Applied Biochemistry and Biotechnology (v.162, #5)

Diversity of Sulfur-Oxidizing Bacteria in Greenwater System of Coastal Aquaculture by Kishore Kumar Krishnani; V. Kathiravan; M. Natarajan; M. Kailasam; S. M. Pillai (1225-1237).
Reduced sulfur compounds produced by the metabolism are the one of the major problems in aquaculture. In the present study, herbivorous fishes have been cultured as biomanipulators for secretions of slime, which enhanced the production of greenwater containing beneficial bacteria. The genes encoding soxB which is largely unique to sulfur-oxidizing bacteria (SOB) due to its hydrolytic function has been targeted for examining the diversity of SOB in the green water system of coastal aquaculture. Novel sequences obtained based on the sequencing of metagenomic clone libraries for soxB genes revealed the abundance of SOB in green water system. Phylogenetic tree constructed from aligned amino acid sequences demonstrated that different clusters have only 82–93% match with Roseobacter sp., Phaeobacter sp., Roseovarius sp., Sulfitobacter sp., Ruegeria sp., and Oceanibulbus sp. The level of conservation of the soxB amino acid sequences ranged from 42% to 71%. 16S rRNA gene analyses of enrichment culture from green water system revealed the presence of Pseudoxanthomonas sp., which has 97% similarity with nutritionally fastidious Indian strain of Pseudoxanthomonas mexicana—a sulfur chemolithotrophic γ-proteobacterium. Our results illustrate the relevance of SOB in the functioning of the green water system of coastal shrimp aquaculture for oxidation of reduced sulfur compounds, which in turn maintain the sulfide concentration well within the prescribed safe levels.
Keywords: Diversity; Sulfur-oxidizing bacteria; soxB gene; 16S rRNA; Greenwater; Coastal aquaculture

In Vitro Renaturation of Alkaline Family G/11 Xylanase via a Folding Intermediate: α-Crystallin Facilitates Refolding in an ATP-Independent Manner by Tanmay Dutta; Arindam Bhattacharjee; Uddalak Majumdar; Saugata Sinha Ray; Rupam Sahoo; Sanjay Ghosh (1238-1248).
In this study, alkaliphilic family G/11 xylanase from alkali-tolerant filamentous fungi Penicillium citrinum MTCC 6489 was used as a model system to gain insight into the molecular aspects of unfolding/refolding of alkaliphilic glycosyl hydrolase protein family. The intrinsic protein fluorescence suggested a putative intermediate state of protein in presence of 2 M guanidium hydrochloride (GdmCl) with an emission maximum of 353 nm. Here we studied the refolding of GdmCl-denatured alkaline xylanase in the presence and the absence of a multimeric chaperone protein α-crystallin to elucidate the molecular mechanism of intramolecular interactions of the alkaliphilic xylanase protein that dictates its extremophilic character. Our results, based on intrinsic tryptophan fluorescence and hydrophobic fluorophore 8-anilino-1- naphthalene sulfonate-binding studies, suggest that α-crystallin formed a complex with a putative molten globule-like intermediate in the refolding pathway of xylanase in an ATP-independent manner. A 2 M GdmCl is sufficient to denature alkaline xylanase completely. The hydrodynamic radius (RH) of a native alkaline xylanase is 4.0, which becomes 5.0 in the presence of 2 M GdmCl whereas in presence of the higher concentration of GdmCl RH value was shifted to 100, indicating the aggregation of denatured xylanase. The α-crystallin·xylanase complex exhibited the recovery of functional activity with the extent of ~43%. Addition of ATP to the complex did not show any significant effect on activity recovery of the denatured protein.
Keywords: Alkaline xylanase; Denaturation; Refolding; α-Crystallin; Guanidium hydrochloride

Generation of a Novel High-Affinity Monoclonal Antibody with Conformational Recognition Epitope on Human IgM by Sina Sarikhani; Manouchehr Mirshahi; Mohammad Reza Gharaati; Tooran Mirshahi (1249-1257).
As IgM is the first isotype of antibody which appears in blood after initial exposure to a foreign antigen in the pattern of primary response, detection, and quantification of this molecule in blood seems invaluable. To approach these goals, generation, and characterization of a highly specific mAb (monoclonal antibody) against human IgM were investigated. Human IgM immunoglobulins were used to immunize Balb/c mice. Spleen cells taken from the immunized animals were fused with SP2/O myeloma cells using PEG (polyethylene glycol, MW 1450) as fusogen. The hybridomas were cultured in HAT containing medium and supernatants from the growing hybrids were screened by enzyme-linked immunosorbent assay (ELISA) using plates coated with pure human IgM and the positive wells were then cloned at limiting dilutions. The best clone designated as MAN-1, was injected intraperitoneally to some Pristane-injected mice. Anti-IgM mAb was purified from the animals’ ascitic fluid by protein-G sepharose followed by DEAE-cellulose ion exchange chromatography. MAN-1 interacted with human IgM with a very high specificity and affinity. The purity of the sample was tested by SDS-PAGE and the affinity constant was measured $$ left( {{K_{ ext{a}}} = { ext{3}}.{ ext{5}} imes { ext{1}}{0^{ ext{9}}}{{ ext{M}}^{{ ext{ - 1}}}}} ight) $$ . Immunoblotting and competitive ELISA were done and the results showed that the harvested antibody recognizes a conformational epitope on the μ chain of human IgM and there was no cross-reactivity with other subclasses of immunoglobulins. Furthermore, isotyping test was done and the results showed the subclass of the obtained mAb which was IgG1κ.
Keywords: Monoclonal antibody; IgM; Anti-human IgM antibody

Fusion toxins are an emerging class of targeted therapeutics for the treatment of cancer. Diphtheria toxin-stem cell factor (DT-SCF) is one such novel fusion toxin designed to target malignancies expressing c-kit. Since, c-kit overexpression has been reported on many types of cancers, it appeared to be a reasonably good molecule to target. In the present study, we report construction, expression, purification, and characterization of DT-SCF. DT-SCF gene coding for 1–387 amino acids of diphtheria toxin, His-Ala linker, 2–141 amino acids of SCF was cloned into expression vector with C terminal His tag. The induced DT-SCF protein was exclusively expressed in insoluble fraction. Purification of DT-SCF was achieved by inclusion body isolation and metal affinity chromatography under denaturing and reducing conditions. Purified DT-SCF was renatured partially on-column by gradually reducing denaturant concentration followed by complete refolding through rapid dilution technique. Cell viability assay provided the evidence that DT-SCF is a potent cytotoxic agent selective to cells expressing c-kit. The novelty of this study lies in employing SCF as a ligand in construction of fusion toxin to target wide range of malignancies expressing c-kit. Efficacy of DT-SCF fusion toxin was demonstrated over a range of malignancies such as chronic myeloid leukemia (K562), acute lymphoblastic leukemia (MOLT4), pancreatic carcinoma (PANC-1), and cervical carcinoma (HeLa 229). This is the first study reporting specificity and efficacy of DT-SCF against tumor cells expressing c-kit. There was significant correlation (P = 0.007) between c-kit expression on cells and their sensitivity to DT-SCF fusion toxin.
Keywords: Diphtheria toxin; SCF; Immunotoxins; Protein expression; Refolding; Cell cycle analysis; Cytotoxicity

Thermal Drying of Lactobacillus delbrueckii subsp. bulgaricus and its Efficient Use as Starter for Whey Fermentation and Unsalted Cheese Making by Eleftheria Katechaki; Theodoros Solomonidis; Argyro Bekatorou; Athanasios A. Koutinas (1270-1285).
Lactobacillus bulgaricus grown on whey was dried by a simple thermal drying method at the range 35–55°C and its efficiency for lactic acid fermentation of whey was evaluated. Drying of cells in whey suspension in the examined temperature range did not affect significantly their viability (82–87% survival), indicating a protective effect of whey as both growth and drying medium. The kinetics of fermentation of whey and mixtures of whey/molasses using the dried culture were comparable to those of non-dried cells, and only low pH had a detrimental effect on the fermentation ability of the dried cells. Furthermore, dried L. bulgaricus, free or immobilized on casein coagulates, was used as starter for the production of unsalted hard-type cheese. The effects of the amount of starter culture and the immobilization technique, the evolution of microbial counts, and the sensory properties of the produced cheeses were evaluated during ripening at various temperatures.
Keywords: Lactobacillus delbrueckii subsp. bulgaricus ; Thermal drying; Whey; Lactic acid fermentation; Volatiles; Unsalted hard-type cheese; Immobilization

The Effect of pH on Continuous Biohydrogen Production from Swine Wastewater Supplemented with Glucose by Yecong Li; Jun Zhu; Xiao Wu; Curtis Miller; Liang Wang (1286-1296).
The effect of pH on hydrogen production from liquid swine manure supplemented with glucose by a mixed culture of fermentative bacteria in an anaerobic sequencing batch reactor was evaluated in this study. At 37 ± 1 °C, five pH values ranging from 4.7 to 5.9 at an increment of 0.3 were tested at a hydraulic retention time (HRT) of 16 h. The results showed that at this HRT, the optimal pH for hydrogen production was 5.0, under which the biogas comprised 33.57 ± 5.65% of hydrogen with a production rate of 8.88 ± 2.94 L-H2/day and a yield of 1.48 ± 0.49 L-H2/L liquid swine manure. The highest biomass concentration, highest butyric acid to acetic acid ratio, lowest propionic acid concentration, and the best stability were all found at pH 5.0, while the highest CH4 productivity was found at pH 5.9. For efficient hydrogen production, oxygen content should be controlled under 2%, beyond which an inverse linear relationship (R 2 = 0.986) was observed.
Keywords: Hydrogen production; Swine manure; pH effect

Cloning, Expression, and Cell Localization of a Novel Small Heat Shock Protein Gene: BmHSP25.4 by Qing Sheng; Jiayin Xia; Zuoming Nie; Yaozhou Zhang (1297-1305).
Using molecular approaches, a new member of the Bombyx mori small heat shock protein family was cloned and characterized. The isolated gene contains an open reading frame of 672 bp, encodes a polypeptide of 223 amino acid residues with a predicted molecular mass of 25.4 kDa, and is therefore named BmHSP25.4. The gene codes for a protein homologous to the previously characterized HSP20.4 and HSP19.9. Western blotting analysis revealed that BmHSP25.4 existed in the fifth-instar larva’s fatty body and blood tissues. Immunohistochemistry assay also showed that BmHSP25.4 was located in the fifth-instar larva’s fatty body. The results of above studies have indicated constitutive expression of BmHSP25.4 in fatty body, blood tissues, and Bm5 cells. Finally, we examined the effect of heat stress on localization of BmHSP25.4 using anti-BmHSP25.4 polyclonal antibody by immunofluorescence. Under normal conditions, BmHSP25.4 was mostly found in the cytoplasm. However, after heat treatment, most of BmHSP25.4 distributed in the cell membrane. After 3 h of recovery following the heat shock treatment, the localization of BmHSP25.4 was the same as that under normal conditions.
Keywords: BmHSP25.4; Prokaryotic expression; Purification; Polyclonal antibody preparation; Tissue localization; Subcellular localization

Rice straw hemicellulosic hydrolysate was used as fermentation medium for ethanol production by Pichia stipitis NRRL Y-7124. Shaking bath experiments were initially performed aiming to establish the best initial xylose concentration to be used in this bioconversion process. In the sequence, assays were carried out under different agitation (100 to 200 rpm) and aeration (V flask/V medium ratio varying from 2.5 to 5.0) conditions, and the influence of these variables on the fermentative parameters values (ethanol yield factor, Y P/S; cell yield factor, Y X/S; and ethanol volumetric productivity, Q P) was investigated through a 22 full-factorial design. Initial xylose concentration of about 50 g/l was the most suitable for the development of this process, since the yeast was able to convert substrate in product with high efficiency. The factorial design assays showed a strong influence of both process variables in all the evaluated responses. The agitation and aeration increase caused a deviation in the yeast metabolism from ethanol to biomass production. The best results (Y P/S = 0.37 g/g and Q P = 0.39 g/l.h) were found when the lowest aeration (2.5 V flask/V medium ratio) and highest agitation (200 rpm) levels were employed. Under this condition, a process efficiency of 72.5% was achieved. These results demonstrated that the establishment of adequate conditions of aeration is of great relevance to improve the ethanol production from xylose by Pichia stipitis, using rice straw hemicellulosic hydrolysate as fermentation medium.
Keywords: Ethanol; Fermentation; Hemicellulosic hydrolysate; Pichia stipitis ; Rice straw

Dry Red Wine Making Using Yeast Immobilized on Cork Pieces by Argyris Tsakiris; Panagiotis Kandylis; Argyro Bekatorou; Yiannis Kourkoutas; Athanasios A. Koutinas (1316-1326).
The commercial Saccharomyces cerevisiae Uvaferme 299 wine yeast was immobilized on cork pieces to produce a biocatalyst for use in dry red wine making. The immobilized biocatalyst was suitable for clarified and non-clarified grape must fermentation at ambient and low temperatures (0–25 °C). Fermentation times were low and very low amounts of residual sugar were detected, showing suitability for dry wine production. The presence of suspended solids in the non-clarified must did not affect the activity of the immobilized cells. Complete fermentation of sugars at 0 °C was possible with immobilized Uvaferme 299, although not a cryotolerant strain, indicating a cryoprotective effect of cork. The presence of cork did not affect the composition of major volatiles with methanol and acetaldehyde kept at low levels. Reduction of amyl alcohols on total volatiles was also observed in wines fermented at low temperatures. Differences in the headspace aroma profile in wines produced by immobilized and free cells were found by GC–MS analysis, but no cork taint compounds were detected.
Keywords: Dry red wine making; Cork; Yeast immobilization; Low temperature fermentation; Aroma volatiles

Isolation and Characterization of Exopolysaccharides Produced by the Cyanobacterium Limnothrix redekei PUPCCC 116 by J. I. S. Khattar; D. P. Singh; Namita Jindal; N. Kaur; Y. Singh; P. Rahi; A. Gulati (1327-1338).
Limnothrix redekei PUPCCC 116, a filamentous cyanobacterium, has been identified through 16S rRNA gene sequencing. Exopolysaccharides (EPS) of this organism have been isolated and characterized chemically, and its rheological properties were compared with commercial xanthan. The organism produced 304 µg EPS/ml culture in 21 days. The rate of EPS production was maximum (313 µg EPS/mg protein/day) during the initial days of growth, and it decreased to 140 µg EPS/mg protein/day during 18-21 days of growth. Chemical analysis of EPS revealed the presence of glucose/mannose, ribose, rhamnose, and uronic acid. Fourier transformed infrared spectrum of EPS further revealed the presence of methyl and carboxyl groups besides C–N groups indicating the presence of peptidyl moieties. Elemental analysis of EPS showed the presence of 4.97% N. The organism under continuous light produced 102% more EPS compared to when grown under a light/dark cycle of 14/10 h. The rheological properties of EPS were comparable with commercial xanthan gum.
Keywords: Cyanobacterium; Exopolysaccharides; Limnothrix redekei ; Monomer composition; Rheology; 16S rRNA gene

Purification and Characterization of a Novel Thermostable Mycelial Lectin from Aspergillus terricola by Ram Sarup Singh; Ranjeeta Bhari; Hemant Preet Kaur; Monika Vig (1339-1349).
Lectin has been isolated from mycelia of Aspergillus terricola by single step purification on porcine stomach mucin-Sepharose 4B affinity column. Lectin could be effectively purified with 75% recovery and 4.47-fold increase in specific activity. Lectin migrated as a single band on SDS-PAGE with an apparent molecular mass of 32.5 kDa. Sugar inhibition assay revealed that the lectin did not strongly interact with most carbohydrates and their derivatives tested while strong binding affinity to d-glucose, d-sucrose, N-acetyl-d-galactosamine, asialofetuin, porcine stomach mucin, and bovine submaxillary mucin was indicated. Neuraminidase and protease treatment to erythrocytes enhanced lectin titre. Lectin activity was stable within the pH range of 7.0–10.5. A. terricola lectin displayed remarkable thermostability and remained unaffected upon incubation at 70 °C for 2.5 h. Lectin did not require metal ions for its activity. Incubation with denaturants (urea, thiourea, and guanidine–HCl) substantially reduced lectin activity. Carbohydrate analysis revealed that it is a glycoprotein with 9.76% total sugars.
Keywords: Aspergillus terricola ; Lectin; Hemagglutination; Inhibition; Purification

Evaluation of Lipase Production by Genetic Algorithm and Particle Swarm Optimization and Their Comparative Study by Vijay Kumar Garlapati; Pandu Ranga Vundavilli; Rintu Banerjee (1350-1361).
This paper presents the nature-inspired genetic algorithm (GA) and particle swarm optimization (PSO) approaches for optimization of fermentation conditions of lipase production for enhanced lipase activity. The central composite non-linear regression model of lipase production served as the optimization problem for PSO and GA approaches. The overall optimized fermentation conditions obtained thereby, when verified experimentally, have brought about a significant improvement (more than 15 U/gds (gram dry substrate)) in the lipase titer value. The performance of both optimization approaches in terms of computational time and convergence rate has been compared. The results show that the PSO approach (96.18 U/gds in 46 generations) has slightly better performance and possesses better convergence and computational efficiency than the GA approach (95.34 U/gds in 337 generations). Hence, the proposed PSO approach with the minimal parameter tuning is a viable tool for optimization of fermentation conditions of enzyme production.
Keywords: Optimization; Lipase; Fermentation; Genetic algorithm; Particle swarm optimization

Isolation and Characterization of a Novel Thermophilic-Organic Solvent Stable Lipase From Acinetobacter baylyi by Sasithorn Uttatree; Pakorn Winayanuwattikun; Jittima Charoenpanich (1362-1376).
The benzene tolerant Acinetobacter baylyi isolated from marine sludge in Angsila, Thailand could constitutively secrete lipolytic enzymes. The enzyme was successfully purified 21.89-fold to homogeneity by ammonium sulfate precipitation and gel-permeable column chromatography with a relative molecular mass as 30 kDa. The enzyme expressed maximum activity at 60°C and pH 8.0 with p-nitrophenyl palmitate as a substrate and found to be stable in pH and temperature ranging from 6.0-9.0 to 60-80°C, respectively. A study on solvent stability revealed that the enzyme was highly resisted to many organic solvents especially benzene and isoamyl alcohol, but 40% inhibited by decane, hexane, acetonitrile, and short-chain alcohols. Lipase activity was completely inhibited in the presence of Fe2+, Mn2+, EDTA, SDS, and Triton X-100 while it was suffered detrimentally by Tween 80. The activity was enhanced by phenylmethylsulfonyl fluoride (PMSF), Na+, and Mg2+ and no significant effect was found in the presence of Ca2+ and Li+. Half of an activity was retained by Ba2+, Ag+, Hg+, Ni2+, Zn2+, and DTT. The enzyme could hydrolyze a wide range of p-nitrophenyl esters, but preferentially medium length acyl chains (C8-C12). Among natural oils and fats, the enzyme 11-folds favorably catalyzed the hydrolysis of rice bran oil, corn oil, sesame oil, and coconut oil in comparison to palm oil. Moreover, the transesterification activity of palm oil to fatty acid methyl esters (FAMEs) revealed 31.64 ± 1.58% after 48 h. The characteristics of novel A. baylyi lipase, as high temperature stability, organic solvent tolerance, and transesterification capacity from palm oil to FAMEs, indicate that it could be a vigorous biocatalyzer in the prospective fields as bioenergy industry or even in organic synthesis and pharmaceutical industry.
Keywords: Solvent-stable lipase; Thermophilic lipase; Acinetobacter baylyi ; Marine sludge; Biodiesel production

Use of Cellulase Inhibitors to Produce Cellobiose by Misook Kim; Donal F. Day (1379-1390).
The economics driving biorefinery development requires high value-added products such as cellobiose for financial feasibility. This research describes a simple technology for increasing cellobiose yields during lignocellulosic hydrolysis. The yield of cellobiose produced during cellulose hydrolysis was maximized by modification of reaction conditions. The addition of an inhibitor from the group that includes glucose oxidase, gluconolactone, and gluconic acid during cellulase hydrolysis of cellulose increased the amount of cellobiose produced. The optimal conditions for cellobiose production were determined for four factors; reaction time, cellulase concentration, cellulose concentration, and inhibitor concentration using a Box-Behnken experimental design. Gluconolactone in the cellulase system resulted in the greatest production of cellobiose (31.2%) from cellulose. The yield of cellobiose was 23.7% with glucose oxidase, similar to 21.9% with gluconic acid.
Keywords: Cellobiose; Cellulase; Glucose oxidase; Gluconolactone; Gluconic acid; Cellulase inhibition

Cloning and Identification of a Novel P-II Class Snake Venom Metalloproteinase from Gloydius halys by Shou-Tao Zhang; Ping Lu; Yun-Fei Qin; San-Jun Chen; Ai-Guang Guo (1391-1402).
Ahpfibrase was a new snake venom metalloproteinase (SVMP) which was cloned from Gloydius halys. The cDNA sequence with 1,891 base pairs encodes an open reading frame of 477 amino acids which includes a 17 amino acid signal peptide, plus a 171 amino acid segment of zymogen-like propeptide, a metalloproteinase domain of 200 amino acids, a spacer of 16 amino acids, and a disintegrin-like peptide of 73 amino acids. The metalloproteinase domain contained a conserved signature zinc-binding motif HEXXHXXGXXH in the catalytic region and a methionine-turn CIM. To determine the activity of ahpfibrase, the coding region including both the metalloproteinase domain and disintegrin region was amplified by PCR, inserted into the pET25b(+) vector, and expressed in Escherichia coli. The recombinant protein was recovered from inclusion bodies with 8 M urea and refolding was performed by fed-batch dilution method, and purified recombinant ahpfibrase showed the fibrinolytic activity and platelet aggregation–inhibition ability.
Keywords: Expression; cDNA; Cloning; Gloydius halys ; Refolding; Snake venom metalloproteinase

Cytokines Expression Induced by Ganoderma sinensis Fungal Immunomodulatory Proteins (FIP-gsi) in Mouse Spleen Cells by Qizhang Li; Xuefei Wang; Yiyuan Chen; Juan Lin; Xuanwei Zhou (1403-1413).
Ganoderma sinensis fungal immunomodulatory protein (FIP-gsi) was a new member of FIPs family. Based on the cloning of FIP-gsi gene from G. sinensis, this paper reported that FIP-gsi gene was expressed in Escherichia coli expression system. Then, the recombinant proteins were analyzed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Finally, the immunomodulatory activity was examined by inducing cytokine genes expression. The results showed that the recombinant FIP-gsi protein could be expressed in E. coli and got the yield of about 25% of the soluble form in the total soluble protein. The FIP-gsi protein was composed of 111 amino acids, and the sequence of homologous rate was 88.6% with FIP-glu (LZ-8). Furthermore, it could enhance the levels of interleukin (IL)-2, IL-3, IL-4, interferon gamma, tumor necrosis factor alpha, and IL-2 receptor (IL-2R) in mouse spleen cells.
Keywords: Ganoderma sinensis ; Fungal immunomodulatory protein; Expression; Cytokine

Estimation of Treatment Time for Microbial Preprocessing of Biomass by Swetha Mahalaxmi; Colin R. Jackson; Clint Williford; Charles L. Burandt (1414-1422).
Biochemical conversion of lignocellulosic biomass to ethanol involves size reduction, preprocessing, pretreatment, enzyme hydrolysis, and fermentation. In recent years, microbial preprocessing has been gaining attention as a means to produce labile biomass for lessening the requirement of pretreatment severity. However, loss of sugars due to microbial consumption is a major consequence, suggesting its minimization through optimization of nutrients, temperature, and preprocessing time. In this work, we emphasized estimation of fungal preprocessing time, at which higher sugar yields can be achieved after preprocessing and enzyme hydrolysis. The estimation is based on the enzymatic activity profile obtained by treating switchgrass with Phanerochaete chrysosporium for 28 days. Enzyme assays were conducted once in every 7 days for 28 days, for activities of phenol oxidase, peroxidase, β-glucosidase, β-xylosidase, and cellobiohydrolase. We found no activity for phenol oxidase and peroxidase, but the greatest activities for cellulases on the seventh day. We then treated switchgrass for 7 days with P. chrysosporium and observed that the preprocessed switchgrass had higher glucan (39%), xylan (17.5%), and total sugar yields (25.5%) than the unpreprocessed switchgrass (34%, 37.5%, and 20.5%, respectively, p < 0.05). This verifies the utility of using enzyme assays for initial estimation of preprocessing time to enhance sugar yields.
Keywords: Microbial preprocessing; Fungal preprocessing; Preprocessing time; Enzyme activity assays; Phanerochaete chrysosporium ; Partial cellulose degradation

2-Deoxyribose-5-phosphate aldolase (DERA) catalyzes a sequential aldol reaction useful in synthetic chemistry. In this work, the effect of a feeding strategy on the production of a thermophilic DERA was investigated in fed-batch cultures of recombinant Escherichia coli BL21 (pET303-DERA008). The predetermined specific growth rate (µ set) was evaluated at 0.20, 0.15, and 0.10 h−1, respectively. The DERA concentration and volumetric productivity were associated with µ set. The cells synthesized the enzyme most efficiently at µ set = 0.15 h−1. The maximum enzyme concentration (5.12 g/L) and total volumetric productivity (0.256 g L−1 h−1) obtained were over 10 and five times higher than that from traditional batch cultures. Furthermore, the acetate concentration remained at a relatively low level, less than 0.4 g/L, under this condition which would not inhibit cell growth and target protein expression. Thus, a specific growth rate control strategy has been successfully applied to induce fed-batch cultures for the maximal production of the thermophilic 2-deoxyribose-5-phosphate aldolase.
Keywords: 2-Deoxyribose-5-phosphate aldolase; Fed-batch culture; Feeding strategy; Enzyme production; Acetic acid

Expression and Immunogenicity of a Recombinant Chimeric Protein of Human Colorectal Cancer Antigen GA733-2 and an Fc Antibody Fragment in Stably Transformed Drosophila melanogaster S2 Cells by Yeon Ju Seok; Kyung Il Kim; Ki Hyun Yoo; Jeon Hwang-Bo; Hyun Ho Lee; Dong Hwa Shon; Ki Sung Ko; Hyung Sik Kang; Kyung Jin Lee; Doo-Byoung Oh; Young Hee Joung; In Sik Chung (1435-1445).
Human colorectal cancer antigen GA733-2 fused to the immunoglobin Fc fragment (GA733-2-Fc) was expressed in stably transformed Drosophila melanogaster S2 cells, and the immunogenicity of recombinant GA733-2-Fc was investigated in mice. Recombinant GA733-2-Fc was secreted into a culture medium with a molecular mass of approximately 65 kDa. Recombinant GA733-2-Fc was purified to homogeneity using affinity fractionation with Protein A sepharose 4 Fast Flow. Recombinant GA733-2-Fc proteins elicited production of specific antibodies against recombinant GA733-2 by immunization through an intraperitoneal route. Recombinant GA733-2-Fc-induced antibodies showed a binding activity to human colorectal carcinoma HCT-116 cells. Secretory recombinant GA733-2-Fc from Drosophila S2 cell systems can be used as an effective experimental antigen for research in cancer vaccine development.
Keywords: Cancer antigen; Drosophila melanogaster S2 cells; Expression; Immunogenicity; Recombinant GA733-2-Fc

Using enrichment procedures, five strains that can utilize soybean phytosterols as the sole carbon source were isolated from steroids-contaminated soil samples. Among the isolated strains, the strain NwIB-01 with the highest steroid degradation ability was identified as Mycobacterium neoaurum by morphological, physiological, biochemical tests and 16S rRNA sequence analysis. Meanwhile, the key enzyme gene, which was involved in steroid metabolism and encoding 395-amino acid 3-ketosteroid 9α-hydroxylase (KSH), was obtained from M. neoaurum NwIB-01 with the assistance of homology analysis and chromosome walking. To our best knowledge, this is the first report to the gene of key enzyme KSH from M. neoaurum. Strain NwIB-01 exhibited powerful ability of cleaving the side chain specifically from soybean phytosterols to accumulate 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD). It was showed that when cultured in 15 g/l phytosterols, the yield of ADD reached 4.23 g/l while accompanied by 1.76 g/l AD in 96-h-old culture (the molar yield of AD + ADD is 64.7%). The strain NwIB-01 can be applied as excellent phytosterols-transformation strains in potential industrial applications.
Keywords: Mycobacterium neoaurum ; Extraction chromatography; Screening; Phytosterols; 4-Androstene-3,17-dione (AD); 1,4-Androstadiene-3,17-dione (ADD); 3-Ketosteroid 9α-hydroxylase (KSH)

An Oxidant- and Organic Solvent-Resistant Alkaline Metalloprotease from Streptomyces olivochromogenes by Jaya Ram Simkhada; Seung Sik Cho; Seong Ju Park; Poonam Mander; Yun Hee Choi; Hyo Jeong Lee; Jin Cheol Yoo (1457-1470).
Organic solvent- and detergent-resistant proteases are important from an industrial viewpoint. However, they have been less frequently reported and only few of them are from actinomycetes. A metalloprotease from Streptomyces olivochromogenes (SOMP) was purified by ion exchange with Poros HQ and gel filtration with Sepharose CL-6B. Apparent molecular mass of the enzyme was estimated to be 51 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and gelatin zymography. The activity was optimum at pH 7.5 and 50 °C and stable between pH 7.0 and 10.0. SOMP was stable below 45 °C and Ca2+ increased its thermostability. Ca2+ enhanced while Co2+, Cu2+, Zn2+, Mn2+, and Fe2+ inhibited the activity. Ethylenediaminetetraacetic acid and ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, but not phenylmethylsulfonyl fluoride, aprotinin, and pefabloc SC, significantly suppressed the activity, suggesting that it might be a metalloprotease. Importantly, it is highly resistant against various detergents, organic solvents, and oxidizing agents, and the activity is enhanced by H2O2. The enzyme could be a novel protease based on its origin and peculiar biochemical properties. It may be useful in biotechnological applications especially for organic solvent-based enzymatic synthesis.
Keywords: Oxidant and solvent resistant; Metalloprotease; Streptomyces olivochromogenes

Kinetic Study on the Pretreatment and Enzymatic Saccharification of Rice Hull for the Production of Fermentable Sugars by Gong-Yuan Wei; You-Jung Lee; Yi-Joon Kim; Il-Hyuck Jin; Jai-Heon Lee; Chung-Han Chung; Jin-Woo Lee (1471-1482).
The production of fermentable sugars from rice hull was studied by dilute acid pretreatment and enzymatic saccharification. Rice hull (15%, w/v) was pretreated by 1% (v/v) sulfuric acid at high temperature (120∼160 °C) for 15, 30, 45, and 60 min, respectively. The maximum sugar concentration from rice hull in the prehydrolysate was obtained at 140 °C for 30 min, but the enzymatic saccharification yield from the corresponding pretreated rice hull is not high. To another aspect, the maximum enzymatic saccharification yield was achieved at 160 °C for 60 min, while the recovery of fermentable sugars was the poorest. To take account of fermentable sugars from pretreatment and enzymatic saccharification, the maximum yield of sugars was obtained only when rice hull was treated at 140 °C for 30 min. Under this condition, 72.5% (w/w) of all sugars generated from the raw material can be recovered. The kinetic study on the enzymatic saccharification of dilute acid pretreated rice hull was also performed in this work by a modified Michaelis–Menten model and a diffusion-limited model. After calculation by a linear and a non-linear regression analysis, both models showed good relation with the experimental results.
Keywords: Rice hull; Pretreatment; Hydrolysis; Saccharification; Cellulase; Kinetics

Purification and Biochemical Characterization of an Acid-Stable Lipase from the Pyloric Caeca of Sardine (Sardinella aurita) by Nabil Smichi; Ahmed Fendri; Raja Chaâbouni; Faouzi Ben Rebah; Youssef Gargouri; Nabil Miled (1483-1496).
A lipolytic activity was located in the sardine digestive glands (pyloric caeca), from which a sardine digestive lipase (SaDL) was purified. Pure SaDL has a molecular mass of 43 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The enzyme was found to be more active on short-chain triacylglycerols than on long-chain ones. SaDL does not present the interfacial activation phenomenon. Control experiments were performed under the same experimental conditions, with dromedary and turkey pancreatic lipases and showed a positive interfacial activation phenomenon. Sodium deoxycholate (NaDC) has an inhibitory effect on the lipase activity. The pure enzyme lost 40% of its activity in presence of 8 mM NaDC. SaDL was found to be mostly stable at low pH values. Interestingly, no colipase was detected in the sardine pyloric caeca. Analogous results were reported for the scorpion and the crab digestive systems. This is in line with the idea that colipase might has evolved in mammal animals simultaneously with the appearance of an exocrine pancreas. No similarity was found between the NH2-terminal amino acid residues of SaDL and those of lipases from the digestive tract of other species. Altogether, these results suggest that SaDL is a member of a new group of lipases belonging to aquatic species.
Keywords: Sardinella aurita ; Pyloric caeca; Acid-stable lipase; Purification; Characterization

As one of the most important groups of industrial enzymes, cold-adapted protease has been studied widely. An extracellular cold-adapted alkaline protease metalloproteinase (MP), produced by a marine bacterium strain YS-80-122, has been purified. The NH2-amino acid sequence of the purified alkaline protease MP was ANGTSSAFTQ, which was identical to that of the serralysin from Pseudomonas sp. “TAC II 18”. The MP structural gene (lupA gene) was cloned by inverse PCR, and the open reading frame of 1,443 bp encoded a 463 amino acid protein (without signal peptide). Sequence alignment reveals that the alkaline protease MP belongs to the serralysin-type metalloproteases. The recombinant protein LupA was expressed in Escherichia coli, and Western blotting confirmed that the LupA was homologous to the cold-adapted alkaline protease MP.
Keywords: Cold-adapted; Alkaline protease; Metalloprotease; lupA ; Serralysin

Reduction of Benzaldehyde Catalyzed by Papain-Based Semisynthetic Enzymes by Chun-xiang Chen; Bo Jiang; E. A. Carrey; Li-min Zhu (1506-1516).
Some features of native enzyme’s active site were used to conjunction with a chemical reagent or modifying group, which would generate new functionality different from the natural enzyme. In order to obtain an efficient catalyst, we have designed four different molecular size N-derivatives of modifiers and introduced them into the active site of papain to obtain new semisynthetic enzymes, which were used as catalyst in reduction of benzaldehyde to yield benzyl alcohol respectively, and the reactions carried out with recycling agent in 0.1 M phosphate buffer pH 6.5 at 37 °C. The results had shown that a longer N-derivative of semisynthetic enzyme had higher catalytic activity. Furthermore, we propose a plausible model for the catalytic mechanism in the semisynthetic enzymes system.
Keywords: Papain; Semisynthetic enzyme; Reduction