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1.
The Vitreoscilla hemoglobin (VHb) gene (vgb) was integrated into the chromosome of Bacillus thuringiensis BMB171 using integrative vector pEG491. The production of VHb was confirmed by CO-difference spectra analysis. Fermentation
experiments results showed that with the production of VHb, the critical oxygen concentration (COC) of the host strain was
reduced from 18 to 12%. The maximum viable cell counts of the VHb+ strain in high, middle, and low aeration/agitation fermentations were 0.94-, 1.23-, and 1.59-fold of those of the VHb− strain, respectively. Under the same conditions, the yields of insecticidal crystal proteins (ICP) by VHb+ strain were 1.22-, 1.63-, and 3.13-fold of those of the VHb− strain. The production of VHb also accelerated the formation of ICP and spores. These results indicated that the production
of VHb could improve the cell density and ICP yield of B. thuringiensis, especially under low aeration/agitation condition. 相似文献
2.
Sanjay Kumar K. Pakshirajan V. Venkata Dasu 《Applied microbiology and biotechnology》2009,84(3):477-486
Glutaminase-free l-asparaginase is known to be an excellent anticancer agent. In the present study, statistically based experimental designs
were applied to maximize the production of glutaminase-free l-asparaginase from Pectobacterium carotovorum MTCC 1428. Nine components of the medium were examined for their significance on the production of l-asparaginase using the Plackett–Burman experimental design. The medium components, viz., glucose, l-asparagine, KH2PO4, and MgSO4·7H2O, were screened based on their high confidence levels (P < 0.04). The optimum levels of glucose, l-asparagine, KH2PO4, and MgSO4·7H2O were found to be 2.076, 5.202, 1.773, and 0.373 g L−1, respectively, using the central composite experimental design. The maximum specific activity of l-asparaginase in the optimized medium was 27.88 U mg−1 of protein, resulting in an overall 8.3-fold increase in the production compared to the unoptimized medium. 相似文献
3.
l-Asparaginase is an anti-neoplastic drug used in lymphoblastic leukemia chemotherapy. Nowadays, this enzyme derived from bacterial
sources, mostly l-asparaginase II from Escherichia coli and in lesser amount l-asparaginase of Erwinia sp. has medical utilization. The long-term usage of these agents leads to allergic reactions and new asparaginase with new
immunological characteristics is required. Halophilic bacteria might contain l-asparaginase with novel immunological properties that can be used in hypersensitive patients. In this experiment, we have
screened moderate Halophilic bacteria for l-asparaginase production ability and showed that Halophilic bacteria produce intra- and extracellular l-asparaginase. Bacillus sp. BCCS 034 was found to produce the highest l-asparaginase (1.64 IU/ml supernatant) extracellularly. 相似文献
4.
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
5.
Fekete E Karaffa L Sándor E Bányai I Seiboth B Gyémánt G Sepsi A Szentirmai A Kubicek CP 《Archives of microbiology》2004,181(1):35-44
The catabolism of d-galactose in yeast depends on the enzymes of the Leloir pathway. In contrast, Aspergillus nidulans mutants in galactokinase (galE) can still grow on d-galactose in the presence of ammonium—but not nitrate—ions as nitrogen source. A. nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading pathway may proceed via this intermediate. The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A. nidulans loss-of-function mutant in this enzyme (araA1) did not show NAD+-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol. The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A. nidulans hexokinase (frA1) mutant. l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A. nidulans was unable to grow on d-galactose. The results therefore provide evidence for an alternative pathway of d-galactose catabolism in A. nidulans that involves reduction of the d-galactose to galactitol and NAD+-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose. 相似文献
6.
Methee Khamduang Kanoktip Packdibamrung Jarun Chutmanop Yusuf Chisti Penjit Srinophakun 《Journal of industrial microbiology & biotechnology》2009,36(10):1267-1274
The production of l-phenylalanine is conventionally carried out by fermentations that use glucose or sucrose as the carbon source. This work
reports on the use of glycerol as an inexpensive and abundant sole carbon source for producing l-phenylalanine using the genetically modified bacterium Escherichia coli BL21(DE3). Fermentations were carried out at 37°C, pH 7.4, using a defined medium in a stirred tank bioreactor at various
intensities of impeller agitation speeds (300–500 rpm corresponding to 0.97–1.62 m s−1 impeller tip speed) and aeration rates (2–8 L min−1, or 1–4 vvm). This highly aerobic fermentation required a good supply of oxygen, but intense agitation (impeller tip speed
~1.62 m s−1) reduced the biomass and l-phenylalanine productivity, possibly because of shear sensitivity of the recombinant bacterium. Production of l-phenylalanine was apparently strongly associated with growth. Under the best operating conditions (1.30 m s−1 impeller tip speed, 4 vvm aeration rate), the yield of l-phenylalanine on glycerol was 0.58 g g−1, or more than twice the best yield attainable on sucrose (0.25 g g−1). In the best case, the peak concentration of l-phenylalanine was 5.6 g L−1, or comparable to values attained in batch fermentations that use glucose or sucrose. The use of glycerol for the commercial
production of l-phenylalanine with E. coli BL21(DE3) has the potential to substantially reduce the cost of production compared to sucrose- and glucose-based fermentations. 相似文献
7.
Blombach B Schreiner ME Moch M Oldiges M Eikmanns BJ 《Applied microbiology and biotechnology》2007,76(3):615-623
Intracellular precursor supply is a critical factor for amino acid productivity of Corynebacterium glutamicum. To test for the effect of improved pyruvate availability on l-lysine production, we deleted the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex (PDHC) in the l-lysine-producer C. glutamicum DM1729 and characterised the resulting strain DM1729-BB1 for growth and l-lysine production. Compared to the host strain, C. glutamicum DM1729-BB1 showed no PDHC activity, was acetate auxotrophic and, after complete consumption of the available carbon sources
glucose and acetate, showed a more than 50% lower substrate-specific biomass yield (0.14 vs 0.33 mol C/mol C), an about fourfold
higher biomass-specific l-lysine yield (5.27 vs 1.23 mmol/g cell dry weight) and a more than 40% higher substrate-specific l-lysine yield (0.13 vs 0.09 mol C/mol C). Overexpression of the pyruvate carboxylase or diaminopimelate dehydrogenase genes
in C. glutamicum DM1729-BB1 resulted in a further increase in the biomass-specific l-lysine yield by 6 and 56%, respectively. In addition to l-lysine, significant amounts of pyruvate, l-alanine and l-valine were produced by C. glutamicum DM1729-BB1 and its derivatives, suggesting a surplus of precursor availability and a further potential to improve l-lysine production by engineering the l-lysine biosynthetic pathway.
This study is dedicated to Prof. Dr. Hermann Sahm on the occasion of his 65th birthday. 相似文献
8.
A major limitation on the expression of some foreign proteins in transgenic plants is the toxic effect of such proteins on
the host plant resulting in inhibition of normal growth and development. A solution to this problem is to control the expression
of genes for such proteins by means of inducible promoters, as is frequently done in microbial systems. A cDNA clone was obtained
from subtractive hybridization of non-harvested and harvested alfalfa leaf tissue, named hi12. The hi12 cDNA was identified as part of the S-adenosyl-l-methionine: trans-caffeoyl-CoA3-O-methyltransferase gene of alfalfa, a gene encoding an essential key enzyme in lignin synthesis. The hi12 gene was strongly induced by harvesting and wounding but not by heat shock. The promoter of the hi12 gene, isolated by genomic walking, contained several stress response cis-elements. Transgenic plants of tobacco and Medicago truncatula containing the GUS gene driven by the promoter showed GUS expression following harvesting, demonstrating the activity of
these regulatory regions in other plant species. 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.
Poonperm W Takata G Okada H Morimoto K Granström TB Izumori K 《Applied microbiology and biotechnology》2007,76(6):1297-1307
The l-rhamnose isomerase gene (L
-rhi) encoding for l-rhamnose isomerase (l-RhI) from Bacillus pallidus Y25, a facultative thermophilic bacterium, was cloned and overexpressed in Escherichia coli with a cooperation of the 6×His sequence at a C-terminal of the protein. The open reading frame of L
-rhi consisted of 1,236 nucleotides encoding 412 amino acid residues with a calculated molecular mass of 47,636 Da, showing a
good agreement with the native enzyme. Mass-produced l-RhI was achieved in a large quantity (470 mg/l broth) as a soluble protein. The recombinant enzyme was purified to homogeneity
by a single step purification using a Ni-NTA affinity column chromatography. The purified recombinant l-RhI exhibited maximum activity at 65°C (pH 7.0) under assay conditions, while 90% of the initial enzyme activity could be
retained after incubation at 60°C for 60 min. The apparent affinity (K
m) and catalytic efficiency (k
cat/K
m) for l-rhamnose (at 65°C) were 4.89 mM and 8.36 × 105 M−1 min−1, respectively. The enzyme demonstrated relatively low levels of amino acid sequence similarity (42 and 12%), higher thermostability,
and different substrate specificity to those of E. coli and Pseudomonas stutzeri, respectively. The enzyme has a good catalyzing activity at 50°C, for d-allose, l-mannose, d-ribulose, and l-talose from d-psicose, l-fructose, d-ribose and l-tagatose with a conversion yield of 35, 25, 16 and 10%, respectively, without a contamination of by-products. These findings
indicated that the recombinant l-RhI from B. pallidus is appropriate for use as a new source of rare sugar producing enzyme on a mass scale production. 相似文献
11.
12.
l-arabinose isomerase (EC5.3.1.4. AI) mediates the isomerization of d-galactose into d-tagatose as well as the conversion of l-arabinose into l-ribulose. The AI from Lactobacillus plantarum SK-2 was purified to an apparent homogeneity giving a single band on SDS–PAGE with a molecular mass of 59.6 kDa. Optimum
activity was observed at 50°C and pH 7.0. The enzyme was stable at 50°C for 2 h and held between pH 4.5 and 8.5 for 1 h. AI
activity was stimulated by Mn2+, Fe3+, Fe2+, Ca2+ and inhibited by Cu2+, Ag+, Hg2+, Pb2+. d-galactose and l-arabinose as substrates were isomerized with high activity. l-arabitol was the strongest competitive inhibitor of AI. The apparent Michaelis–Menten constant (K
m), for galactose, was 119 mM. The first ten N-terminal amino acids of the enzyme were determined as MLSVPDYEFW, which is identical to L. plantarum (Q88S84). Using the purified AI, 390 mg tagatose could be converted from 1,000 mg galactose in 96 h, and this production
corresponds to a 39% equilibrium. 相似文献
13.
Wenjuan Yao Xiaozhao Deng Hui Zhong Miao Liu Pu Zheng Zhihao Sun Yun Zhang 《Journal of industrial microbiology & biotechnology》2009,36(7):911-921
Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis.
The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC
13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by
the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in
ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production
strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis. 相似文献
14.
Li Y Kawakami N Ogola HJ Ashida H Ishikawa T Shibata H Sawa Y 《Applied microbiology and biotechnology》2011,90(6):1953-1962
l-aspartate dehydrogenase (EC 1.4.1.21; l-AspDH) is a rare member of amino acid dehydrogenase superfamily and so far, two thermophilic enzymes have been reported.
In our study, an ORF PA3505 encoding for a putative l-AspDH in the mesophilic bacterium Pseudomonas aeruginosa PAO1 was identified, cloned, and overexpressed in Escherichia coli. The homogeneously purified enzyme (PaeAspDH) was a dimeric protein with a molecular mass of about 28 kDa exhibiting a very
high specific activity for l-aspartate (l-Asp) and oxaloacetate (OAA) of 127 and 147 U mg−1, respectively. The enzyme was capable of utilizing both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine
dinucleotide phosphate (NADP) as coenzyme. PaeAspDH showed a T
m value of 48°C for 20 min that was improved to approximately 60°C by the addition of 0.4 M NaCl or 30% glycerol. The apparent
K
m values for OAA, NADH, and ammonia were 2.12, 0.045, and 10.1 mM, respectively; comparable results were observed with NADPH.
The l-Asp production system B consisting of PaeAspDH, Bacillus subtilis malate dehydrogenase and E. coli fumarase, achieved a high level of l-Asp production (625 mM) from fumarate in fed-batch process with a molar conversion yield of 89.4%. Furthermore, the fermentative
production system C released 33 mM of l-Asp after 50 h by using succinate as carbon source. This study represented an extensive characterization of the mesophilic
AspDH and its potential applicability for efficient and attractive production of l-Asp. Our novel production systems are also hopeful for developing the new processes for other compounds production. 相似文献
15.
An NAD+-dependent l-arabinitol 4-dehydrogenase (LAD, EC 1.1.1.12) from Neurospora crassa was cloned and expressed in Escherichia coli and purified to homogeneity. The enzyme was a homotetramer and contained two Zn2+ ions per subunit, displaying similar characteristics to medium-chain sorbitol dehydrogenases (SDHs). High enzymatic activity
was observed for substrates l-arabinitol, adonitol, and xylitol and no activity for d-mannitol, d-arabinitol, or d-sorbitol. The enzyme showed strong preference for NAD+ but also displayed a very low yet detectable activity with NADP+. Mutational analysis of residue F59, the single different substrate-binding residue between LADs and d-SDHs, failed to confer the enzyme the ability to accept d-sorbitol as a substrate, suggesting that the amino acids flanking the active site cleft may be responsible for the different
activity and affinity patterns between LADs and SDHs. This enzyme should be useful for in vivo and in vitro production of
xylitol and ethanol from l-arabinose.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
16.
Gui-Hye Hwang Jae-Yong Cho 《Journal of industrial microbiology & biotechnology》2010,37(11):1131-1136
We recently proposed a metabolic engineering strategy for l-ornithine production based on the hypothesis that an increased intracellular supply of N-acetylglutamate may further enhance l-ornithine production in a well-defined recombinant strain of Corynebacterium
glutamicum. In this work, an argJ-deficient arginine auxotrophic mutant of C. glutamicum is suppressed by a different locus of C. glutamicum ATCC13032. Overexpression of the NCgl1469 open reading frame (ORF), exhibiting N-acetylglutamate synthase (NAGS) activity, was able to complement the C. glutamicum arginine-auxotrophic argJ strain and showed increased NAGS activity from 0.03 to 0.17 units mg−1 protein. Additionally, overexpression of the NCgl1469 ORF resulted in a 39% increase in excreted l-ornithine. These results indicate that the intracellular supply of N-acetylglutamate is a rate-limiting step during l-ornithine production in C. glutamicum. 相似文献
17.
The rpiB gene, encoding ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum, was cloned and expressed in Escherichia coli. RpiB converted d-psicose into d-allose but it did not convert d-xylose, l-rhamnose, d-altrose or d-galactose. The production of d-allose by RpiB was maximal at pH 7.5 and 65°C for 30 min. The half-lives of the enzyme at 50°C and 65°C were 96 h and 4.7 h,
respectively. Under stable conditions of pH 7.5 and 50°C, 165 g d-allose l−1
was produced without by-products from 500 g d-psicose l−1 after 6 h. 相似文献
18.
The effect of pH, aeration and mixing on the growth and production of carbonyl reductase by Candida viswanathii was investigated in a 6.6-l fermentor. Controlling the pH at 8.0 had a very significant effect on the enzyme production.
Aeration and agitation influenced the dissolved oxygen concentration which in turn affected growth as well as enzyme production.
A maximum carbonyl reductase activity (53 Umg−1) was attained in 24 h under the optimal cultivation conditions of controlled pH at 8.0, aeration rate 1 vvm and an agitation
speed of 250 rpm at 25°C. The enzyme activity was twice as high (56 Umg−1) in the fermentor as compared to a shake flask. Further, the duration of growth and enzyme production in the fermentor was
shortened. Cells cultivated under the optimized conditions were used for the preparative scale reduction of N, N-dimethyl-(3-keto)-2-thienyl-propanamine
to (S)-N, N-dimethyl-(3-hydroxy)-2-thienyl-propanamine, a key intermediate in the production of the important antidepressant drug
(S)-duloxetine. 相似文献
19.
Jin Zhou Ju Chu Yong-Hong Wang Si-Liang Zhang Ying-Ping Zhuang Zhong-Yi Yuan 《World journal of microbiology & biotechnology》2008,24(6):789-796
An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH4)2SO4 fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed
the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular
weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric
point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5
and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature
stability was up to 45 °C. Metal ions, such as, Mn2+ and K+ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg2+ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu2+ and Ag2+) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K
m of 120 and 330 μM and V
max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively. 相似文献
20.
Corynebacterium acetoacidophilum RYU3161 was cultivated in al-histidine-limited fed-batch culture. To investigate the effect of cell growth on thel-proline production, 5l fed-batch culture was performed using an exponential feeding rate to obtain the specific growth rates (μ) of 0.04, 0.06,
0.08, and 0.1 h−1. The results show that the highest production ofl-proline was obtained at μ=0.04 h−1. The specificl-proline production rate (Qp) increased proportionally as a function of the specific growth rate, but decreased after it revealed the maximum value at
μ=0.08 h−1. Thus, the highest productivity ofl-proline was 1.66 g L−1 h−1 at μ=0.08 h−1. The results show that the production of L-proline inC. acetoacidophilum RYU3161 has mixed growth-associated characteristics. 相似文献