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Applied Microbiology and Biotechnology (v.71, #2)
Systems for the detection and analysis of protein–protein interactions by Kouichi Kuroda; Michiko Kato; Joji Mima; Mitsuyoshi Ueda (pp. 127-136).
The analysis of protein–protein interactions is important for developing a better understanding of the functional annotations of proteins that are involved in various biochemical reactions in vivo. The discovery that a protein with an unknown function binds to a protein with a known function could provide a significant clue to the cellular pathway concerning the unknown protein. Therefore, information on protein–protein interactions obtained by the comprehensive analysis of all gene products is available for the construction of interactive networks consisting of individual protein–protein interactions, which, in turn, permit elaborate biological phenomena to be understood. Systems for detecting protein–protein interactions in vitro and in vivo have been developed, and have been modified to compensate for limitations. Using these novel approaches, comprehensive and reliable information on protein–protein interactions can be determined. Systems that permit this to be achieved are described in this review.
Simultaneous P-solubilizing and biocontrol activity of microorganisms: potentials and future trends by Nikolay Vassilev; Maria Vassileva; Iana Nikolaeva (pp. 137-144).
Phosphate (P)-solubilizing microorganisms as a group form an important part of the microorganisms, which benefit plant growth and development. Growth promotion and increased uptake of phosphate are not the only mechanisms by which these microorganisms exert a positive effect on plants. Microbially mediated solubilization of insoluble phosphates through release of organic acids is often combined with production of other metabolites, which take part in biological control against soilborne phytopathogens. In vitro studies show the potential of P-solubilizing microorganisms for the simultaneous synthesis and release of pathogen-suppressing metabolites, mainly siderophores, phytohormones, and lytic enzymes. Further trends in this field are discussed, suggesting a number of biotechnological approaches through physiological and biochemical studies using various microorganisms.
Influence of extrinsic factors on granulation in UASB reactor by Manoj K. Tiwari; Saumyen Guha; C. S. Harendranath; Shweta Tripathi (pp. 145-154).
The aim of this mini-review is to synthesize and analyze information on how the process of granulation is affected by environmental and operational conditions in the reactor. The factors reviewed are temperature, pH, alkalinity, organic loading rate, upflow velocity, nature and strength of substrate, nutrients, multivalent cations and heavy metals, microbial ecology of seed sludge, exo-cellular polymer, and addition of natural and synthetic polymers. Careful temperature control and adequate alkalinity is required for generation and maintenance of granules. Nature and strength of substrate in conjunction with intra-granular diffusion to a large extent determines the microstructure of the granules. The divalent cations such as calcium and iron may enhance granulation by ionic bridging and linking exo-cellular polymers. However, their presence in excess may lead to cementation due to precipitation leading to increased ash content and mass transfer limitation. The addition of external additives such as ionic polymers may enhance granulation in the upflow anaerobic sludge blanket reactors.
Continuous production of l(+)-tartaric acid from cis-epoxysuccinate using a membrane recycle reactor by Ronnie Willaert; Luc De Vuyst (pp. 155-163).
The one-step bioconversion of cis-epoxysuccinate (CES) to l(+)-tartaric acid by dried Rhodococcus rhodochrous cells containing CES hydrolase activity was studied by using a continuous bioconversion process. The influence of the pH and the temperature was assessed. A mathematical model was used to quantify the CES hydrolase activity and stability. The optimal pH, which resulted in a maximal CES hydrolase activity and stability, was pH 8.0. A large increase in stability (half-life time) could be obtained when the temperature was decreased from 37 to 14°C during the continuous bioconversion. A total bioconversion was maintained for more than 100 days. This resulted in a large value for the specific productivity since the effect of the large increase in stability was much more important than the decrease of activity at the lower temperature. This continuous bioconversion process was further optimised by calculating the productivity for several continuously stirred tank reactors in series. The specific productivity could be nearly doubled when the number of reactors in the series was increased from 1 to 4.
Novel synthetic 2,6-dichloroisonicotinate derivatives as effective elicitors for inducing the biosynthesis of plant secondary metabolites by Zhi-Gang Qian; Zhen-Jiang Zhao; Yufang Xu; Xuhong Qian; Jian-Jiang Zhong (pp. 164-167).
Two novel 2,6-dichloroisonicotinic acid (INA) derivatives [trifluoroethyl 2,6-dichloroisonicotinate (TFINA) and 2-(2,6-dichloro-pyridine-4-carbonyloxy)-ethyl jasmonate (DPCEJ)] were chemically synthesized and evaluated by bioassay as potential elicitors for inducing the biosynthesis of plant secondary metabolites. A suspension culture of Taxus chinensis, which stably produces a high level of bioactive taxuyunnanine C (Tc), was taken as a model plant cell system. A significant increase in Tc accumulation was observed in the presence of TFINA or DPCEJ. For example, addition of 100 μM TFINA or DPCEJ on day 7 led to a high Tc content of 21.6±2.0 or 27.7±1.0 mg g−1 (on day 21), while the Tc content was 13.7±1.0 and 17.1±0.9 mg g−1 for the control and that with addition of 100 μM INA, respectively. To the best of our knowledge, this is the first report on the use of synthetic INA derivatives for inducing the biosynthesis of plant secondary metabolites. The results indicate that the newly synthesized INA analogues can act as promising elicitors for secondary metabolism induction in plant cell cultures.
Active-site engineering of biphenyl dioxygenase: effect of substituted amino acids on substrate specificity and regiospecificity by Hikaru Suenaga; Masatoshi Goto; Kensuke Furukawa (pp. 168-176).
Biphenyl dioxygenase (Bph Dox) catalyzes the initial dioxygenation step in the metabolism of biphenyl. The large subunit (BphA1) of Bph Dox plays a crucial role in the determination of the substrate specificity of biphenyl-related compounds including polychlorinated biphenyls (PCBs). Previously, the substitution of Asn at Thr-376 near the active-site iron in the BphA1 of Pseudomonas pseudoalcaligenes KF707 expanded the oxidation range and altered the regiospecificity of Bph Dox for PCBs. In this study, we replaced Thr-376 with Gly, Ser, Gln, Tyr, Val, Phe, Asp, and Lys and expressed these enzymes in Escherichia coli. Bph Dox mutants of Thr376Asn, Thr376Val, Thr376Phe, and Thr376Lys showed novel degradation activity for dibenzofuran, which is a poor substrate for KF707 Bph Dox. All active Bph Dox mutants showed altered regiospecificity with 2,2′-dichlorobiphenyl and 2,5,4′-trichlorobiphenyl. The Thr376Gly, Thr376Val, Thr376Phe, and Thr376Asp Bph Dox mutants introduced molecular oxygen at the 2,3 position of 2,2′-dichlorobiphenyl, forming 2-chloro-2′,3′-dihydroxybiphenyl with concomitant dechlorination. The Bph Dox mutants of Thr376Gly, Thr376Ser, Thr376Asp, and Thr376Lys attacked 2,5,4′-trichlorobiphenyl via both 2′,3′- and 3,4-dioxygenation activities. In particular, the Thr376Phe Bph Dox mutant exhibited enhanced and expanded degradation activities toward all of the compounds tested. Further site-directed mutation was induced to change the oxidizing character of KF707 Bph Dox to that of the Bph Dox of Burkholderia xenovorans LB400 by the substitution of two amino acids, Ile335Phe and Thr376Asn, near the active-site.
A unique polypeptide from the C-terminus of the exocellular esterase of Acinetobacter venetianus RAG-1 modulates the emulsifying activity of the polymeric bioemulsifier apoemulsan by Horacio Bach; David L. Gutnick (pp. 177-183).
An exocellular esterase from the oil-degrading Acinetobacter venetianus RAG-1 was previously shown to enhance the emulsification and emulsion stabilization properties of the amphipathic, aminopolysaccharide bioemulsifier, emulsan [Bach H, Berdichevsky Y, Gutnick D (2003) An exocellular protein from the oil-degrading microbe Acinetobacter venetianus RAG-1 enhances the emulsifying activity of the polymeric bioemulsifier emulsan. Appl Environ Microbiol 69:2608–15]. This enhancement was specific for the RAG-1 esterase and was independent of catalytic activity. In this report, fragments from both the N′- and C′-termini were cloned as fusions to the C-terminus of the maltose-binding protein (MBP) and were tested for enhancement activity in the presence of the deproteinated form of emulsan, apoemulsan. The activity could be localized to the C-terminal third of the protein which exhibited the same activity as the intact enzyme. MBP itself was completely inactive and could be cleaved from the fusion without affecting the subsequent emulsification. However, the enhancement completely depended on the presence of a unique C-terminal 20 amino acid peptide not found in any other protein in the databases. In addition, progressive removal of amino acids from the N-terminus of the active MBP polypeptide resulted in a concomitant loss of activity, indicating that enhancement is also proportional to the size of the peptide fragment. The middle third and the C-terminal third of the enzyme each contained a copy of the conserved Cardin–Weintraub consensus sequence for protein binding to heparin. These sequences were not detected in homologous esterases from a closely related strain, Acinetobacter calcoaceticus BD413.
Optimization of culture on the overproduction of TRAIL in high-cell-density culture by recombinant Escherichia coli by Qingping Luo; Ya-Ling Shen; Dong-Zhi Wei; Wei Cao (pp. 184-191).
Different nutrient-feeding cultures were carried out in producing recombinant protein of truncated tumor necrosis factor related apoptosis-inducing ligand (TRAIL) (114–281 amino acids of TRAIL) in Escherichia coli strain C600/pBV-TRAIL. The effects of preinduction specific growth rate, postinduction carbon source (glucose and glycerol), and feeding strategies were investigated. The higher preinduction specific growth rate (μ=0.22 h−1) contributed to the increase in the TRAIL production, at which TRAIL was accumulated in bacterial cells as 7.2% of total cellular protein, corresponding to 1.99 g l−1 in contrast with 5.1% (1.29 g l−1) at preinduction specific growth rate (μ=0.1 h−1) during high-cell-density culture. Glycerol was superior to glucose as the postinduction carbon source for TRAIL production. Under similar culture conditions, the final concentration of TRAIL was produced 1.59-fold more when glycerol was used as postinduction carbon source than when glucose was used. At the same time, the results showed that it is efficient to adopt the pH-stat feeding strategy at postinduction for the overproduction of TRAIL. The TRAIL production was increased up to 4.51 g l−1, approximately 16.1% of total cellular protein. The mechanisms behind the preinduction specific growth rate effect on the expression level may be ascribed to the leakage secretion of acetate.
Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system by Yungen Miao; Yuansong Zhang; Koichi Nakagaki; Tianfu Zhao; Aichun Zhao; Yan Meng; Masao Nakagaki; Enoch Y. Park; Katsumi Maenaka (pp. 192-199).
Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) has a lot of advantages such as high expression efficiency, convenience, and low feeding cost. In this report, we used a recently developed BmNPV bacmid, which could infect both B. mori cell lines and silkworm larvae. The results showed it takes only 7 to 10 days to generate recombinant baculovirus and permit the rapid isolation from small-scale cultures and then use it to transfect B. mori cell lines, compared to traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses. Using this BES, we expressed a recombinant spider flagelliform protein in BmN cell line, which was around 37 kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The BmNPV bacmid system using silkworm would be very attractive for expression of target proteins.
Expression of laccase gene lcc1 in Coprinopsis cinerea under control of various basidiomycetous promoters by Sreedhar Kilaru; Patrik J. Hoegger; Andrzej Majcherczyk; Claire Burns; Kazuo Shishido; Andy Bailey; Gary D. Foster; Ursula Kües (pp. 200-210).
Coprinopsis cinerea laccase gene lcc1 was expressed in this basidiomycete under naturally non-inductive conditions using various homologous and heterologous promoters. Laccase expression was achieved in solid and liquid media with promoter sequences from the C. cinerea tub1 gene, the Agaricus bisporus gpdII gene, the Lentinus edodes priA gene and the Schizophyllum commune Sc3 gene. As measured by enzyme activity in liquid cultures, a 277-bp gpdII promoter fragment, followed by a 423-bp priA fragment, was most efficient. A shorter priA sequence of 372 bp was inactive. tub1 promoter fragments were reasonably active, whereas the S. commune Sc3 promoter sequence was less active, in comparison. Irrespective of the promoter used, addition of copper to the medium increased enzymatic activities for highly active transformants by 10- to 50-fold and for less active transformants for 2- to 7-fold. The highest enzymatic activities (3 U/ml) were reached with the gpdII promoter in the presence of 0.1 mM CuSO4.
Recombinant Candida utilis for the production of biotin by Yi-Ren Hong; Ya-Lei Chen; Lynn Farh; Wen-Jen Yang; Chen-Hua Liao; David Shiuan (pp. 211-221).
Biotin is an important nutritional supplement but is difficult to manufacture effectively. Here we present a trial of biotin production using the food yeast Candida utilis. In this system, we cloned the C. utilis biotin synthase (BIO2) gene, the gene of the rate-limiting enzyme for biotin biosynthesis, and assembled it under the control of a strong promoter. A series of plasmids were constructed to direct the integration of the BIO2 gene, either high-copy integration with 18S rDNA fragment or low-copy integration with URA3 or HIS3 fragment. The BIO2 gene can be successfully integrated into the C. utilis chromosome and can drive biotin production using these plasmids. The biotin yield in this system can reach 100-fold above the endogenous level in a small-scale culture. Although the biotin production is not stable if the selection pressure is removed, this system has the potential to produce biotin-rich feed or food additives directly without the requirement of further purification.
Effect of expressing polyhydroxybutyrate synthesis genes (phbCAB) in Streptococcus zooepidemicus on production of lactic acid and hyaluronic acid by Jinyu Zhang; Ning Hao; Guo-Qiang Chen (pp. 222-227).
Hyaluronic acid (HA) has been industrially produced using the gram-positive bacterium Streptococcus zooepidemicus. Large amount of lactic acid formation was one of the important factors that restricted cell growth and HA productivity and lowered the substrate to HA conversion efficiency in a fermentor. In this study, polyhydroxybutyrate (PHB) synthesis genes (phbCAB) of Ralstonia eutropha were cloned from the plasmid pBHR68 and were inserted into the plasmid pEU308, an expression vector for gram-positive bacteria. The plasmid was transformed into S. zooepidemicus by electroporation. β-Ketothiolase (PhbA), acetoacetyl-CoA reductase (PhbB), and polyhydroxyalkanoate (PHA) synthase (PhbC) activity assays were carried out to demonstrate the expression of these genes. The PhbA and PhbB activities were 3.13 and 1.23 U mg−1, respectively. No PhbC activities were detected. In shake flask studies, there was no obvious difference between the wild-type and recombinant S. zooepidemicus harboring phbCAB genes in terms of lactic acid and HA formation. However, in fermentor studies, the recombinant produced only 40 g L−1 lactic acid and 7.5 g L−1 HA, whereas the wild type produced 65 g L−1 lactic acid and 5.5 g L−1 HA. These results suggested that expression of phbCAB genes in S. zooepidemicus could help regulate HA production metabolism. Because the lactic acid formation in S. zooepidemicus was sensitive to cellular oxidation/reduction potential, it is proposed that the PHB synthesis pathway could act as a regulator to adjust the cellular oxidation/reduction potential. This is the first study demonstrating that PHA synthesis related to energy and carbon metabolism could be employed as a pathway to regulate other cellular metabolism and possibly to regulate the production of other metabolic products.
Enzymatic degradation of nitriles by Klebsiella oxytoca by C. M. Kao; K. F. Chen; J. K. Liu; S. M. Chou; S. C. Chen (pp. 228-233).
Klebsiella oxytoca, isolated from cyanide-containing wastewater, was able to utilize many nitriles as sole source of nitrogen. The major objective of this study was to explore the ability of K. oxytoca to utilize some nitriles and then further evaluate the pathways of transformation of cyanide compounds by K. oxytoca. Results from this study indicate that succinonitrile and valeronitrile were the most optimal sources of nitrogen for the growth of K. oxytoca. The biodegradation of acetonitrile proceeded with the formation of acetamide followed by acetic acid. The production of ammonia was also detected in this biodegradation experiment. Similar results were observed in the propionitrile biodegradation experiments. Collectively, this study suggests that the breakdown of acetonitrile or propionitrile by this bacterium was via a two-step enzymatic hydrolysis with amides as the intermediates and organic acids plus with ammonia as the end products.
Extraction of chitin from red crab shell waste by cofermentation with Lactobacillus paracasei subsp. tolerans KCTC-3074 and Serratia marcescens FS-3 by W. J. Jung; G. H. Jo; J. H. Kuk; K. Y. Kim; R. D. Park (pp. 234-237).
For one-step extraction of chitin from red crab shell waste, cofermentation with Lactobacillus paracasei subsp. tolerans KCTC-3074, a lactic-acid-producing bacterium, and Serratia marcescens FS-3, a protease-producing bacterium, was conducted. Fermentation with single strain (L. 3074 or FS-3) was also conducted. At day 7, the pH in L. 3074, FS-3, and L. 3074+FS-3 (1:1) treatment decreased from 6.90 to 3.30, 5.88, and 3.48, respectively. Ash content in the residue after fermentation treatment of crab shells in L. 3074 and L. 3074+FS-3 (1:1) treatment drastically decreased from 41.2% to 3.19 and 1.15%, respectively. In L. 3074+FS-3 (1:1) cofermentation, the level of demineralization was the highest value of 97.2%, but the level of deproteinization in the cofermentation was 52.6% at day 7. Protein content in the treatment of FS-3 alone reduced from 22.4 to 3.62%. These results indicate that cofermentation of the shells using the two strains is efficient and applicable for the one-step extraction of crude chitin from red crab shell waste.
The effects of Lactobacillus-fermented milk on lipid metabolism in hamsters fed on high-cholesterol diet by Chiu-Hsia Chiu; Tzu-Yu Lu; Yun-Yu Tseng; Tzu-Ming Pan (pp. 238-245).
The objective of this study was to evaluate the effects of local Lactobacillus strains (NTU 101 and 102) on cholesterol-lowering effects in vivo. Thirty male hamsters were housed, divided into five groups, and fed on a cholesterol diet (5 g/kg diet) to induce hypercholesterolemia. Milk fermented by Lactobacillus paracasei subsp. paracasei NTU 101, Lactobacillus plantarum NTU 102, and Lactobacillus acidophilus BCRC 17010 was administrated for this study. After treatment with different fermented milk, blood was taken and liver was removed for the determination of lipoproteins, including total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglyceride. Lactobacilli and bifidobacteria decreased (105) in the control group; when hamsters were fed on fermented milk, the number of lactobacilli (107–108) and bifidobacteria (105–107) was increased. Serum and liver total cholesterol levels were significantly reduced by about 26.4, 23.5, and 30.1% and by about 17.7, 15.9, and 13.4% when hamsters were given fermented milk. However, serum HDL-C and LDL-C were also reduced. The results of this study showed that the hypocholesterolemic effect of local Lactobacillus strains was attributed to its ability to lower serum and liver total cholesterol levels. Thus, local Lactobacillus strains could significantly increase probiotic count.
Decolorization of anthraquinone dye by Shewanella decolorationis S12 by Meiying Xu; Jun Guo; Guoqu Zeng; Xiaoyan Zhong; Guoping Sun (pp. 246-251).
A new species of genus Shewanella, Shewanella decolorationis S12, from activated sludge of a textile-printing wastewater treatment plant, can decolorize Reactive Brilliant Blue K-GR, one kind of anthraquinone dye, with flocculation first. Although S. decolorationis displayed good growth in an aerobic condition, color removal was the best in an anaerobic condition. For color removal, the most suitable pH values and temperatures were pH 6.0–8.0 and 30–37°C under anaerobic culture. More than 99% of Reactive Brilliant Blue K-GR was removed in color within 15 h at a dye concentration of 50 mg/l. Lactate was the suitable carbon source for the dye decolorization. A metal compound, HgCl2, had the inhibitory effect on decolorization of Reactive Brilliant Blue K-GR, but a nearly complete decolorization also could be observed at a HgCl2 concentration of 10 mg/l. The enzyme activities, which mediate the tested dye decolorization, were not significantly affected by preadaptation of the bacterium to the dye.