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1.
Bacillus coagulans has been of great commercial interest over the past decade owing to its strong ability of producing optical pure l-lactic acid from both hexose and pentose sugars including l-arabinose with high yield, titer and productivity under thermophilic conditions. The l-arabinose isomerase (L-AI) from Bacillus coagulans was heterologously over-expressed in Escherichia coli. The open reading frame of the L-AI has 1,422 nucleotides encoding a protein with 474 amino acid residues. The recombinant
L-AI was purified to homogeneity by one-step His-tag affinity chromatography. The molecular mass of the enzyme was estimated
to be 56 kDa by SDS-PAGE. The enzyme was most active at 70°C and pH 7.0. The metal ion Mn2+ was shown to be the best activator for enzymatic activity and thermostability. The enzyme showed higher activity at acidic
pH than at alkaline pH. The kinetic studies showed that the K
m, V
max and k
cat/K
m for the conversion of l-arabinose were 106 mM, 84 U/mg and 34.5 mM−1min−1, respectively. The equilibrium ratio of l-arabinose to l-ribulose was 78:22 under optimal conditions. l-ribulose (97 g/L) was obtained from 500 g/l of l-arabinose catalyzed by the enzyme (8.3 U/mL) under the optimal conditions within 1.5 h, giving at a substrate conversion
of 19.4% and a production rate of 65 g L−1 h−1. 相似文献
2.
Mark S. Ou Lonnie O. Ingram K. T. Shanmugam 《Journal of industrial microbiology & biotechnology》2011,38(5):599-605
Lactic acid is used as an additive in foods, pharmaceuticals, and cosmetics, and is also an industrial chemical. Optically
pure lactic acid is increasingly used as a renewable bio-based product to replace petroleum-based plastics. However, current
production of lactic acid depends on carbohydrate feedstocks that have alternate uses as foods. The use of non-food feedstocks
by current commercial biocatalysts is limited by inefficient pathways for pentose utilization. B. coagulans strain 36D1 is a thermotolerant bacterium that can grow and efficiently ferment pentoses using the pentose-phosphate pathway
and all other sugar constituents of lignocellulosic biomass at 50°C and pH 5.0, conditions that also favor simultaneous enzymatic
saccharification and fermentation (SSF) of cellulose. Using this bacterial biocatalyst, high levels (150–180 g l−1) of lactic acid were produced from xylose and glucose with minimal by-products in mineral salts medium. In a fed-batch SSF
of crystalline cellulose with fungal enzymes and B. coagulans, lactic acid titer was 80 g l−1 and the yield was close to 80%. These results demonstrate that B. coagulans can effectively ferment non-food carbohydrates from lignocellulose to l(+)-lactic acid at sufficient concentrations for commercial application. The high temperature fermentation of pentoses and
hexoses to lactic acid by B. coagulans has these additional advantages: reduction in cellulase loading in SSF of cellulose with a decrease in enzyme cost in the
process and a reduction in contamination of large-scale fermentations. 相似文献
3.
Livingstone JR Yoshida I Tarui Y Hirooka K Yamamoto Y Tsutui N Hirasawa E 《Journal of plant research》2002,115(5):393-400
NAD-dependent aminoaldehyde dehydrogenase (AMADH, EC 1.2.1.-) from Avena shoots was purified by DEAE Sephacel, hydroxyapatite, 5′-AMP Sepharose 4B, Mono Q, and TSK-GEL column chromatographies to
homogeneity by the criterion of native PAGE. SDS–PAGE yielded a single band at a molecular mass of 55 kDa. IEF studies showed
a band with a pI value of 5.3. In contrast to AMADHs from other species, the TSK-GEL chromatography showed that Avena AMADH exists as a monomer in the native state. The purified enzyme catalyzed the oxidations of 3-aminopropionaldehyde (APAL),
4-aminobutyraldehyde (ABAL) N-(3-aminopropyl)-4-aminobutyraldehyde (APBAL), and 4-guanidinobutyraldehyde (GBAL), but not of betaine aldehyde or indoleacetaldehyde.
The K
m values for APAL, ABAL, and GBAL were 1.5×10–6, 2.2×10–6, and 1.3×10–5 M, respectively. Although N-terminal amino acid sequence of Avena AMADH could not be determined due to a modification of the amino residue, the sequence of the fragment of AMADH cleaved by
V8 protease showed greater similarity to the barley BADH than to the pea AMADH.
Electronic Publication 相似文献
4.
Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes. 相似文献
5.
The gene encoding malate dehydrogenase (MDH) was overexpressed in a pflB ldhA double mutant of Escherichia coli, NZN111, for succinic acid production. With MDH overexpression, NZN111/pTrc99A-mdh restored the ability to metabolize glucose anaerobically and 0.55 g/L of succinic acid was produced from 3 g/L of glucose
in shake flask culture. When supplied with 10 g/L of sodium bicarbonate (NaHCO3), the succinic acid yield of NZN111/pTrc99A-mdh reached 1.14 mol/mol glucose. Supply of NaHCO3 also improved succinic acid production by the control strain, NZN111/pTrc99A. Measurement of key enzymes activities revealed
that phosphoenolpyruvate (PEP) carboxykinase and PEP carboxylase in addition to MDH played important roles. Two-stage culture
of NZN111/pTrc99A-mdh was carried out in a 5-L bioreactor and 12.2 g/L of succinic acid were produced from 15.6 g/L of glucose. Fed-batch culture
was also performed, and the succinic acid concentration reached 31.9 g/L with a yield of 1.19 mol/mol glucose. 相似文献
6.
Skory CD 《Journal of industrial microbiology & biotechnology》2003,30(1):22-27
This work demonstrates the first example of a fungal lactate dehydrogenase (LDH) expressed in yeast. A L(+)-LDH gene, ldhA, from the filamentous fungus Rhizopus oryzae was modified to be expressed under control of the Saccharomyces cerevisiae adh1 promoter and terminator and then placed in a 2μ-containing yeast-replicating plasmid. The resulting construct, pLdhA68X,
was transformed and tested by fermentation analyses in haploid and diploid yeast containing similar genetic backgrounds. Both
recombinant strains utilized 92 g glucose/l in approximately 30 h. The diploid isolate accumulated approximately 40% more
lactic acid with a final concentration of 38 g lactic acid/l and a yield of 0.44 g lactic acid/g glucose. The optimal pH for
lactic acid production by the diploid strain was pH 5. LDH activity in this strain remained relatively constant at 1.5 units/mg
protein throughout the fermentation. The majority of carbon was still diverted to the ethanol fermentation pathway, as indicated
by ethanol yields between 0.25–0.33 g/g glucose. S. cerevisiae mutants impaired in ethanol production were transformed with pLdhA68X in an attempt to increase the lactic acid yield by
minimizing the conversion of pyruvate to ethanol. Mutants with diminished pyruvate decarboxylase activity and mutants with
disrupted alcohol dehydrogenase activity did result in transformants with diminished ethanol production. However, the efficiency
of lactic acid production also decreased.
Electronic Publication 相似文献
7.
Zhengfang Zhang Yanming Sheng Keyi Jiang Zhao Wang Yuguo Zheng Qing Zhu 《Biotechnology letters》2010,32(4):513-516
A newly isolated Bacillus megaterium with epoxide hydrolase activity resolved racemic glycidyl (o, m, p)-methylphenyl ethers to give enantiopure epoxides in 84–99% enantiomeric excess and with 21–73 enantiomeric ratios. The (S)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ether while the (R)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained at the level by enzyme-substrate docking studies. 相似文献
8.
A genetic transformation system has been developed for callus cells of Crataegus
aronia using Agrobacterium
tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with
5 mg l−1 Indole-3-butyric acid (IBA) and 0.5 mg l−1 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different
types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red
colored callus was obtained on MS medium supplemented with 2 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l−1 kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli
were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l−1 hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this
is the first time to report an Agrobacterium-mediated transformation system in Crataegus
aronia. 相似文献
9.
Kenro Tokuhiro Nobuhiro Ishida Eiji Nagamori Satoshi Saitoh Toru Onishi Akihiko Kondo Haruo Takahashi 《Applied microbiology and biotechnology》2009,82(5):883-890
Expression of a heterologous l-lactate dehydrogenase (l-ldh) gene enables production of optically pure l-lactate by yeast Saccharomyces cerevisiae. However, the lactate yields with engineered yeasts are lower than those in the case of lactic acid bacteria because there
is a strong tendency for ethanol to be competitively produced from pyruvate. To decrease the ethanol production and increase
the lactate yield, inactivation of the genes that are involved in ethanol production from pyruvate is necessary. We conducted
double disruption of the pyruvate decarboxylase 1 (PDC1) and alcohol dehydrogenase 1 (ADH1) genes in a S. cerevisiae strain by replacing them with the bovine l-ldh gene. The lactate yield was increased in the pdc1/adh1 double mutant compared with that in the single pdc1 mutant. The specific growth rate of the double mutant was decreased on glucose but not affected on ethanol or acetate compared
with in the control strain. The aeration rate had a strong influence on the production rate and yield of lactate in this strain.
The highest lactate yield of 0.75 g lactate produced per gram of glucose consumed was achieved at a lower aeration rate. 相似文献
10.
Ali Dehestani Kamal Kazemitabar Gholamreza Ahmadian Nadali Babaeian Jelodar Ali Hatef Salmanian Mehdi Seyedi Heshmat Rahimian Seyedhadi Ghasemi 《Biotechnology letters》2010,32(4):539-546
The Bacillus pumilus SG2 chitinase gene (ChiS) and its truncated form lacking chitin binding (ChBD) and fibronectin type III (FnIII) domains were transformed to Arabidopsis plants and the expression, functionality and antifungal activity of the recombinant proteins were investigated. Results showed
that while the two enzyme forms showed almost equal hydrolytic activity toward colloidal chitin, they exhibited a significant
difference in antifungal activity. Recombinant ChiS in plant protein extracts displayed a high inhibitory effect on spore
germination and radial growth of hyphae in Alternaria brassicicola, Fusarium graminearum and Botrytis cinerea, while the activity of the truncated enzyme was strongly abolished. These findings demonstrate that ChBD and FnIII domains
are not necessary for hydrolysis of colloidal chitin but play an important role in hydrolysis of chitin–glucan complex of
fungal cell walls. Twenty microgram aliquots of protein extracts from ChiS transgenic lines displayed strong antifungal activity
causing up to 80% decrease in fungal spore germination. This is the first report of a Bacillus pumilus chitinase expressed in plant system. 相似文献
11.
Noel H. Holmgren 《Brittonia》2018,70(1):115-139
A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations. 相似文献
12.
13.
Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner.
The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate
larvae Galleria
mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host. 相似文献
14.
15.
The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae. 相似文献
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19.
The foodborne pathogen Bacillus cereus can form biofilms on various food contact surfaces, leading to contamination of food products. To study the mechanisms of
biofilm formation by B. cereus, a Tn5401 library was generated from strain UW101C. Eight thousand mutants were screened in EPS, a low nutrient medium. One mutant
(M124), with a disruption in codY, developed fourfold less biofilm than the wild-type, and its defective biofilm phenotype was rescued by complementation.
Addition of 0.1% casamino acids to EPS prolonged the duration of biofilms in the wild-type but not codY mutant. When decoyinine, a GTP synthesis inhibitor, was added to EPS, biofilm formation was decreased in the wild-type but
not the mutant. The codY mutant produced three times higher protease activity than the wild-type. Zymogram and SDS-PAGE data showed that production
of the protease (∼130 kDa) was repressed by CodY. Addition of proteinase K to EPS decreased biofilm formation by the wild-type.
Using a dpp-lacZ fusion reporter system, it was shown that that the B. cereus CodY can sense amino acids and GTP levels. These data suggest that by responding to amino acids and intracellular GTP levels
CodY represses production of an unknown protease and is involved in biofilm formation. 相似文献
20.
An SJ Pandeya D Park SW Li J Kwon JK Koeda S Hosokawa M Paek NC Choi D Kang BC 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2011,122(3):459-470
A temperature-sensitive mutant of Capsicum chinense, sy-2, shows a normal developmental phenotype when grown above 24°C. However, when grown at 20°C, sy-2 exhibits developmental defects, such as chlorophyll deficiency and shrunken leaves. To understand the underlying mechanism
of this temperature-dependent response, phenotypic characterization and genetic analysis were performed. The results revealed
abnormal chloroplast structures and cell collapse in leaves of the sy-2 plants grown at 20°C. Moreover, an excessive accumulation of reactive oxygen species (ROS) resulting in cell death was detected
in the chlorophyll-deficient sectors of the leaves. However, the expression profile of the ROS scavenging genes did not alter
in sy-2 plants grown at 20°C. A further analysis of fatty acid content in the leaves showed the impaired pathway of linoleic acid
(18:2) to linolenic acid (18:3). Additionally, the Cafad7 gene was downregulated in sy-2 plants. This change may lead to dramatic physiological disorder and alteration of leaf morphology in sy-2 plants by losing low-temperature tolerance. Genetic analysis of an F2 population from a cross between C. chinense ‘sy-2’ and wild-type C. chinense ‘No. 3341’ showed that the sy-2 phenotype is controlled by a single recessive gene. Molecular mapping revealed that the sy-2 gene is located at a genomic region of the pepper linkage group 1, corresponding to the 300 kb region of the Ch1_scaffold
00106 in tomato chromosome 1. Candidate genes in this region will reveal the identity of sy-2 and the underlying mechanism of the temperature-dependent plant response. 相似文献