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Applied Microbiology and Biotechnology (v.70, #1)
Phage display systems and their applications by Matthias Paschke (pp. 2-11).
Screening phage display libraries of proteins and peptides has, for almost two decades, proven to be a powerful technology for selecting polypeptides with desired biological and physicochemical properties from huge molecular libraries. The scope of phage display applications continues to expand. Recent applications and technical improvements driving further developments in the field of phage display are discussed.
Phage display systems and their applications by Matthias Paschke (pp. 2-11).
Screening phage display libraries of proteins and peptides has, for almost two decades, proven to be a powerful technology for selecting polypeptides with desired biological and physicochemical properties from huge molecular libraries. The scope of phage display applications continues to expand. Recent applications and technical improvements driving further developments in the field of phage display are discussed.
Perspectives for synthesis and production of polyurethanes and related polymers by enzymes directed toward green and sustainable chemistry by Shuichi Matsumura; Yasuyuki Soeda; Kazunobu Toshima (pp. 12-20).
Enzyme-catalyzed polymerization and degradation will play an important role in both the synthesis and chemical recycling of green and sustainable polyurethane. This minireview covers the new synthetic routes to polyurethane without using diisocyanate, the biodegradation of polyurethane, and the enzymatic synthesis and the chemical recycling of poly(ester-urethane) (PEU) and poly(carbonate-urethane) (PCU). The lipase-catalyzed polymerization of low molecular weight and biodegradable urethanediols with short-chain dialkyl carbonate and alkanedioates produced PCU and PEU, respectively. They were readily degraded in an organic solvent into the repolymerizable cyclic oligomers by lipase as a novel chemical recycling. These results will be applicable for the production strategies of green and sustainable polyurethanes.
Perspectives for synthesis and production of polyurethanes and related polymers by enzymes directed toward green and sustainable chemistry by Shuichi Matsumura; Yasuyuki Soeda; Kazunobu Toshima (pp. 12-20).
Enzyme-catalyzed polymerization and degradation will play an important role in both the synthesis and chemical recycling of green and sustainable polyurethane. This minireview covers the new synthetic routes to polyurethane without using diisocyanate, the biodegradation of polyurethane, and the enzymatic synthesis and the chemical recycling of poly(ester-urethane) (PEU) and poly(carbonate-urethane) (PCU). The lipase-catalyzed polymerization of low molecular weight and biodegradable urethanediols with short-chain dialkyl carbonate and alkanedioates produced PCU and PEU, respectively. They were readily degraded in an organic solvent into the repolymerizable cyclic oligomers by lipase as a novel chemical recycling. These results will be applicable for the production strategies of green and sustainable polyurethanes.
Microbial keratinases and their prospective applications: an overview by Rani Gupta; Priya Ramnani (pp. 21-33).
Microbial keratinases have become biotechnologically important since they target the hydrolysis of highly rigid, strongly cross-linked structural polypeptide “keratin” recalcitrant to the commonly known proteolytic enzymes trypsin, pepsin and papain. These enzymes are largely produced in the presence of keratinous substrates in the form of hair, feather, wool, nail, horn etc. during their degradation. The complex mechanism of keratinolysis involves cooperative action of sulfitolytic and proteolytic systems. Keratinases are robust enzymes with a wide temperature and pH activity range and are largely serine or metallo proteases. Sequence homologies of keratinases indicate their relatedness to subtilisin family of serine proteases. They stand out among proteases since they attack the keratin residues and hence find application in developing cost-effective feather by-products for feed and fertilizers. Their application can also be extended to detergent and leather industries where they serve as specialty enzymes. Besides, they also find application in wool and silk cleaning; in the leather industry, better dehairing potential of these enzymes has led to the development of greener hair-saving dehairing technology and personal care products. Further, their prospective application in the challenging field of prion degradation would revolutionize the protease world in the near future.
Microbial keratinases and their prospective applications: an overview by Rani Gupta; Priya Ramnani (pp. 21-33).
Microbial keratinases have become biotechnologically important since they target the hydrolysis of highly rigid, strongly cross-linked structural polypeptide “keratin” recalcitrant to the commonly known proteolytic enzymes trypsin, pepsin and papain. These enzymes are largely produced in the presence of keratinous substrates in the form of hair, feather, wool, nail, horn etc. during their degradation. The complex mechanism of keratinolysis involves cooperative action of sulfitolytic and proteolytic systems. Keratinases are robust enzymes with a wide temperature and pH activity range and are largely serine or metallo proteases. Sequence homologies of keratinases indicate their relatedness to subtilisin family of serine proteases. They stand out among proteases since they attack the keratin residues and hence find application in developing cost-effective feather by-products for feed and fertilizers. Their application can also be extended to detergent and leather industries where they serve as specialty enzymes. Besides, they also find application in wool and silk cleaning; in the leather industry, better dehairing potential of these enzymes has led to the development of greener hair-saving dehairing technology and personal care products. Further, their prospective application in the challenging field of prion degradation would revolutionize the protease world in the near future.
Stability validation of seeding cell control parameters in large-scale hybridoma cell culture by Ling Li; Li Mi; Jun Qin; Qiang Feng; Rong Liu; Xiaoling Yu; Liqing Xu; Zhinan Chen (pp. 34-39).
Stability and reproducibility of seeding cell performance in large-scale hybridoma cell culture has been reported by controlling only initial cell seeding density. The aim of the current study was to integrate multiple seeding cell control parameters to maintain stable and consistent cell physiological status for HAb18 cell expansion. Three parameters and their ranges were investigated, including initial cell seeding density in the range of 0.075–0.5×106 cells ml−1, “timepost” after cell passage between 8 and 36 h, and duration of subculture up to 6 months after cell revival. Cell performance was tested at the 1 L, 5 L, and 75 L scales. Desirable performance was found within the following parameter ranges: initial cell seeding density of 0.1–0.3×106 cells ml−1, “timepost” after cell passage between 14 and 22 h, and duration of subculture within 3 months of cell revival. Our results showed that cell growth rate and antibody productivity of three batches at 1 L, 5 L, and 75 L scale were found to be stably maintained within a range of 0.036–0.047 h−1 and 0.577–0.747 pg cell−1 h−1, with the positivity rate of antigen-binding activity within 97–99.75%, and the intensity of fluorescence around 200. This study may provide a simple but effective method to maintain seeding cell physiological status stable and consistent by combining seeding cell control parameters.
Stability validation of seeding cell control parameters in large-scale hybridoma cell culture by Ling Li; Li Mi; Jun Qin; Qiang Feng; Rong Liu; Xiaoling Yu; Liqing Xu; Zhinan Chen (pp. 34-39).
Stability and reproducibility of seeding cell performance in large-scale hybridoma cell culture has been reported by controlling only initial cell seeding density. The aim of the current study was to integrate multiple seeding cell control parameters to maintain stable and consistent cell physiological status for HAb18 cell expansion. Three parameters and their ranges were investigated, including initial cell seeding density in the range of 0.075–0.5×106 cells ml−1, “timepost” after cell passage between 8 and 36 h, and duration of subculture up to 6 months after cell revival. Cell performance was tested at the 1 L, 5 L, and 75 L scales. Desirable performance was found within the following parameter ranges: initial cell seeding density of 0.1–0.3×106 cells ml−1, “timepost” after cell passage between 14 and 22 h, and duration of subculture within 3 months of cell revival. Our results showed that cell growth rate and antibody productivity of three batches at 1 L, 5 L, and 75 L scale were found to be stably maintained within a range of 0.036–0.047 h−1 and 0.577–0.747 pg cell−1 h−1, with the positivity rate of antigen-binding activity within 97–99.75%, and the intensity of fluorescence around 200. This study may provide a simple but effective method to maintain seeding cell physiological status stable and consistent by combining seeding cell control parameters.
Construction of a two-strain system for asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to (S)-4-chloro-3-hydroxybutanoate ethyl ester by Zhinan Xu; Ying Liu; Limei Fang; Xiaoxia Jiang; Keju Jing; Peilin Cen (pp. 40-46).
Escherichia coli M15 (pQE30-car0210) was constructed to express carbonyl reductase (CAR) by cloning the car gene from Candida magnoliae and inserting it into pQE30. By cultivating E. coli M15 (pQE30-car0210) and M15 (pQE30-gdh0310), 8.2-fold and 12.3-fold enhancements in specific enzymatic activity over the corresponding original strain were achieved, respectively. After separate cultivations, these two strains were then mixed together at appropriate ratio to construct a novel two-strain system, in which M15 (pQE30-car0210) expressed CAR for ethyl 4-chloro-3-oxobutanoate (COBE) bioreduction and M15 (pQE30-gdh0310) expressed glucose dehydrogenase (GDH) for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration. In this complex system, the effects of substrate concentration, the biomass ratio between two strains as well as reaction temperature were investigated for efficient bioreduction. The results showed that the bioreduction reaction could be completed effectively without any addition of GDH or NADPH/NADP+. An optical purity of 99% (enantiometric efficiency) was obtained, and the yield of (S)-4-chloro-3-hydroxybutanoate ethyl ester reached 96.6% when initial concentration of COBE was 36.9 mM. The coupling reactions between two different strains were further explored by determining the profile of NADPH in the reaction broth.
Construction of a two-strain system for asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to (S)-4-chloro-3-hydroxybutanoate ethyl ester by Zhinan Xu; Ying Liu; Limei Fang; Xiaoxia Jiang; Keju Jing; Peilin Cen (pp. 40-46).
Escherichia coli M15 (pQE30-car0210) was constructed to express carbonyl reductase (CAR) by cloning the car gene from Candida magnoliae and inserting it into pQE30. By cultivating E. coli M15 (pQE30-car0210) and M15 (pQE30-gdh0310), 8.2-fold and 12.3-fold enhancements in specific enzymatic activity over the corresponding original strain were achieved, respectively. After separate cultivations, these two strains were then mixed together at appropriate ratio to construct a novel two-strain system, in which M15 (pQE30-car0210) expressed CAR for ethyl 4-chloro-3-oxobutanoate (COBE) bioreduction and M15 (pQE30-gdh0310) expressed glucose dehydrogenase (GDH) for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration. In this complex system, the effects of substrate concentration, the biomass ratio between two strains as well as reaction temperature were investigated for efficient bioreduction. The results showed that the bioreduction reaction could be completed effectively without any addition of GDH or NADPH/NADP+. An optical purity of 99% (enantiometric efficiency) was obtained, and the yield of (S)-4-chloro-3-hydroxybutanoate ethyl ester reached 96.6% when initial concentration of COBE was 36.9 mM. The coupling reactions between two different strains were further explored by determining the profile of NADPH in the reaction broth.
Growth characteristic of anaerobic ammonium-oxidizing bacteria in an anaerobic biological filtrated reactor by Kazuichi Isaka; Yasuhiro Date; Tatsuo Sumino; Sachiko Yoshie; Satoshi Tsuneda (pp. 47-52).
The doubling time of anaerobic ammonium-oxidizing (anammox) bacteria in an anaerobic biological filtrated (ABF) reactor was determined. Fluorescence in situ hybridization analysis was used to detect and count anammox bacteria cells in anammox sludge. As a result, the populations of anammox bacteria at 14th and 21st days were 1.1×106 and 1.7×107 cells/ml reactor, respectively. From these results, the doubling time of anammox bacteria was calculated as 1.8 days, and the specific growth rate (μ) was 0.39 day−1. This result indicated that the anammox bacteria have higher growth rate than the reported value (doubling time, 11 days). Furthermore, it was clearly demonstrated that nitrogen conversion rate was proportional to the population of anammox bacteria. Maintaining the ideal environment for the growth of anammox bacteria in the ABF reactor might lead to faster growth. This is the first report of the growth rate of anammox bacteria based on the direct counting of anammox bacteria.
Growth characteristic of anaerobic ammonium-oxidizing bacteria in an anaerobic biological filtrated reactor by Kazuichi Isaka; Yasuhiro Date; Tatsuo Sumino; Sachiko Yoshie; Satoshi Tsuneda (pp. 47-52).
The doubling time of anaerobic ammonium-oxidizing (anammox) bacteria in an anaerobic biological filtrated (ABF) reactor was determined. Fluorescence in situ hybridization analysis was used to detect and count anammox bacteria cells in anammox sludge. As a result, the populations of anammox bacteria at 14th and 21st days were 1.1×106 and 1.7×107 cells/ml reactor, respectively. From these results, the doubling time of anammox bacteria was calculated as 1.8 days, and the specific growth rate (μ) was 0.39 day−1. This result indicated that the anammox bacteria have higher growth rate than the reported value (doubling time, 11 days). Furthermore, it was clearly demonstrated that nitrogen conversion rate was proportional to the population of anammox bacteria. Maintaining the ideal environment for the growth of anammox bacteria in the ABF reactor might lead to faster growth. This is the first report of the growth rate of anammox bacteria based on the direct counting of anammox bacteria.
Biotransformation of β-ionone by engineered cytochrome P450 BM-3 by Vlada B. Urlacher; Akhmadjan Makhsumkhanov; Rolf D. Schmid (pp. 53-59).
Wild-type cytochrome P450 monooxygenase from Bacillus megaterium (P450 BM-3) has a low hydroxylation activity for β-ionone (<1 min−1). Substitution of phenylalanine by valine at position 87 led to a more than 100-fold increase in β-ionone hydroxylation activity (115 min−1). Enzyme activity could be further increased by both site-directed and random mutagenesis. The mutant R47L Y51F F87V, designed by site-directed mutagenesis, and the mutant A74E F87V P386S, obtained after two rounds of error-prone polymerase chain reaction, exhibited an increase in activity of up to 300-fold compared to the wild-type enzyme. The triple mutant R47 LY51F F87V exhibited moderate enantioselectivity, forming (R)-4-hydroxy-β-ionone with an optical purity of 39%. All mutants regioselectively converted β-ionone into 4-hydroxy-β-ionone. The regioselectivity is determined amongst others by the absolute configuration of the substrate.
Biotransformation of β-ionone by engineered cytochrome P450 BM-3 by Vlada B. Urlacher; Akhmadjan Makhsumkhanov; Rolf D. Schmid (pp. 53-59).
Wild-type cytochrome P450 monooxygenase from Bacillus megaterium (P450 BM-3) has a low hydroxylation activity for β-ionone (<1 min−1). Substitution of phenylalanine by valine at position 87 led to a more than 100-fold increase in β-ionone hydroxylation activity (115 min−1). Enzyme activity could be further increased by both site-directed and random mutagenesis. The mutant R47L Y51F F87V, designed by site-directed mutagenesis, and the mutant A74E F87V P386S, obtained after two rounds of error-prone polymerase chain reaction, exhibited an increase in activity of up to 300-fold compared to the wild-type enzyme. The triple mutant R47 LY51F F87V exhibited moderate enantioselectivity, forming (R)-4-hydroxy-β-ionone with an optical purity of 39%. All mutants regioselectively converted β-ionone into 4-hydroxy-β-ionone. The regioselectivity is determined amongst others by the absolute configuration of the substrate.
Glucose-1-phosphatase (AgpE) from Enterobacter cloacae displays enhanced phytase activity by T. Herter; O. V. Berezina; N. V. Zinin; G. A. Velikodvorskaya; R. Greiner; R. Borriss (pp. 60-64).
Using a screening procedure developed for detection of phytate hydrolysing enzymes, the gene agpE encoding glucose-1-phosphatase was cloned from an Enterobacter cloacae VKPM B2254 plasmid library. Sequence analysis revealed 78% identity on nucleotide and 79% identity on peptide level to Escherichia coli glucose-1-phosphatase characterising the respective gene product as a representative of acid histidine phosphatases harbouring the RH(G/N)RXRP motif. The purified recombinant protein displayed maximum specific activity of 196 U mg−1 protein against glucose-1-phosphate but was also active against other sugar phosphates and p-nitrophenyl phosphate. High-performance ion chromatography of hydrolysis products revealed that AgpE can act as a 3-phytase but is only able to cleave off the third phosphate group from the myo-inositol sugar ring. Based on sequence comparison and catalytic behaviour against phytate, we propose to classify bacterial acid histidine phosphatases/phytases in the three following subclasses: (1) AppA-related phytases, (2) PhyK-related phytases and (3) Agp-related phytases. A distinguished activity of 32 U mg−1 of protein towards myo-inositol-hexa-phosphate, which is two times higher than that of E. coli Agp, suggests that possibly functional differences in terms of phytase activity between Agp- and AppA-like acid histidine phosphatases are fluent.
Glucose-1-phosphatase (AgpE) from Enterobacter cloacae displays enhanced phytase activity by T. Herter; O. V. Berezina; N. V. Zinin; G. A. Velikodvorskaya; R. Greiner; R. Borriss (pp. 60-64).
Using a screening procedure developed for detection of phytate hydrolysing enzymes, the gene agpE encoding glucose-1-phosphatase was cloned from an Enterobacter cloacae VKPM B2254 plasmid library. Sequence analysis revealed 78% identity on nucleotide and 79% identity on peptide level to Escherichia coli glucose-1-phosphatase characterising the respective gene product as a representative of acid histidine phosphatases harbouring the RH(G/N)RXRP motif. The purified recombinant protein displayed maximum specific activity of 196 U mg−1 protein against glucose-1-phosphate but was also active against other sugar phosphates and p-nitrophenyl phosphate. High-performance ion chromatography of hydrolysis products revealed that AgpE can act as a 3-phytase but is only able to cleave off the third phosphate group from the myo-inositol sugar ring. Based on sequence comparison and catalytic behaviour against phytate, we propose to classify bacterial acid histidine phosphatases/phytases in the three following subclasses: (1) AppA-related phytases, (2) PhyK-related phytases and (3) Agp-related phytases. A distinguished activity of 32 U mg−1 of protein towards myo-inositol-hexa-phosphate, which is two times higher than that of E. coli Agp, suggests that possibly functional differences in terms of phytase activity between Agp- and AppA-like acid histidine phosphatases are fluent.
Biobleach boosting effect of recombinant xylanase B from the hyperthermophilic Thermotoga maritima on wheat straw pulp by Z. Q. Jiang; X. T. Li; S. Q. Yang; L. T. Li; Y. Li; W. Y. Feng (pp. 65-71).
The recombinant xylanase B (XynB) of Thermotoga maritima MSB8 was found to be highly specific towards xylans and exhibit very low activity towards carboxymethylcellulose in previous study. XynB was thermostable at neutral to alkaline pH region at 90°C and retained more than 90% activity after 1 h over the pH range of pH 6.1 to 11.1. The suitability of XynB for use in the biobleaching of wheat straw pulp was investigated. Pretreatment of the pulp with XynB resulted in a substantial improvement in the bleachability of wheat straw pulp. When XynB at 10 U g−1 was used to treat wheat straw pulp, it reduced pulp kappa number by 1.1 point, enhanced pulp brightness by 5.5% (% ISO) and improved other pulp properties, such as tensile index and breaking length. Biobleaching of wheat straw pulp with XynB saved active chlorine up to 34.5% while still maintaining the brightness at the control level. Besides, pretreatment of pulp with XynB was also effective at an alkaline pH as high as pH 10.1. This is the first report on the potential application of XynB from T. maritima MSB8 in the pulp and paper sector.
Biobleach boosting effect of recombinant xylanase B from the hyperthermophilic Thermotoga maritima on wheat straw pulp by Z. Q. Jiang; X. T. Li; S. Q. Yang; L. T. Li; Y. Li; W. Y. Feng (pp. 65-71).
The recombinant xylanase B (XynB) of Thermotoga maritima MSB8 was found to be highly specific towards xylans and exhibit very low activity towards carboxymethylcellulose in previous study. XynB was thermostable at neutral to alkaline pH region at 90°C and retained more than 90% activity after 1 h over the pH range of pH 6.1 to 11.1. The suitability of XynB for use in the biobleaching of wheat straw pulp was investigated. Pretreatment of the pulp with XynB resulted in a substantial improvement in the bleachability of wheat straw pulp. When XynB at 10 U g−1 was used to treat wheat straw pulp, it reduced pulp kappa number by 1.1 point, enhanced pulp brightness by 5.5% (% ISO) and improved other pulp properties, such as tensile index and breaking length. Biobleaching of wheat straw pulp with XynB saved active chlorine up to 34.5% while still maintaining the brightness at the control level. Besides, pretreatment of pulp with XynB was also effective at an alkaline pH as high as pH 10.1. This is the first report on the potential application of XynB from T. maritima MSB8 in the pulp and paper sector.
Location of glucose oxidase during production by Aspergillus niger by K. G. Clarke; M. Johnstone-Robertson; B. Price; S. T. L. Harrison (pp. 72-77).
The production of the enzyme glucose oxidase by Aspergillus niger is well documented. However, its distribution within the fungal culture is less well defined. Since the enzyme location impacts significantly on enzyme recovery, this study quantifies the enzyme distribution between the extracellular fluid, cell wall, cytoplasm and slime mucilage fractions in an A. niger NRRL-3. The culture was separated into the individual fractions and the glucose oxidase activity was determined in each. The extracellular fluid contained 38% of the total activity. The remaining 62% was associated with the mycelia and was distributed between the cell wall, cytoplasm and slime mucilage in the proportions of 34, 12 and 16%, respectively. Intracellular cytoplasmic and cell wall sites were confirmed using immunocytochemical labelling of the mycelia. In the non-viable cell, the mycelial-associated enzyme was distributed between these sites, whereas in the viable cell, it was predominantly associated with the cell wall. The distribution of the enzyme activity indicates that recovery from the solids would result in a 38% loss, whereas recovery from the extracellular fluid would result in a 62% loss. The results also suggest, however, that this 62% loss could be reduced to around 34% by disintegrating the solids prior to separation due to the contribution of the enzyme in the cytoplasm and slime mucilage. This was confirmed by independently establishing the percentage activity in the liquid and solid portions of a disintegrated culture as 62 and 38%, respectively.
Location of glucose oxidase during production by Aspergillus niger by K. G. Clarke; M. Johnstone-Robertson; B. Price; S. T. L. Harrison (pp. 72-77).
The production of the enzyme glucose oxidase by Aspergillus niger is well documented. However, its distribution within the fungal culture is less well defined. Since the enzyme location impacts significantly on enzyme recovery, this study quantifies the enzyme distribution between the extracellular fluid, cell wall, cytoplasm and slime mucilage fractions in an A. niger NRRL-3. The culture was separated into the individual fractions and the glucose oxidase activity was determined in each. The extracellular fluid contained 38% of the total activity. The remaining 62% was associated with the mycelia and was distributed between the cell wall, cytoplasm and slime mucilage in the proportions of 34, 12 and 16%, respectively. Intracellular cytoplasmic and cell wall sites were confirmed using immunocytochemical labelling of the mycelia. In the non-viable cell, the mycelial-associated enzyme was distributed between these sites, whereas in the viable cell, it was predominantly associated with the cell wall. The distribution of the enzyme activity indicates that recovery from the solids would result in a 38% loss, whereas recovery from the extracellular fluid would result in a 62% loss. The results also suggest, however, that this 62% loss could be reduced to around 34% by disintegrating the solids prior to separation due to the contribution of the enzyme in the cytoplasm and slime mucilage. This was confirmed by independently establishing the percentage activity in the liquid and solid portions of a disintegrated culture as 62 and 38%, respectively.
Gene expression and function study of fusion immunotoxin anti-Her-2-scFv—SEC2 in Escherichia coli by Xu Ming-Kai; Zhang Cheng-Gang (pp. 78-84).
The whole sec2 DNA fragment was obtained by polymerase chain reaction with total genomic DNA extracted from Staphylococcus aureus. The newly discovered gene contains 717 bp (GenBank Accession number AY450554) and encodes 239 amino acids in accordance with the protein sequence reported in GenBank. The sec2 gene was fused to anti-Her-2 scFv gene with a DNA linker at the upstream to construct the fusion gene of immunotoxin which was subcloned to expression vector pET-32a(+) and expressed efficiently in Escherichia coli. The purified fusion immunotoxin could target the HER-2 overexpressed by breast tumor cell SK-Br-3 in vitro and demonstrate tumor-inhibition effect on SK-Br-3.
Gene expression and function study of fusion immunotoxin anti-Her-2-scFv—SEC2 in Escherichia coli by Xu Ming-Kai; Zhang Cheng-Gang (pp. 78-84).
The whole sec2 DNA fragment was obtained by polymerase chain reaction with total genomic DNA extracted from Staphylococcus aureus. The newly discovered gene contains 717 bp (GenBank Accession number AY450554) and encodes 239 amino acids in accordance with the protein sequence reported in GenBank. The sec2 gene was fused to anti-Her-2 scFv gene with a DNA linker at the upstream to construct the fusion gene of immunotoxin which was subcloned to expression vector pET-32a(+) and expressed efficiently in Escherichia coli. The purified fusion immunotoxin could target the HER-2 overexpressed by breast tumor cell SK-Br-3 in vitro and demonstrate tumor-inhibition effect on SK-Br-3.
Expression of a soluble flavone synthase allows the biosynthesis of phytoestrogen derivatives in Escherichia coli by Effendi Leonard; Joseph Chemler; Kok Hong Lim; Mattheos A. G. Koffas (pp. 85-91).
Flavones are plant secondary metabolites with potent pharmacological properties. We report the functional expression of FSI, a flavonoid 2-oxoglutarate-dependent dioxygenase-encoding flavone synthase from parsley in Escherichia coli. This expression allows the biosynthesis of various flavones from phenylpropanoid acids in recombinant E. coli strains simultaneously expressing five plant-specific flavone biosynthetic genes. The gene ensemble consists of 4CL-2 (4-coumarate:CoA ligase) and FSI (flavone synthase I) from parsley, chsA (chalcone synthase) and chiA (chalcone isomerase) from Petunia hybrida, and OMT1A (7-O-methyltransferase) from peppermint. After a 24-h cultivation, the recombinant E. coli produces significant amounts of apigenin (415 μg/l), luteolin (10 μg/l), and genkwanin (208 μg/l). The majority of the flavone products are excreted in the culture media; however, 25% is contained within the cells. The metabolic engineering strategy presented demonstrates that plant-specific flavones are successfully produced in E. coli for the first time by incorporating a soluble flavone synthase confined only in Apiaceae.
Expression of a soluble flavone synthase allows the biosynthesis of phytoestrogen derivatives in Escherichia coli by Effendi Leonard; Joseph Chemler; Kok Hong Lim; Mattheos A. G. Koffas (pp. 85-91).
Flavones are plant secondary metabolites with potent pharmacological properties. We report the functional expression of FSI, a flavonoid 2-oxoglutarate-dependent dioxygenase-encoding flavone synthase from parsley in Escherichia coli. This expression allows the biosynthesis of various flavones from phenylpropanoid acids in recombinant E. coli strains simultaneously expressing five plant-specific flavone biosynthetic genes. The gene ensemble consists of 4CL-2 (4-coumarate:CoA ligase) and FSI (flavone synthase I) from parsley, chsA (chalcone synthase) and chiA (chalcone isomerase) from Petunia hybrida, and OMT1A (7-O-methyltransferase) from peppermint. After a 24-h cultivation, the recombinant E. coli produces significant amounts of apigenin (415 μg/l), luteolin (10 μg/l), and genkwanin (208 μg/l). The majority of the flavone products are excreted in the culture media; however, 25% is contained within the cells. The metabolic engineering strategy presented demonstrates that plant-specific flavones are successfully produced in E. coli for the first time by incorporating a soluble flavone synthase confined only in Apiaceae.
Molecular and enzymatic analysis of the “aldoxime–nitrile pathway” in the glutaronitrile degrader Pseudomonas sp. K-9 by Yasuo Kato; Yasuhisa Asano (pp. 92-101).
A gene cluster responsible for aldoxime metabolism in the glutaronitrile degrader Pseudomonas sp. K-9 was analyzed genetically and enzymatically. The cluster was composed of genes coding for aldoxime dehydratase (Oxd), nitrile hydratase (NHase), NHase activator, amidase, acyl-CoA ligase, and some regulatory and functionally unknown proteins, which were similar to proteins appearing in the “aldoxime–nitrile pathway” gene cluster from strains having Fe-containing NHase. A key enzyme in the cluster, OxdK, which has 32.7–90.3 % identity with known Oxds, was overexpressed in Escherichia coli cells under the control of a T7 promoter in its His6-tagged form, purified, and characterized. The enzyme showed similar characteristics with the known Oxds coexisting with an Fe-containing NHase in its subunit structure, substrate specificity, and effects on various compounds. The enzyme can be classified into a group of “aliphatic aldoxime dehydratase (EC 4.99.1.5).” The existence of a gene cluster of enzymes responsible for aldoxime metabolism via the aldoxime–nitrile pathway (aldoxime→nitrile→amide→acid→acyl-CoA) in Pseudomonas sp. K-9, and the fact that the proteins comprising the cluster are similar to those acting on aliphatic type substrates, evidently clarified the alkylaldoxime-degrading pathway in that strain.
Molecular and enzymatic analysis of the “aldoxime–nitrile pathway” in the glutaronitrile degrader Pseudomonas sp. K-9 by Yasuo Kato; Yasuhisa Asano (pp. 92-101).
A gene cluster responsible for aldoxime metabolism in the glutaronitrile degrader Pseudomonas sp. K-9 was analyzed genetically and enzymatically. The cluster was composed of genes coding for aldoxime dehydratase (Oxd), nitrile hydratase (NHase), NHase activator, amidase, acyl-CoA ligase, and some regulatory and functionally unknown proteins, which were similar to proteins appearing in the “aldoxime–nitrile pathway” gene cluster from strains having Fe-containing NHase. A key enzyme in the cluster, OxdK, which has 32.7–90.3 % identity with known Oxds, was overexpressed in Escherichia coli cells under the control of a T7 promoter in its His6-tagged form, purified, and characterized. The enzyme showed similar characteristics with the known Oxds coexisting with an Fe-containing NHase in its subunit structure, substrate specificity, and effects on various compounds. The enzyme can be classified into a group of “aliphatic aldoxime dehydratase (EC 4.99.1.5).” The existence of a gene cluster of enzymes responsible for aldoxime metabolism via the aldoxime–nitrile pathway (aldoxime→nitrile→amide→acid→acyl-CoA) in Pseudomonas sp. K-9, and the fact that the proteins comprising the cluster are similar to those acting on aliphatic type substrates, evidently clarified the alkylaldoxime-degrading pathway in that strain.
Inhibition of sortase-mediated Staphylococcus aureus adhesion to fibronectin via fibronectin-binding protein by sortase inhibitors by Ki-Bong Oh; Mi-Na Oh; Jae-Gyu Kim; Dong-Sun Shin; Jongheon Shin (pp. 102-106).
The sortase enzymes are a family of Gram-positive transpeptidases responsible for anchoring surface protein virulence factors to the peptidoglycan cell wall layer. In Staphylococcus aureus, deletion of the sortase isoforms results in marked reduction in virulence and infection potential, making it an important antivirulence target. Recombinant sortase A (SrtA) and sortase B (SrtB) were incubated with peptide substrate containing either the LPETG or NPQTN motifs. (Z)-3-(2,5-dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile, β-sitosterol-3-O-glucopyranoside, berberine chloride, and psammaplin A1 showed potent inhibitory activity against SrtA and SrtB. These compounds also exhibited potent inhibitory activity against S. aureus cell adhesion to fibronectin. The fibronectin-binding activity data highlight the potential of these compounds for the treatment of S. aureus infections via inhibition of sortase activity.
Inhibition of sortase-mediated Staphylococcus aureus adhesion to fibronectin via fibronectin-binding protein by sortase inhibitors by Ki-Bong Oh; Mi-Na Oh; Jae-Gyu Kim; Dong-Sun Shin; Jongheon Shin (pp. 102-106).
The sortase enzymes are a family of Gram-positive transpeptidases responsible for anchoring surface protein virulence factors to the peptidoglycan cell wall layer. In Staphylococcus aureus, deletion of the sortase isoforms results in marked reduction in virulence and infection potential, making it an important antivirulence target. Recombinant sortase A (SrtA) and sortase B (SrtB) were incubated with peptide substrate containing either the LPETG or NPQTN motifs. (Z)-3-(2,5-dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile, β-sitosterol-3-O-glucopyranoside, berberine chloride, and psammaplin A1 showed potent inhibitory activity against SrtA and SrtB. These compounds also exhibited potent inhibitory activity against S. aureus cell adhesion to fibronectin. The fibronectin-binding activity data highlight the potential of these compounds for the treatment of S. aureus infections via inhibition of sortase activity.
Antihyperglycemic effect of polysaccharide from fermented broth of Pleurotus citrinopileatus by Shu-Hui Hu; Jinn-Chyi Wang; Juang-Lin Lien; Ean-Tun Liaw; Min-Yen Lee (pp. 107-113).
Pleurotus citrinopileatus is an edible mushroom, which has recently become very popular, with a consequent increase in industrial production. Water-soluble polysaccharides (WSPS), extracted from edible mushrooms, have been found to have antitumor and immunoenhancing effects. In this study, we investigate the effects of WSPS extracted from submerged fermented medium of P. citrinopileatus on hyperglycemia and damaged pancreatic cells in rats with streptozotocin (STZ)-induced diabetes. The diabetic rats fed with water-soluble polysaccharide of P. citrinopileatus (SPPC) lost less body weight than those fed SPPC-free regular diet. Serum total cholesterol and triglyceride levels in the diabetic rats fed with SPPC at a dose of 0.4 g/kg bw daily was lower than in the groups fed with SPPC at doses of 0.04 and 0.12 g/kg bw. The fasting blood glucose levels of diabetic rats fed with SPPC were 44% lower than the negative controls. The degree of damage to the islets of Langerhans of the rats fed with the highest dosage of SPPC was significantly lower than those fed with SPPC at doses of 0.04 and 0.12 g/kg bw. The results showed that STZ-induced diabetic rats fed with SPPC might help alleviate the elevation of the level of that in fasting blood glucose.
Antihyperglycemic effect of polysaccharide from fermented broth of Pleurotus citrinopileatus by Shu-Hui Hu; Jinn-Chyi Wang; Juang-Lin Lien; Ean-Tun Liaw; Min-Yen Lee (pp. 107-113).
Pleurotus citrinopileatus is an edible mushroom, which has recently become very popular, with a consequent increase in industrial production. Water-soluble polysaccharides (WSPS), extracted from edible mushrooms, have been found to have antitumor and immunoenhancing effects. In this study, we investigate the effects of WSPS extracted from submerged fermented medium of P. citrinopileatus on hyperglycemia and damaged pancreatic cells in rats with streptozotocin (STZ)-induced diabetes. The diabetic rats fed with water-soluble polysaccharide of P. citrinopileatus (SPPC) lost less body weight than those fed SPPC-free regular diet. Serum total cholesterol and triglyceride levels in the diabetic rats fed with SPPC at a dose of 0.4 g/kg bw daily was lower than in the groups fed with SPPC at doses of 0.04 and 0.12 g/kg bw. The fasting blood glucose levels of diabetic rats fed with SPPC were 44% lower than the negative controls. The degree of damage to the islets of Langerhans of the rats fed with the highest dosage of SPPC was significantly lower than those fed with SPPC at doses of 0.04 and 0.12 g/kg bw. The results showed that STZ-induced diabetic rats fed with SPPC might help alleviate the elevation of the level of that in fasting blood glucose.
The degradation of α-quaternary nonylphenol isomers by Sphingomonas sp. strain TTNP3 involves a type II ipso-substitution mechanism by P. F. X. Corvini; J. Hollender; R. Ji; S. Schumacher; J. Prell; G. Hommes; U. Priefer; R. Vinken; A. Schäffer (pp. 114-122).
The degradation of radiolabeled 4(3′,5′-dimethyl-3′-heptyl)-phenol [nonylphenol (NP)] was tested with resting cells of Sphingomonas sp. strain TTNP3. Concomitantly to the degradation of NP, a metabolite identified as hydroquinone transiently accumulated and short-chain organic acids were then produced at the expense of hydroquinone. Two other radiolabeled isomers of NP, 4(2′,6′-dimethyl-2′-heptyl)-phenol and 4(3′,6′-dimethyl-3′-heptyl)-phenol, were synthesized. In parallel experiments, the 4(2′,6′-dimethyl-2′-heptyl)-phenol was degraded more slowly than the other isomers of NP by strain TTNP3, possibly because of effects of the side-chain structure on the kinetics of degradation. Alkylbenzenediol and alkoxyphenol derivatives identified as metabolites during previous studies were synthesized and tested as substrates. The derivatives were not degraded, which indicated that the mineralization of NP does not proceed via alkoxyphenol as the principal intermediate. The results obtained led to the elucidation of the degradation pathway of NP isomers with a quaternary α-carbon. The proposed mechanism is a type II ipso substitution, leading to hydroquinone and nonanol as the main metabolites and to the dead-end metabolites alkylbenzenediol or alkoxyphenol, depending on the substitution at the α-carbon of the carbocationic intermediate formed.
The degradation of α-quaternary nonylphenol isomers by Sphingomonas sp. strain TTNP3 involves a type II ipso-substitution mechanism by P. F. X. Corvini; J. Hollender; R. Ji; S. Schumacher; J. Prell; G. Hommes; U. Priefer; R. Vinken; A. Schäffer (pp. 114-122).
The degradation of radiolabeled 4(3′,5′-dimethyl-3′-heptyl)-phenol [nonylphenol (NP)] was tested with resting cells of Sphingomonas sp. strain TTNP3. Concomitantly to the degradation of NP, a metabolite identified as hydroquinone transiently accumulated and short-chain organic acids were then produced at the expense of hydroquinone. Two other radiolabeled isomers of NP, 4(2′,6′-dimethyl-2′-heptyl)-phenol and 4(3′,6′-dimethyl-3′-heptyl)-phenol, were synthesized. In parallel experiments, the 4(2′,6′-dimethyl-2′-heptyl)-phenol was degraded more slowly than the other isomers of NP by strain TTNP3, possibly because of effects of the side-chain structure on the kinetics of degradation. Alkylbenzenediol and alkoxyphenol derivatives identified as metabolites during previous studies were synthesized and tested as substrates. The derivatives were not degraded, which indicated that the mineralization of NP does not proceed via alkoxyphenol as the principal intermediate. The results obtained led to the elucidation of the degradation pathway of NP isomers with a quaternary α-carbon. The proposed mechanism is a type II ipso substitution, leading to hydroquinone and nonanol as the main metabolites and to the dead-end metabolites alkylbenzenediol or alkoxyphenol, depending on the substitution at the α-carbon of the carbocationic intermediate formed.
Biosynthesis of radiolabeled cellodextrins by the Clostridium thermocellum cellobiose and cellodextrin phosphorylases for measurement of intracellular sugars by Y. -H. P. Zhang; L. R. Lynd (pp. 123-129).
The Clostridium thermocellum cellobiose and cellodextrin phosphorylases (glucosyl transferases) in the cell extract were used to synthesize radiolabeled cellodextrins with a degree of polymerization (DP=2–6) from nonradioactive glucose-1-phosphate and radioactive glucose. Chain lengths of synthesized cellodextrin were controlled by the absence or presence of dithiothreitol and by reaction conditions. All cellodextrins have the sole radioactive glucose unit located at the reducing ends. Mixed cellodextrins (G2–G6) were separated efficiently by size-exclusion chromatography or less efficiently by thin-layer chromatography. A new rapid sampling device was developed using disposable syringes containing an ultracold methanol-quenching buffer. It was simple, less costly, and especially convenient for anaerobic fermentation. After an impulse feed of radiolabeled cellobiose, the intracellular sugar levels were measured after a series of operations—sampling, extracting, concentrating, separating, and reading. Results showed that the largest amount of radioactivity was cellobiose with lesser amounts of glucose, cellotriose, and cellotetraose, and an average DP of intracellular cellodextrins was ca. 2.
Biosynthesis of radiolabeled cellodextrins by the Clostridium thermocellum cellobiose and cellodextrin phosphorylases for measurement of intracellular sugars by Y. -H. P. Zhang; L. R. Lynd (pp. 123-129).
The Clostridium thermocellum cellobiose and cellodextrin phosphorylases (glucosyl transferases) in the cell extract were used to synthesize radiolabeled cellodextrins with a degree of polymerization (DP=2–6) from nonradioactive glucose-1-phosphate and radioactive glucose. Chain lengths of synthesized cellodextrin were controlled by the absence or presence of dithiothreitol and by reaction conditions. All cellodextrins have the sole radioactive glucose unit located at the reducing ends. Mixed cellodextrins (G2–G6) were separated efficiently by size-exclusion chromatography or less efficiently by thin-layer chromatography. A new rapid sampling device was developed using disposable syringes containing an ultracold methanol-quenching buffer. It was simple, less costly, and especially convenient for anaerobic fermentation. After an impulse feed of radiolabeled cellobiose, the intracellular sugar levels were measured after a series of operations—sampling, extracting, concentrating, separating, and reading. Results showed that the largest amount of radioactivity was cellobiose with lesser amounts of glucose, cellotriose, and cellotetraose, and an average DP of intracellular cellodextrins was ca. 2.