate; they are capable of dissolving a variety of substrates and are also lipase-compatible (Green Chem., 2008, 10, 696–705). In the present study, we carried out the lipase-catalyzed transesterifications of Miglyol® oil 812 and soybean oil in these novel ILs. These ILs are capable of dissolving oils at the reaction temperature (50 °C); meanwhile, lipases maintained high catalytic activities in these media even in high concentrations of methanol (up to 50% v/v). High conversions of Miglyol oil were observed in mixtures of IL and methanol (70/30, v/v) when the reaction was catalyzed by a variety of lipases and different enzyme preparations (free and immobilized), especially with the use of two alkylammonium ILs 2 and 3. The preliminary study on the transesterification of soybean oil in IL/methanol mixtures further confirms the potential of using oil-dissolving and lipase-stabilizing ILs in the efficient production of biodiesels.
Keywords: Biodiesel; Enzymatic transesterification; Ionic liquid; Lipase; Vegetable oil; Triglyceride

Immobilization of Aspergillus niger Xylanase on Chitosan Using Dialdehyde Starch as a Coupling Agent by Hongge Chen; Liangwei Liu; Shuai Lv; Xinyu Liu; Mingdao Wang; Andong Song; Xincheng Jia (24-32).
Dialdehyde starch (DAS) was used as a novel coupling agent to prepare chitosan carrier to immobilize the xylanase from Aspergillus niger A-25. Compared with glutaraldehyde-cross-linked chitosan (CS-GA) and pure chitosan beads, the DAS-cross-linked chitosan (CS-DAS) beads exhibited the highest xylanase activity recovery. The DAS adding amount and cross-linking time in CS-DAS preparation process were optimized with respect to activity recovery to the values of 1.0 g (6.7% w/v concentration) and 16 h, respectively. The optimum temperature of both the CS-DAS- and CS-GA-immobilized xylanase was observed to be 5 °C higher than that of free enzyme (50 °C). The CS-DAS-immobilized xylanase had the highest thermal and storage stability as compared to the CS-GA-immobilized and free xylanase. The apparent K m and V max values of the CS-DAS-immobilized xylanase were estimated to be 1.29 mg/ml and 300.7 μmol/min/mg protein, respectively. The CS-DAS-immobilized xylanase could produce from birchwood xylan high-quality xylo-oligosaccharides, mainly composed of xylotriose, as free xylanase did. The proposed CS-DAS carrier was more advantageous over the CS-GA or pure chitosan carrier for xylanase immobilization application.
Keywords: Dialdehyde starch; Xylanase; Chitosan; Immobilization

Feasibility of Hydrothermal Pretreatment on Maize Silage for Bioethanol Production by Jian Xu; Mette Hedegaard Thomsen; Anne Belinda Thomsen (33-42).
The potential of maize silage as a feedstock to produce bioethanol was evaluated in the present study. The hydrothermal pretreatment with five different pretreatment severity factors (PSF) was employed to pretreat the maize silage and compared in terms of sugar recovery, toxic test, and ethanol production by prehydrolysis and simultaneous saccharification and fermentation. After pretreatment, most of the cellulose remained in the residue, ranging between 85.87% by the highest PSF (185°C, 15 min) and 92.90% obtained at the lowest PSF (185°C, 3 min). A larger part of starch, varying from 71.64% by the highest PSF to 78.28% by the lowest, was liberated into liquor part, leaving 8.05–11.74% in the residues. Xylan recovery in the residues increased from 44.25% at the highest PSF to 82.95% at the lowest. The recovery of xylan in liquor changed from 20.13% to 50.33%. Toxic test indicated that all the liquors from the five conditions were not toxic to the Baker’s yeast. Pretreatment under 195°C for 7 min had the similar PSF with that of 185°C for 15 min, and both gave the higher ethanol concentration of 19.92 and 19.98 g/L, respectively. The ethanol concentration from untreated maize silage was only 7.67 g/L.
Keywords: Maize silage; Bioethanol; Hydrothermal pretreatment; Pretreatment severity factor (PSF); Toxic test; Sugar recovery

The present work explores suspicious consequence of low molecular weight glycosaminoglycan (LMW-GAG) on oxidative stress and cellular abnormalities in isoproterenol (ISO)-induced myocardial infarction in an experimental model. Group-III male Wistar rats (140 ± 10 g) were administrated by ISO (85 mg ISO/ml subcutaneously (SC) injected at the last two days of a 2–week period). Group-IV rats were treated LMW-GAG plus ISO (300 μg/day per rat SC for 1 week followed by 85 mg/kg ISO on the end last two days of the 2 - weeks). Untreated control (Group-I) and LMW-GAG drug control (Group-II) were also included. Serum and tissue lactate dehydrogenase, aminotransferases, and creatine kinase activities were increased in ISO group, which were normalized by LMW-GAG pretreatment rats. Antioxidant enzymes – superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) activities and non-enzymatic enzyme reduced glutathione (GSH) were decreased in the ISO induced rats, and this was increased by LMW-GAG pretreatment. Increased level of thiobarbituric acid reactive substances (TBARS) in plasma and the heart of ISO treated rats; pre s.c. injected with LMW-GAG to ISO-induced rats decreased the levels of TBARS. Histological examination revealed that the ISO-induced deleterious changes in the heart tissues were offset by LMW-GAG treatment. LMW-GAG affords considerable protection to the tissues challenged by cardiotoxicity, evidenced by its correction and restoration of serum and tissue indices of injury, to normalcy.
Keywords: LMW-GAG; Isoproterenol; Myocardial infarction; Lipid peroxidation; Antioxidants

Response of Cellulase Activity in pH-Controlled Cultures of the Filamentous Fungus Acremonium cellulolyticus by Joni Prasetyo; Shyuuhei Sumita; Naoyuki Okuda; Enoch Y. Park (52-61).
Cellulase production was investigated in pH-controlled cultures of Acremonium cellulolyticus. The response to culture pH was investigated for three cellulolytic enzymes, carbomethyl cellulase (CMCase), avicelase, and β-glucosidase. Avicelase and β-glucosidase showed similar profiles, with maximum activity in cultures at pH 5.5–6. The CMCase activity was highest in a pH 4 culture. At an acidic pH, the ratios of CMCase and avicelase activity to cellulase activity defined by filter paper unit were high, but at a neutral pH, the β-glucosidase ratio was high. The pH 6.0 culture showed the highest cellulase activity within the range of pH 3.5–6.5 cultures. The saccharification activity from A. cellulolyticus was compared to those of the cellulolytic enzymes from other species. The A. cellulolyticus culture broth had a saccharification yield comparable to those of the Trichoderma enzymes GC220 and Cellulosin T2, under conditions with the same cellulase activity. The saccharification yields from Solka floc, Avicel, and waste paper, measured as the percent of released reducing sugar to dried substrate, were greater than 80% after 96 h of reaction. The yields were 16% from carboxymethylcellulose and 26% from wood chip refiner. Thus, the A. cellulolyticus enzymes were suitable for converting cellulolytic biomass to reducing sugars for biomass ethanol production. This study is a step toward the establishment of an efficient system to reutilize cellulolytic biomass.
Keywords: Cellulase; Acremonium cellulolyticus ; Biomass; Saccharification; Lignocellulose

To reduce the recalcitrance and enhance enzymatic activity, dilute H2SO4 pretreatment was carried out on Alamo switchgrass (Panicum virgatum). Ball-milled lignin was isolated from switchgrass before and after pretreatment. Its structure was characterized by 13C, HSQC, and 31P NMR spectroscopy. It was confirmed that ball-milled switchgrass lignin is of HGS type with a considerable amount of p-coumarate and felurate esters of lignin. The major ball-milled lignin interunit was the β-O-4 linkage, and a minor amount of phenylcoumarin, resinol, and spirodienone units were also present. As a result of the acid pretreatment, there was 36% decrease of β-O-4 linkage observed. In addition to these changes, the S/G ratio decreases from 0.80 to 0.53.
Keywords: Pretreatment; Switchgrass; Ball-milled lignin; HSQC; 13C and 31P NMR spectroscopy

Fibrinolytic Serine Protease Isolation from Bacillus amyloliquefaciens An6 Grown on Mirabilis jalapa Tuber Powders by Rym Agrebi; Noomen Hmidet; Mohamed Hajji; Nawrez Ktari; Anissa Haddar; Nahed Fakhfakh-Zouari; Moncef Nasri (75-88).
In this study, Mirabilis jalapa tuber powder (MJTP) was used as a new complex organic substrate for the growth and production of fibrinolytic enzymes by a newly isolated Bacillus amyloliquefaciens An6. Maximum protease activity (1,057 U/ml) with casein as a substrate was obtained when the strain was grown in medium containing (grams per liter) MJTP 30, yeast extract 6, CaCl2 1, K2HPO4 0.1, and K2HPO4 0.1. The strain was also found to grow and produce extracellular proteases in a medium containing only MJTP, indicating that it can obtain its carbon, nitrogen, and salts requirements directly from MJTP. The B. amyloliquefaciens An6 fibrinase (BAF1) was partially purified, and fibrinolytic activity was assayed in a test tube with an artificial fibrin clot. The molecular weight of the partially purified BAF1 fibrinolytic protease was estimated to be 30 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. The optimum temperature and pH for the caseinolytic activity were 60 °C and 9.0, respectively. The enzyme was highly stable from pH 6.0 to 11.0 and retained 62% of its initial activity after 1 h incubation at 50 °C. However, the enzyme was inactivated at higher temperatures. The activity of the enzyme was totally lost in the presence of phenylmethylsulfonyl fluoride, suggesting that BAF1 is a serine protease.
Keywords: Bacillus amyloliquefaciens ; Mirabilis jalapa ; Protease; Fibrinase

Isomaltulose was obtained from sucrose solution by immobilized cells of Erwinia sp. D12 using a batch and a continuous process. Parameters for sucrose conversion into isomaltulose were evaluated using both experimental design and response surface methodology. Erwinia sp. D12 cells were immobilized in different alginates, and the influence of substrate flow rate and concentration parameters to produce isomaltulose from sucrose were observed. Response surface methodology demonstrated that packed bed columns containing cells immobilized in low-viscosity sodium alginate (250 cP) presented a mean isomaltulose conversion rate of 47%. In a continuous process, both sucrose substrate concentration and substrate flow rate parameters had a significant effect (p < 0.05) and influenced the conversion of sucrose into isomaltulose. Higher conversion rates of sucrose into isomaltulose, from 53–75% were obtained using 75 g of immobilized cells at a substrate flow rate of 0.6 mL/min.
Keywords: Calcium alginate; Immobilized cells; Isomaltulose; Response surface

Cloning and Heterologous Expression of a Novel Endoglucanase Gene egVIII from Trichoderma viride in Saccharomyces cerevisiae by Xiao-Mei Huang; Qian Yang; Zhi-Hua Liu; Jin-Xia Fan; Xiu-Ling Chen; Jin-Zhu Song; Yun Wang (103-115).
Endoglucanase is a major cellulolytic enzyme produced by the fungus Trichoderma viride. The 1,317 bp cDNA of endoglucanase gene egVIII was cloned from T. viride AS3.3711, encoding a 438 amino acid protein with a calculated molecular mass of 46.86 kDa and isoelectric point of 4.32. Sequence analysis suggested that EGVIII belonged to the glycosyl hydrolase family 5. The N-terminal region of EGVIII contains a signal peptide sequence of 19 amino acid residues, indicating that it is an extracellular enzyme. Transcription of the egVIII gene in T. viride AS3.3711 can be induced by carboxymethyl cellulose sodium (CMC-Na), sucrose, microcrystalline cellulose, and corn stalk, and inhibited by glucose and fructose. The α-mating factor signal can effectively enhance the secretion of the recombinant EGVIII in Saccharomyces cerevisiae, as demonstrated by the enzymatic activity of recombinant yeast IpYEMα-xegVIII in the supernatant, which was 0.86 times higher than that of the IpYES2-egVIII. Recombinant endoglucanase EGVIII showed optimal activity at a temperature of 60°C and pH of 6.0. It was stable when incubated from 35°C to 70°C for 1 h. The enzymatic activity of recombinant EGVIII was stable at a pH 3.0 to 7.5 at 50°C and reached the highest level at 0.174U when activated by 75 mM of Zn2+. The Michaelis–Menten constant (Km) and Kcat values for CMC-Na and cellotriose hydrolysis were 3.82 mg/ml, 9.56 s−1 and 1.75 mg/ml, 7.08 s−1, respectively. Transgenic yeast strain IpYEMα-xegVIII might be useful for renewable fuels industries.
Keywords: Trichoderma viride ; Endoglucanase; Gene cloning; Yeast expression

Rational Design of Catechol-2, 3-dioxygenase for Improving the Enzyme Characteristics by Jiashi Wei; Ying Zhou; Tao Xu; Baorong Lu (116-126).
Catechol-2, 3-dioxygenase (C23O) from Pseudomonas sp. CGMCC2953 identified in our laboratory, which is one of the key enzymes responsible for phenanthrene biodegradation, was expected to get better characteristics tolerant to environment for its further application. With the aim of improving the enzyme properties by introducing intermolecular disulfide bonds, X-ray structure of a C23O from Pseudomonas putida MT-2, a highly conserved homologous with the C23O from Pseudomonas sp. CGMCC2953, was directly used to find the potential sites for forming disulfide bonds between two monomers of the target C23O. Two sites, Ala229 and His294, were identified and mutated to cysteine, respectively, by using site mutagenesis. The expected disulfide bond between these two CYS residues was confirmed with both molecular modeling and experimental results. The optimum temperature of the mutated enzyme was widened from 40 to 40∼50 °C. The mutated C23O became more alkalescency stable compared with the wild-type enzyme, e.g., 75% of the maximal enzyme activity retained even under pH 9.5 while 50% residue for the wild-type one. Improvement of thermostability of the mutated C230 with the redesigned disulfide was also confirmed.
Keywords: Catechol-2,3-oxygenase (C23O); Computer simulation; Disulfide bond; Gene site mutagenesis; Stability

The Antifungal Activity of Sarcococca saligna Ethanol Extract and its Combination Effect with Fluconazole against Different Resistant Aspergillus Species by Kamyar Mollazadeh Moghaddam; Mohammad Arfan; Jamal Rafique; Sassan Rezaee; Parisa Jafari Fesharaki; Ahmad Reza Gohari; Ahmad Reza Shahverdi (127-133).
Microbial resistance is a major drawback in chemotherapy of microbial or fungal infection disease. In this study, the antifungal activity of ethanol extract of a selected plant (Sarcococca saligna) has been investigated against clinical isolates of Aspergillus niger, Aspergillus treus, Aspergillus flavus, and Aspergillus fumigatus. Also, the enhancement of the antifungal activity of fluconazole by this extract was further evaluated against mentioned test strains. Conventional disk diffusion method was used to assay the antifungal activity of S. saligna ethanol extract in the absence and presence of fluconazole. The highest antifungal activity was observed against A. treus. The ethanol extract of S. saligna enhanced the antifungal activity of fluconazole against A. niger and A. treus and A. flavus. At the highest tested contents (4 mg/disk), 1.15-, 0.64-, and 2.47-fold increases in inhibition zone surface area were observed for A. niger, A. treus, and A. flavus, respectively. However, no enhancing effect was observed for this plant extract against Aspergillus fumigates at tested contents (0.5, 1, 2, 3, and 4 mg/disk). In a separate experiment, the general cytotoxicity of the ethanol extract of S. saligna was examined with brine shrimp assay. This plant extract showed low cytotoxicity against Artemia salina (LC50 = 186 µg/ml).
Keywords: Sarcococca saligna ; Antifungal activity; Combination effect; Fluconazole; Aspergillus spp.

Concanavalin A (ConA), a mannose/glucose-binding legume lectin, has been reported to induce tumor cell death via a mitochondria-mediated autophagic pathway; however, the precise mechanism by which induces cell death remains to be discovered. In this study, we simulated the three-dimensional structure of ConA monomer, its dimer, and tetramer forms and reported its molecular dynamics simulations and phylogenetic analysis. Subsequently, we showed that ConA possessed remarkable antiproliferative effects on HepG2 cells. Further data showed that there was a link among its hemagglutinating, sugar-binding, and antiproliferative activities. In addition, we found that ConA induced apoptosis in HepG2 cells. Then, we demonstrated that the treatment of ConA caused mitochondrial transmembrane potential (MMP) collapse, cytochrome c release, and activation of caspase. In conclusion, we demonstrate that there is a positive correlation between carbohydrate-binding activity and antiproliferative activity of ConA. In addition, we confirm that ConA induces HepG2 cell death through a mitochondrial apoptotic pathway.
Keywords: Concanavalin A (ConA); Molecular structure; Phylogenetic analysis; Antiproliferative; Apoptosis; Mitochondrial pathway

Partial Purification and Characterization of Glutaminase from Lactobacillus reuteri KCTC3594 by Jeong-Min Jeon; Hae-In Lee; Sang-Hyun Han; Chung-Soon Chang; Jae-Seong So (146-154).
In this study, we attempted to purify and characterize glutaminase (EC. 3.5.1.2) from Lactobacillus reuteri KCTC3594. The glutaminase was purified approximately 21-fold from the cell-free extract of L. reuteri KCTC3594 by protamine sulfate treatment and chromatography methods including anion exchange and gel filtration. The sizes of two major bands of the enzyme were presumed to be 70 and 50 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The glutaminase activity of L. reuteri KCTC3594 was assayed in various ranges of pH, temperature, and salt concentrations. The enzyme activity was optimal at 40 °C and pH of 7.5. It was shown that the glutaminase was salt-tolerant because the enzyme activity was maintained 50% at 15% (w/v) salt concentrations. On the other hand, the enzyme was strongly inhibited up to 80% by 6-diazo-5-oxo-l-norleucine (10 mM) and iodoacetate (50 mM) indicating that the purified enzyme represents typical characteristics of glutaminase.
Keywords: Lactobacillus spp.; Glutaminase; Purification; Salt tolerance; Natural flavor

Expression, Purification, and C-terminal Site-Specific PEGylation of Cysteine-Mutated Glucagon-Like Peptide-1 by Mingming Gao; Hong Tian; Chen Ma; Xiangdong Gao; Wei Guo; Wenbing Yao (155-165).
Glucagon-like peptide-1 (GLP-1) is attracting increasing interest on account of its prominent benefits in type 2 diabetes. However, its clinical application is limited because of short biological half-life. This study was designed to produce a C-terminal site-specific PEGylated analog of cysteine-mutated GLP-1 (cGLP-1) to prolong its action. The gene of cGLP-1 was inserted into pET32a to construct a thioredoxinA fusion protein. After expression in BL21 (DE3) strain, the fusion protein was purified with Ni-affinity chromatography and then was PEGylated with methoxy-polyethylene glycol-maleimide (mPEG10K-MAL). The PEGylated fusion protein was purified with anion exchange chromatography and then was cleaved by enterokinase. The digested product was further purified with reverse-phase chromatography. Finally, 8.7 mg mPEG10K–cGLP-1 with a purity of up to 98% was obtained from the original 500 ml culture. The circular dichroism spectra indicated that mPEG10K–cGLP-1 maintained the secondary structure of native GLP-1. As compared with that of native GLP-1, the plasma glucose lowering activity of mPEG10K–cGLP-1 was significantly extended. These results suggest that our method will be useful in obtaining a large quantity of mPEG10K–cGLP-1 for further study and mPEG10K–cGLP-1 might find a role in the therapy of type 2 diabetes through C-terminal site-specific PEGylation.
Keywords: Glucagon-like peptide-1; Incretin; PEGylation; Diabetes; Enterokinase

Probiotic Bile Salt Hydrolase: Current Developments and Perspectives by Anil K. Patel; Reeta R. Singhania; Ashok Pandey; Sudhir B. Chincholkar (166-180).
Probiotic has modernized the current dietetic sense with novel therapeutic and nutritional benefits to the consumers. The presence of bile salt hydrolase (BSH) in probiotics renders them more tolerant to bile salts, which also helps to reduce the blood cholesterol level of the host. This review focuses on the occurrence of bile salt hydrolase among probiotics and its characterization, importance, applications, and genetics involved with recent updates. Research on bile salt hydrolase is still in its infancy. The current perspective reveals a huge market potential of probiotics with bile salt hydrolase. Intensive research in this field is desired to resolve some of the lacunae.
Keywords: Probiotics; Bile acids; Bile salt hydrolase; Cholesterol

Response surface methodology was used to optimize the fermentation medium for enhancing naringinase production by Staphylococcus xylosus. The first step of this process involved the individual adjustment and optimization of various medium components at shake flask level. Sources of carbon (sucrose) and nitrogen (sodium nitrate), as well as an inducer (naringin) and pH levels were all found to be the important factors significantly affecting naringinase production. In the second step, a 22 full factorial central composite design was applied to determine the optimal levels of each of the significant variables. A second-order polynomial was derived by multiple regression analysis on the experimental data. Using this methodology, the optimum values for the critical components were obtained as follows: sucrose, 10.0%; sodium nitrate, 10.0%; pH 5.6; biomass concentration, 1.58%; and naringin, 0.50% (w/v), respectively. Under optimal conditions, the experimental naringinase production was 8.45 U/mL. The determination coefficients (R 2) were 0.9908 and 0.9950 for naringinase activity and biomass production, respectively, indicating an adequate degree of reliability in the model.
Keywords: Naringinase; Response surface methodology; Central composite rotatable design; Staphylococcus xylosus ; Naringin

Polyol Additives Modulate the In Vitro Stability and Activity of Recombinant Human Phenylalanine Hydroxylase by Cátia Nascimento; João Leandro; Paulo Roque Lino; Luís Ramos; António José Almeida; Isabel Tavares de Almeida; Paula Leandro (192-207).
Phenylketonuria (PKU; OMIM 261600), the most common disorder of amino acid metabolism, is caused by a deficient activity of human phenylalanine hydroxylase (hPAH). Although the dietetic treatment has proven to be effective in preventing the psycho-motor impairment, much effort has been made to develop new therapeutic approaches. Enzyme replacement therapy with hPAH could be regarded as a potential form of PKU treatment if the reported in vitro hPAH instability could be overcome. In this study, we investigated the effect of different polyol compounds, e.g. glycerol, mannitol and PEG-6000 on the in vitro stability of purified hPAH produced in a heterologous prokaryotic expression system. The recombinant human enzyme was stored in the presence of the studied stabilizing agents at different temperatures (4 and −20 °C) during a 1-month period. Protein content, degradation products, specific activity, oligomeric profile and conformational characteristics were assessed during storage. The obtained results showed that the use of 50% glycerol or 10% mannitol, at −20 °C, protected the enzyme from loss of its enzymatic activity. The determined ΔG 0 and quenching parameters indicate the occurrence of conformational changes, which may be responsible for the observed increase in catalytic efficiency.
Keywords: Additives; Enzyme activity preservation; Human phenylalanine hydroxylase; Phenylketonuria; Protein stabilization

Hydrogen Generation Through Indirect Biophotolysis in Batch Cultures of the Nonheterocystous Nitrogen-Fixing Cyanobacterium Plectonema boryanum by Michael H. Huesemann; Tom S. Hausmann; Blaine M. Carter; Jared J. Gerschler; John R. Benemann (208-220).
The nitrogen-fixing nonheterocystous cyanobacterium Plectonema boryanum was used as a model organism to study hydrogen generation by indirect biophotolysis in nitrogen-limited batch cultures that were continuously illuminated and sparged with argon/CO2 to maintain anaerobiosis. The highest hydrogen-production rate (i.e., 0.18 mL/mg day or 7.3 µmol/mg day) was observed in cultures with an initial medium nitrate concentration of 1 mM at a light intensity of 100 µmol/m2 s. The addition of photosystem II (PSII) inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) did not reduce hydrogen-production rates relative to unchallenged controls for 50 to 150 h, and intracellular glycogen concentrations decreased significantly during the hydrogen generation period. The insensitivity of the hydrogen-production process to DCMU is indicative of the fact that hydrogen was not derived from water splitting at PSII (i.e., direct biophotolysis) but rather from electrons provided by intracellular glycogen reserves (i.e., indirect biophotolysis). It was shown that hydrogen generation could be sustained for long time periods by subjecting the cultures to alternating cycles of aerobic, nitrogen-limited growth and anaerobic hydrogen production.
Keywords: Indirect biophotolysis; Cyanobacteria; Glycogen; DCMU

Solid-Phase Synthesis of a Biotin Derivative and its Application to the Development of Anti-Biotin Antibodies by Ilias Papasarantos; Persefoni Klimentzou; Vassiliki Koutrafouri; Maria Anagnostouli; Christos Zikos; Maria Paravatou-Petsotas; Evangelia Livaniou (221-232).
A biotin derivative, namely biotin–aminocaproic acid–lysine (BAL), was synthesized with solid-phase chemistry, conjugated to a carrier-protein, and used for rabbit immunization. The aminocaproic acid–lysine “long-arm” was used in order to project the biotin-hapten above the carrier-protein surface. Lysine was selected due to its Nε-amino group, through which BAL was conjugated to the carrier-protein. BAL was synthesized on a commercially available resin with the Fmoc-solid-phase strategy; this has simplified the experimental procedure, overcome the need for intermediate purification steps, and led to a final product of high purity, with high yield. The anti-BAL antibodies recognized free biotin, as shown with an in-house-developed ELISA, in which biotin conjugated to a synthetic “lysine–dendrimer” was used to coat the ELISA microwells. In immunocytology and Western-blot experiments, the anti-BAL antibodies led to similar results with those obtained with streptavidin. Synthetic derivatives of hapten molecules that can be easily prepared with solid-phase chemistry, such as BAL, may be used for the development of specific antibodies for the corresponding hapten.
Keywords: Solid-phase synthesis; Anti-biotin antibodies; Streptavidin; ELISA; “Lysine–dendrimer”; Immunocytology; Western blot; Anti-hapten antibodies

The Utilization of Gum Tragacanth to Improve the Growth of Rhodotorula aurantiaca and the Production of γ-Decalactone in Large Scale by Mohamed Alchihab; Jacqueline Destain; Mario Aguedo; Jean-Paul Wathelet; Philippe Thonart (233-241).
The production of γ-decalactone and 4-hydroxydecanoic acid by the psychrophilic yeast R. aurantiaca was studied. The effect of both compounds on the growth of R. aurantiaca was also investigated and our results show that γ-decalactone must be one of the limiting factors for its production. The addition of gum tragacanth to the medium at concentrations of 3 and 4 g/l seems to be an adequate strategy to enhance γ-decalactone production and to reduce its toxicity towards the cell. The production of γ-decalactone and 4-hydroxydecanoic acid was significantly higher in 20-l bioreactor than in 100-l bioreactor. By using 20 g/l of castor oil, 6.5 and 4.5 g/l of γ-decalactone were extracted after acidification at pH 2.0 and distillation at 100 °C for 45 min in 20- and 100-l bioreactors, respectively. We propose a process at industrial scale using a psychrophilic yeast to produce naturally γ-decalactone from castor oil which acts also as a detoxifying agent; moreover the process was improved by adding a natural gum.
Keywords: γ-Decalactone; Gum tragacanth; 4-Hydroxydecanoic acid; R. aurantiaca ; Toxicity

Two DNA fragments containing the entire coding sequences of lactate dehydrogenase (LDH; ldhL1 and ldhD), whose enzymes have high activity for bioconversion of phenylpyruvate (PPA) to phenyllactate (PLA), were amplified from Lactobacillus plantarum SK002 using PCR. Sequencing showed open reading frames of 963 bp (ldhL1) and 999 bp (ldhD) encoding putative proteins of 320 and 332 amino acid residues, respectively. The LDH genes were cloned into an expression vector pET-22b(+) and expressed in Escherichia coli BL21(DE3). The purified recombinant l1-LDH and d-LDH had approximate (SDS-PAGE) molecular weights of 35 and 40 kDa, respectively. l1-LDH and d-LDH had PPA bioconversion specific activities of 71.06 and 215.84 U/mg with K m values of 3.96 and 5.4 mM, respectively. The rl1-LDH and rd-LDH showed maximum enzyme activity at 30 and 40 °C while both had optimum activity at pH 6.0. l1-LDH exhibited a higher pH and temperature stability than d-LDH. The results show that the his-tagged L. plantarum SK002 d- and l1-LDHs are efficient catalysts for bioconversion of PPA to PLA.
Keywords: Lactate dehydrogenase (LDH); Lactobacillus plantarum SK002; Phenyllactate (PLA); Phenylpyruvate (PPA); Gene cloning; Enzymatic characterization

The optimization of process parameters for high amylase production by Saccharomycopsis fibuligera A11 in solid-state fermentation was carried out using central composite design. Finally, the optimal parameters obtained with the response surface methodology (RSM) were moisture 610.0 ml/kg, inoculum 30.0 ml (OD600 nm = 20.0)/kg, the amount ratio of wheat bran to rice husk 0.42, cassava starch concentration 20.0 g/kg, temperature 28 °C, and natural pH. Under the optimized conditions, 4,296 U/g of dry substrate of amylase activity was reached in the solid-state fermentation culture of the yeast strain A11 within 160 h, whereas the predicted maximum amylase activity of 4,222 U/g of dry substrate of amylase activity was derived from the RSM regression. It was found that cassava starch can be actively converted into monosaccharides and oligosaccharides by the crude amylase.
Keywords: Amylase; Saccharomycopsis fibuligera ; Cassava starch; Solid-state fermentation; Response surface methodology; Hydrolysis

Pretreatment is being the first and most expensive step, it has pervasive impacts on all other steps in overall conversion process. There are several pretreatment methods using physical, chemical, and biological principles which are under various stages of investigation. Extrusion can be used as one of the physical pretreatment methods towards biofuel production. The objective of this study was to evaluate the effect of barrel temperature and screw speed on sugar recovery from corn stover, to select a suitable enzyme combination and its ratio. Corn stover was pretreated in a single screw extruder with five screw speeds (25, 50, 75, 100, and 125 rpm) and five barrel temperatures (25, 50, 75, 100, and 125 °C). In order to select a suitable enzyme combination and ratio, different levels of cellulase and β-glucosidase, multienzyme complex and β-glucosidase were used during saccharification of pretreated corn stover. From the statistical analysis, it was found that screw speed and temperature had a significant effect on sugar recovery from corn stover. Higher glucose, xylose, and combined sugar recovery of 75, 49, and 61%, respectively, were recorded at 75 rpm and 125 °C. This pretreatment condition resulted in 2.0, 1.7, and 2.0 times higher than the control sample using 1:4 cellulase and β-glucosidase combination.
Keywords: Biomass; Pretreatment; Extrusion; Screw speed; Temperature; Enzymatic hydrolysis; Sugar recovery

Production and Characterization of a Novel Laccase with Cold Adaptation and High Thermal Stability from an Isolated Fungus by Zhixin Wang; Yujie Cai; Xiangru Liao; Feng Zhang; Dabing Zhang; Zhiling Li (280-294).
A new white-rot fungus SYBC-L1, which could produce an extracellular laccase, was isolated from a decayed Elaeocarpus sylvestris. The strain was identified as Pycnoporus sp. SYBC-L1 according to the morphological characteristics and ribosomal ITS1-5.8S-ITS2 RNA genomic sequence analysis. The highest laccase activity of 24.1 U ml−1, which was approximately 40-fold than that in basal medium, was achieved in optimal culture medium in submerged fermentation. The laccase produced by Pycnoporus sp. SYBC-L1 was not only a cold adaptation enzyme with a relative catalytic activity of 30.2% at 0°C but also a high thermostable enzyme. The half-lives at 60, 70 and 80°C were 85.5, 37.2, and 2.6 h, respectively. The laccase could effectively decolorize weak acid blue AS and diamond black PV up to 88% and 74.7%, respectively, within 2 h in the absence of any redox mediators. The results suggested Pycnoporus sp. SYBC-L1 was a potential candidate for laccase production and industrial application.
Keywords: Laccase; Pycnoporus sp. SYBC-L1; Submerged fermentation; Cold adaptation; Thermal stability; Decolorization

Horticultural waste in wood chips form collected from a landscape company in Singapore was utilized as the substrate for the production of cellulase and hemicellulase under solid-state fermentation by Trichoderma reesei RUT-C30. The effects of substrate pretreatment methods, substrate particle size, incubation temperature and time, initial medium pH value, and moisture content on cellulase and hemicellulase production were investigated. Enzyme complex was obtained at the optimal conditions. This enzyme mixture contained FPase (15.0 U/g substrate dry matter, SDM), CMCase (90.5 U/g SDM), β-glucosidase (61.6 U/g SDM), xylanase (52.1 U/g SDM), and β-xylosidase (10.4 U/g SDM). The soluble protein concentration in the enzyme complex was 26.1 mg/g SDM. The potential of the crude enzyme complex produced was demonstrated by the hydrolysis of wood chips, wood dust, palm oil fiber, and waste newspaper. The performance of the crude enzyme complex was better than the commercial enzyme blend.
Keywords: Horticultural waste; Trichoderma reesei ; Cellulase; Hemicellulase; Solid-state fermentation; Saccharification