Applied Biochemistry and Biotechnology (v.164, #3)

Development of a Temperature Shift Strategy for Efficient Docosahexaenoic Acid Production by a Marine Fungoid Protist, Schizochytrium sp. HX-308 by Yan Zeng; Xiao-Jun Ji; Min Lian; Lu-Jing Ren; Li-Jing Jin; Ping-Kai Ouyang; He Huang (249-255).
Temperature was one of the important environmental factors affecting the biosynthesis of docosahexaenoic acid (DHA; C22:6, ω−3). Generally, a low temperature would slow the strain growth, but promote the accumulation of unsaturated fatty acids. According to this information, the effects of temperature and different two-stage temperature shifting strategies on fatty acid production and DHA content of the marine fungoid protist, Schizochytrium sp. HX-308, were investigated in this study. Finally, the highest DHA percentage was up to 51.98% (per total fatty acids) with the DHA production of 6.05% (per dry cell weight), which was obtained with the method of shifting temperature from 30 °C for 32 h to 20 °C for 12 h.
Keywords: Schizochytrium sp.; Docosahexaenoic acid; Temperature shift

Cellulase Production by Streptomyces viridobrunneus SCPE-09 Using Lignocellulosic Biomass as Inducer Substrate by Fábio Nuno Marques Da Vinha; Mônica Pires Gravina-Oliveira; Marcella Novaes Franco; Andrew Macrae; Elba Pinto da Silva Bon; Rodrigo Pires Nascimento; Rosalie Reed Rodrigues Coelho (256-267).
An actinomycete strain, isolated from a soil sample under a sugar cane plantation in Brazil and identified as Streptomyces viridobrunneus SCPE-09, was selected as a promising cellulolytic strain, and tested for its ability to produce cellulases from agro-industrial residues. Sugar cane bagasse or wheat bran was tested as carbon source, and corn steep liquor tested as nitrogen source. Different concentrations of carbon and nitrogen were tested using factorial design to identify optimal cellulose production. The results showed that media containing wheat bran 2.0% (w/v) and corn steep liquid 0.19% (w/v) lead to the highest production, 2.0 U mL−1 of CMCase, obtained on the fifth day of fermentation. The pH and temperature profile showed optimal activity at pH 4.9 and 50°C. As for thermostability, endoglucanases were most tolerant at 50°C, retaining more than 80% of maximal activity even after 2 h of incubation. Zymogram analyses using supernatant from growth under optimized conditions revealed the presence of two CMCase bands with apparent molecular masses of 37 and 119 kDa. The combination of pH tolerance and CMCase production from agro-industrial residues by S. viridobrunneus SCPE-09 offers promise for future bioethanol biotechnologies.
Keywords: Streptomyces viridobrunneus ; CMCase; Corn steep liquor; Sugar cane bagasse; Wheat bran

Digested slurry samples from twenty-one large-scale anaerobic digestion plants together with intensive pig and dairy farms in Jiangsu Province of China were collected and analyzed for total and dissolved concentrations of Zn, Cu and As, as well as chemical characteristics. The results showed that total concentrations of Zn, Cu and As in digested pig slurries were concentrated to <10, <5 and 0.02–0.1 mg/l, respectively; while <2 and 10–30, <1, and 0.02–0.1 mg/l, respectively, in digested dairy slurries. Lowering the dietary supply of these elements to pig and dairy would be the most effective way to control heavy metal contents in digested manure slurries. Dissolved fractions of Zn, Cu and As accounted for 1–74%, 1–33% and 2–53% of the total concentrations, respectively, in digested pig slurries; and 18–65%, 12–58% and 3–68% in digested dairy slurries. The chemical fractions of heavy metals in digested slurries were not only dependent on the total concentrations of heavy metals in raw manures but also on conditions of digestion and storage. Oxidation pond systems could significantly cripple the total contents of heavy metals in digested slurries, and the removal effect was better in multi-oxidation-pond systems than that in primary-oxidation-pond systems. However, the chemical fractions of heavy metals in digested slurries changed in a complicated manner when stored in oxidation ponds, due to the suspended solid deposition, elements reduction, as well as variations of pH values and oxidation-reduction potential.
Keywords: Digested slurry; Large-scale anaerobic digestion plant; Total concentration of heavy metal; Chemical fraction; Oxidation pond system

Production and Characterization of Polyclonal and Monoclonal Abs Against the RNA-Binding Protein QKI by Jie Zhang; Bo Huang; Fang Yu; Mengying Wei; Guodong Yang; Haiyan Fu; Liang Jin; Liyuan Bai; Xianli He; Zifan Lu (283-293).
RNA-binding protein QKI, a member of the Signal Transduction and Activation of RNA family, is found to be essential in the blood vessel development and postnatal myelination in central nervous system (Woo et al., Oncogene 28:1176–1186, 2009; Lu et al., Nucleic Acids Res 31(15):4616–4624, 2003; Bohnsack et al., Genesis 44(2):93–104, 2006). However, its wide expression pattern suggests other fundamental roles in vivo (Kondo et al., Mamm Genome 10(7):662–669, 1999). To facilitate the understanding of QKI function in various systems, we prepared the polyclonal and monoclonal antibodies against QKI. To obtain the antigen, recombinant His-tagged QKI was expressed in Escherichia coli and highly purified by Ni2+-chelated column combined with hydrophobic and ion exchange methods. Following three types of immunizations with different adjuvants, including Freund’s, PAGE gel, and nitrocellulose membrane, only the antiserum produced with Freund’s adjuvant is effective for Western blot detection. Several McAb clones are able to recognize both endogenous and over-expressed QKI with high affinity in Western blot and immunofluorescence. The specificity of Ab was validated as weakening, and no specific signals were observed in cells with QKI knocking down. Immunohistochemistry analysis further showed positive staining of QKI in kidney where QKI mRNA was abundantly expressed, ensuring the wide applications of the QKI Abs in the ongoing mechanistic studies.
Keywords: QKI; Prokaryotic expression; Immunization; Protein purification; Antibody

Cell Surface Display of Cellulase Activity–Free Xylanase Enzyme on Saccharomyces Cerevisiae EBY100 by Shabina Yeasmin; Chul Hawn Kim; Hyeon Jin Park; Mominul Islam Sheikh; Ji Yong Lee; Jae Won Kim; Kyung Kil Back; Sung Ho Kim (294-304).
Cellulase-free xylanase has potential for its application in the selective removal of hemicellulose from kraft pulp to give good strength to paper. In this study, a gene (xyn) encoding cellulase activity–free xylanase enzyme (Xyn) was isolated from Paenibacillus polymyxa PPL-3. The xyn gene encoded a protein of 221 amino acids, and the purified Xyn was about 22.5 kDa measured by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Moreover, the cellulase activity–free xylanase enzyme (Xyn) was displayed on the cell surface of Saccharomyces cerevisiae EBY100 using Aga2p as an anchor protein. Cell surface display of xylanase enzyme (Xyn) on S. cerevisiae EBY100 was confirmed by immunofluorescence microscopy. Optimum cell surface display of xylanase enzyme (Xyn) was observed at pH 7 and 40 °C. Therefore, cell surface–displayed xylanase enzyme (Xyn) can be used in the paper industry.
Keywords: Cellulase-free xylanase; Yeast surface display; Kraft pulp; Biobleaching; Paenibacillus polymyxa PPL-3

Three Alginate Lyases from Marine Bacterium Pseudomonas fluorescens HZJ216: Purification and Characterization by Liyan Li; Xiaolu Jiang; Huashi Guan; Peng Wang; Hong Guo (305-317).
Three alginate lyases (A, B, and C) from an alginate-degrading marine bacterium strain HZJ216 isolated from brown seaweed in the Yellow Sea of China and identified preliminarily as Pseudomonas fluorescens are purified, and their biochemical properties are described. Molecular masses of the three enzymes are determined by SDS-PAGE to be 60.25, 36, and 23 kDa with isoelectric points of 4, 4.36, and 4.59, respectively. Investigations of these enzymes at different pH and temperatures show that they are most active at pH 7.0 and 35 °C. Alginate lyases A and B are stable in the pH range of 5.0–9.0, while alginate lyase C is stable in the pH range of 5.0–7.0. Among the metal ions tested, additions of Na+, K+, and Mg2+ ions can enhance the enzyme activities while Fe2+, Fe3+, Ba2+, and Zn2+ ions show inhibitory effects. The substrate specificity results demonstrate that alginate lyase C has the specificity for G block while alginate lyases A and B have the activities for both M and G blocks. It is the first report about extracellular alginate lyases with high alginate-degrading activity from P. fluorescens.
Keywords: Pseudomonas fluorescens ; Alginate lyase; Purification; Characterization; Enzyme activity

The alterations of organic acids citrate, α-ketoglutarate, succinate, fumarate, malate production together with isocitrate lyase activity as a glyoxalate shunt enzyme, and antibiotic production of Streptomyces sp M4018 were investigated in relation to changes in the glucose, glycerol and starch concentrations (5–20 g/L) after identification as a strain of Streptomyces hiroshimensis based on phenotypic and genotypic characteristics. The highest intracellular citrate and α-ketoglutarate levels in 20 g/l of glucose, glycerol, and starch mediums were 399.47 ± 4.78, 426.93 ± 6.40, 355.84 ± 5.38 ppm and 444.81 ± 5.12, 192.96 ± 2.26, 115.20 ± 2.87 ppm, respectively. The highest succinate, malate, and fumarate levels were also determined in 20 g/l of glucose medium as 548.9 ± 11.21, 596.15 ± 8.26, and 406.42 ± 6.59 ppm and the levels were significantly higher than the levels in glycerol and starch. Extracellular organic acid levels measured also showed significant correlation with carbon source concentrations by showing negative correlation with pH levels of the growth medium. The antibiotic production of Streptomyces sp. M4018 was also higher in glucose medium as was the case also for organic acids when compared with glycerol. On the other hand, there is no production in starch.
Keywords: Streptomyces sp.; TCA cycle; Glyoxalate cycle; Antibiotic production

A method to exploit hybrid Petri nets for modeling and simulating biochemical processes in a systematic way was introduced. Both molecular biology and biochemical engineering aspects are manipulated. With discrete and continuous elements, the hybrid Petri nets can easily handle biochemical factors such as metabolites concentration and kinetic behaviors. It is possible to translate both molecular biological behavior and biochemical processes workflow into hybrid Petri nets in a natural manner. As an example, penicillin production bioprocess is modeled to illustrate the concepts of the methodology. Results of the dynamic of production parameters in the bioprocess were simulated and observed diagrammatically. Current problems and post-genomic perspectives were also discussed.
Keywords: Biochemical engineering; Modeling and simulation; Multiple-scale; Petri nets; Fed-batch bioprocess; Penicillin fermentation

The results of this study indicate that an increase in CO2 percentage to 30% can enhance Scenedesmus sp. growth in autotrophic cultivation to a maximum of 0.85 g/l as compared with 0.6 g/l obtained in the batch with air (after 6 days of cultivation). However, while the CO2 was higher than 30%, it showed a negative impact on cell growth. A mixotrophic cultivation with 3 g/l of glycerol can achieve 0.38 g l−1 day−1 of the maximum biomass productivity compared with that of 0.21 g l−1 day−1 in autotrophic cultivation. Nevertheless, the lutein content of the mixotrophic cultivation was 0.08–0.1% lower than 0.2–0.25% obtained in autotrophic cultivation, which led to a lower lutein productivity of 0.36 mg l−1 day−1 in the mixotrophic batch compared with 0.44 mg l−1 day−1 obtained in the autotrophic batch. The limitation of cell growth in the mixotrophic cultivation would be the contributing factor regarding the lower lutein productivity. The mixotrophic cultivation of repeated batch to remove potential inhibitive metabolic products from glycerol catabolism does not show an obvious improvement on biomass. Conclusively, mixotrophic cultivation achieves higher biomass productivity with lower lutein content than that of autotrophic cultivation, which leads to lower lutein productivity. Therefore, the autotrophic cultivation is preferred in the lutein production.
Keywords: Mixotrophic; Luedeking–Piret; Lutein; Autotrophic; Scenedesmus

Reductive Alkylation Causes the Formation of a Molten Globule-Like Intermediate Structure in Geobacillus zalihae Strain T1 Thermostable Lipase by Kok Whye Cheong; Thean Chor Leow; Raja Noor Zaliha Raja Abd Rahman; Mahiran Basri; Mohd Basyaruddin Abdul Rahman; Abu Bakar Salleh (362-375).
A thermostable lipase from Geobacillus zalihae strain T1 was chemically modified using propionaldehyde via reductive alkylation. The targeted alkylation sites were lysines, in which T1 lipase possessed 11 residues. Far-UV circular dichroism (CD) spectra of both native and alkylated enzyme showed a similar broad minimum between 208 and 222 nm, thus suggesting a substantial amount of secondary structures in modified enzyme, as compared with the corresponding native enzyme. The hydrolytic activity of the modified enzymes dropped drastically by nearly 15-fold upon chemical modification, despite both the native and modified form showed distinctive α-helical bands at 208 and 222 nm in CD spectra, leading us to the hypothesis of formation of a molten globule (MG)-like structure. As cooperative unfolding transitions were observed, the modified lipase was distinguished from the native state, in which the former possessed a denaturation temperature (T m) in lower temperature range at 61 °C while the latter at 68 °C. This was further supported by 8-anilino-1-naphthalenesulfonic acid (ANS) probed fluorescence which indicated higher exposure of hydrophobic residues, consequential of chemical modification. Based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, a small number of lysine residues were confirmed to be alkylated.
Keywords: Chemical modification; Reductive alkylation; Circular dichroism; Thermostable lipase; Molten globule

Discovery of new bacterial strains with fast identification in a miniaturized system was performed for the synthesis of optically active L-tert-butyl leucine. With tert-butyl leucine amide as nitrogen source, one bacterial strain with high conversion and high enantioselectivity was discovered among 120 isolated microorganisms from local soils and identified as Mycobacterium sp. JX009. Glucose and ammonium chloride were examined as the good carbon source and nitrogen source for the cells’ growth separately. The cells grew better at 30 °C and at pH 7.5 with higher activity of 2,650 U/l in comparison with other conditions. Cells’ stability was improved by immobilization on synthetic resin 0730 without pretreatment. Tert-butyl leucine amide (30 mM) was successfully hydrolyzed by immobilized cells and examined as the highest chemical concentration that cells could endure. After six reaction cycles, the immobilized cells retained 90% activity with production of L-tert-butyl leucine in 98% ee. The results firstly reported the application of new bacterial strain in the hydrolysis of tert-butyl leucine amide to produce optically active L-tert-butyl leucine in an efficient way with investigation in detail.
Keywords: L-tert-butyl leucine; Screening; Mycobacterium sp. JX009; Hydrolysis; Immobilized on resins; Repeated usage

Bacillus spp. of Human Origin: A Potential Siderophoregenic Probiotic Bacteria by Jayesh J. Ahire; Kanchankumar P. Patil; Bhushan Liladhar Chaudhari; Sudhir B. Chincholkar (386-400).
Bacillus spp. ST13, isolated from human stool, was evaluated for siderophoregenic and probiotic qualities prior to its possible application for iron nutrition in humans and animals. It was tested for siderophore production in iron-limiting conditions and found to produce catecholate type of siderophore on the basis of high-performance liquid chromatography (HPLC), FT-IR, NMR, and mass spectra analysis. The isolate was screened for probiotic properties as per WHO and FAO guidelines. The strain ST13 can survive stomach acidity, bile salt and partially simulated gastrointestinal tract conditions. It was susceptible to most of the antibiotic tested and showed antimicrobial activity against enteric pathogens like Salmonella typhimurium, Streptococcus pyogenes, and Staphylococcus aureus. Strain ST13 showed close similarity with Bacillus subtilis using 16S r-RNA gene sequence analysis and biochemical characterization. The methanolic extract of ST13 siderophore was evaluated for DPPH radical scavenging activity, which showed 94.55 ± 0.9% of radical scavenging effect.
Keywords: Bacillus spp.; Probiotic; Siderophore; Catecholate; DPPH radical scavenging