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1.
An actinomycete strain IBL-14 isolated from soil by utilizing diosgenin as the sole carbon and energy source was identified
as Streptomyces virginiae. S. virginiae IBL-14 can transform diosgenin to isonuatigenone. To our knowledge, this is the first reported case of producing rare nuatigenin-type
spirosteroid (isonuatigenone) from pyrano-spirosteroid (diosgenin) by microbial transformation. From diosgenin to isonuatigenone,
the pathway has been confirmed in this study that diosgenin was first converted to diosgenone, and then diosgenone was transformed
to isonuatigenone by the C25 tertiary hydroxylation reaction. It appeared to be favorable to accumulate isonuatigenone when
diosgenin was added to the onset of the stationary phase of cell growth, and the yield of isonuatigenone was about 28.4% during
48 h from 1.5 mM diosgenin. The C25 tertiary hydroxylation of diosgenone by S. virginiae IBL-14 is a novel and interesting reaction and will be a practical tool in producing natural nuatigenin-type steroids.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
2.
Production of diosgenin from Dioscorea zingiberensis tubers through enzymatic saccharification and microbial transformation 总被引:1,自引:0,他引:1
Yu-Ling Zhu Wen Huang Jin-Ren Ni Wei Liu Hui Li 《Applied microbiology and biotechnology》2010,85(5):1409-1416
In order to develop a clean and effective approach for producing the valuable drug diosgenin from Dioscorea zingiberensis tubers, two successive processes, enzymatic saccharification and microbial transformation, were used. With enzymatic saccharification,
98.0% of starch was excluded from the raw herb, releasing saponins from the network structure of starch. Subsequently, the
treated tubers were fermented with Trichoderma reesei under optimal conditions for 156 h. During microbial transformation, glycosidic bonds, which link β-d-glucose or α-l-rhamnose with aglycone at the C-3 position in saponins, were broken down effectively to give a diosgenin yield of 90.6 ± 2.45%,
42.4% higher than that obtained from bioconversion of raw tubers directly. Scaled up fermentation was conducted in a 5.0-l
bioreactor and gave a diosgenin yield of 91.2 ± 3.21%. This is the first report on the preparation of diosgenin from herbs
through microbial transformation as well as utilizing other available components in the raw material, providing an environmentally
friendly alternative to diosgenin production. 相似文献
3.
Hartinger D Schwartz H Hametner C Schatzmayr G Haltrich D Moll WD 《Applied microbiology and biotechnology》2011,91(3):757-768
Fumonisins are carcinogenic mycotoxins that are frequently found as natural contaminants in maize from warm climate regions
around the world. The aminotransferase FumI is encoded as part of a gene cluster of Sphingopyxis sp. MTA144, which enables this bacterial strain to degrade fumonisin B1 and related fumonisins. FumI catalyzes the deamination of the first intermediate of the catabolic pathway, hydrolyzed fumonisin
B1. We used a preparation of purified, His-tagged FumI, produced recombinantly in Escherichia coli in soluble form, for enzyme characterization. The structure of the reaction product was studied by NMR and identified as
2-keto hydrolyzed fumonisin B1. Pyruvate was found to be the preferred co-substrate and amino group receptor (K
M = 490 μM at 10 μM hydrolyzed fumonisin B1) of FumI, but other α-keto acids were also accepted as co-substrates. Addition of the co-enzyme pyridoxal phosphate to the
enzyme preparation enhanced activity, and saturation was already reached at the lowest tested concentration of 10 μM. The
enzyme showed activity in the range of pH 6 to 10 with an optimum at pH 8.5, and in the range of 6°C to 50°C with an optimum
at 35°C. The aminotransferase worked best at low salt concentration. FumI activity could be recovered after preincubation
at pH 4.0 or higher, but not lower. The aminotransferase was denatured after preincubation at 60°C for 1 h, and the residual
activity was also reduced after preincubation at lower temperatures. At optimum conditions, the kinetic parameters K
M = 1.1 μM and k
cat = 104/min were determined with 5 mM pyruvate as co-substrate. Based on the enzyme characteristics, a technological application
of FumI, in combination with the fumonisin carboxylesterase FumD for hydrolysis of fumonisins, for deamination and detoxification
of hydrolyzed fumonisins seems possible, if the enzyme properties are considered. 相似文献
4.
Dominic D. W. S. Wong Victor J. Chan Amanda A. McCormack Sarah B. Batt 《Applied microbiology and biotechnology》2010,86(5):1463-1471
A novel xyloglucan-specific endo-β-1,4-glucanase gene (xeg5A) was isolated, cloned, and expressed in Esherichia coli. The enzyme XEG5A consisted of a C-terminal catalytic domain and N-terminal sequence of ~90 amino acid residues with unknown
function. The catalytic domain assumed an (α/β)8-fold typical of glycoside hydrolase (GH) family 5, with the two catalytic residues Glu240 and Glu362 located on opposite
sides of the surface groove of the molecule. The recombinant enzyme showed high specificity towards tamarind xyloglucan and
decreasing activity towards xyloglucan oligosaccharide (HDP-XGO), carboxymethyl cellulose, and lichenan. Tamarind xyloglucan
was hydrolyzed to three major fragments, XXXG, XXLG/XLXG, and XLLG. The hydrolysis followed the Michaelis–Menten kinetics,
yielding K
m and V
max of 3.61 ± 0.23 mg/ml and 0.30 ± 0.01 mg/ml/min, respectively. However, the hydrolysis of HDP-XGO showed a decrease in the
rate at high concentrations suggesting appearance of excess substrate inhibition. The addition of XXXG resulted in linear
noncompetitive inhibition on the hydrolysis of tamarind xyloglucan giving a K
i of 1.46 ± 0.13 mM. The enzyme was devoid of transglycosylase activities. 相似文献
5.
H. Yamazaki Y. Ohnishi K. Takeuchi N. Mori N. Shiraishi Y. Sakata H. Suzuki S. Horinouchi 《Applied microbiology and biotechnology》1999,52(3):401-409
Rhizomucor pusillus 1116R3 has a defect in alg2 encoding a mannosyltransferase in the asparagine (N)-linked oligosaccharide biosynthetic pathway and produces proteins in less-glycosylated forms. For development of a genetic
transformation system for this zygomycete, an uracil auxotroph (mutant 1116U17) as the host strain was derived by ultraviolet
(UV) mutagenesis as 5-fluoroorotic acid-resistant colonies and the orotidine-5′-monophosphate (OMP) decarboxylase (pyr4) gene as a selection marker was cloned from the wild-type strain R. pusillus F27 by the polymerase chain reaction with primers designed on the basis of the pyr4 sequences from other fungi. The amino acid sequence of R. pusillus Pyr4 deduced from the nucleotide sequence showed high homology with the OMP decarboxylases from various fungi. The pyr4 gene on pUC19 (plasmid pRPPyr4) was introduced into protoplasts of R. pusillus 1116U17 by polyethylene glycol-assisted transformation. Transformation under optimized conditions yielded 5 Ura+ transformants with 1 μg pRPPyr4 DNA and 1 × 107 viable protoplasts. Southern blot analysis of the genomic DNA from the transformants showed that multiple copies of the pRPPyr4
sequence were integrated into the genome by homologous recombination at the pyr4 locus. For the purpose of production of a milk-clotting aspartic proteinase (MPP) in a less-glycosylated form, mpp from the wild-type strain was cloned in pRPPyr4 and introduced into protoplasts of R. pusillus 1116U17. Transformants obtained in this way contained multiple copies of mpp at the chromosomal mpp locus and produced MPP as a mixture of molecules having no sugar chains and Man0∼1GlcNAc2 at the two N-linked glycosylation sites in an amount about 12 times larger than the parent strain. The transformation system for R. pusillus 1116U17 would be useful for production of proteins with truncated N-linked oligosaccharide chains.
Received: 1 February 1999 / Received revision: 26 February 1999 / Accepted: 20 March 1999 相似文献
6.
We have cloned a glucansucrase from the type strain of Leuconostoc mesenteroides (NRRL B-1118; ATCC 8293) and successfully expressed the enzyme in Escherichia coli. The recombinant processed enzyme has a putative sequence identical to the predicted secreted native enzyme (1,473 amino
acids; 161,468 Da). This enzyme catalyzed the synthesis of a water-insoluble α-D-glucan from sucrose (K
M 12 mM) with a broad pH optimum between 5.0 and 5.7 in the presence of calcium. Removal of calcium with dialysis resulted
in lower activity in the acidic pH range, effectively shifting the pH optimum to 6.0–6.2. The enzyme was quickly inactivated
at temperatures above approximately 45°C. The presence of dextran offered some protection from thermal inactivation between
room temperature and 40°C but had little effect above 45°C. NMR and methylation analysis of the water-insoluble α-d-glucan revealed that it had approximately equal amounts of α(1 → 3)-linked and α(1 → 6)-linked d-glucopyranosyl units and a low degree of branching. 相似文献
7.
Borzyszkowska J Stanislawska-Sachadyn A Wirtwein M Sobiczewski W Ciecwierz D Targonski R Gruchala M Rynkiewicz A Limon J 《Journal of applied genetics》2012,53(2):175-182
This study examines whether renin-angiotensin-aldosterone system gene polymorphisms: ACE (encoding for angiotensin converting enzyme) c.2306-117_404 I/D, AGTR1 (encoding for angiotensin II type-1 receptor) c.1080*86A>C and CYP11B2 (encoding for aldosterone synthase) c.-344C>T are associated with the extension of coronary atherosclerosis in a group of
647 patients who underwent elective coronary angiography. The extension of CAD was evaluated using the Gensini score. The
polymorphisms were determined by PCR and RFLP assays. The associations between genotypes and the extent of coronary atherosclerosis
were tested by the Kruskal-Wallis test, followed by pairwise comparisons using Wilcoxon test. The population has been divided
into groups defined by: sex, smoking habit, past myocardial infarction, BMI (>, ≤ 25), age (>, ≤ 55), diabetes mellitus, level
of total cholesterol (>, ≤ 200 mg/dl), LDL cholesterol (>, ≤ 130 mg/dl), HDL cholesterol (>, ≤ 40 mg/dl), triglycerides (>,
≤ 150 mg/dl). Significant associations between the ACE c.2306-117_404 I/D polymorphism and the Gensini score in men with high total cholesterol levels (PKruskal-Wallis = 0.008; Padjusted = 0.009), high level of LDL cholesterol (PKruskal-Wallis = 0.016; Padjusted = 0.028) and low level of HDL cholesterol (PKruskal-Wallis = 0.04; Padjusted = 0.055) have been found. No association between the AGTR1 c.1080*86A>C and CYP11B2 c.-344C>T and the Gensini score has been found. These results suggest that men who carry ACE c.2306-117_404 DD genotype and have high total cholesterol, high LDL cholesterol and low HDL cholesterol levels may be predisposed
to the development of more severe CAD. 相似文献
8.
The enzyme oxaloacetate hydrolase (EC 3.7.1.1), which is involved in oxalate formation, was purified from Aspergillus niger. The native enzyme has a molecular mass of 360–440 kDa, and the denatured enzyme has a molecular mass of 39 kDa, as determined
by gel electrophoresis. Enzyme activity is maximal at pH 7.0 and 45 °C. The fraction containing the enzyme activity contained
at least five proteins. The N-terminal amino acid sequences of four of these proteins were determined. The amino acid sequences
were aligned with EST sequences from A. niger, and an EST sequence that showed 100% identity to all four sequences was identified. Using this EST sequence the gene encoding
oxaloacetate hydrolase (oah) was cloned by inverse PCR. It consists of an ORF of 1227 bp with two introns of 92 and 112 bp, respectively. The gene encodes
a protein of 341 amino acids with a molecular mass of 37 kDa. Under the growth conditions tested, the highest oah expression was found for growth on acetate as carbon source. The gene was expressed only at pH values higher than 4.0.
Received: 9 May 1999 / Accepted: 30 November 1999 相似文献
9.
N. Frankenberg T. Kittel C. Hungerer U. R?mling D. Jahn 《Molecular & general genetics : MGG》1998,257(4):485-489
During tetrapyrrole biosynthesis 5-aminolevulinic acid dehydratase (ALAD) catalyzes the condensation of two molecules of
5-aminolevulinic acid (ALA) to form one molecule of the pyrrole derivative porphobilinogen. In Escherichia coli, the enzyme is encoded by the gene hemB. The hemB gene was cloned from Pseudomonas aeruginosa by functional complementation of an E. coli hemB mutant. An open reading frame of 1011 bp encoding a protein of 336 amino acids (Mr = 37 008) was identified. The gene was mapped to SpeI fragment G and DpnI fragment G of the P. aeruginosa chromosome, corresponding to the 10 to 12 min region of the new map or 19 to 22 min interval of the old map. The 5′ end of
the hemB mRNA was determined and the −10 and −35 regions of a potential σ70-dependent promoter were localized. No obvious regulation of the hemB gene by oxygen, nitrate, heme or iron was detected. Alignment of the amino acid sequences deduced from hemB revealed a potential metal-binding site and indicated that the enzyme is Mg2+-dependent. P. aeruginosa hemB was overexpressed in an E. coli hemB mutant using the phage T7 RNA polymerase system and its Mg2+-dependent activity was directly demonstrated.
Received: 11 July 1997 / Accepted: 9 October 1997 相似文献
10.
11.
Hoyt MA Williams-Abbott LJ Pitkin JW Davis RH 《Molecular & general genetics : MGG》2000,263(4):664-673
S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the formation of decarboxylated AdoMetDC, a precursor of the polyamines
spermidine and spermine. The enzyme is derived from a proenzyme by autocatalytic cleavage. We report the cloning and regulation
of the gene for AdoMetDC in Neurospora crassa, spe-2, and the effect of putrescine on enzyme maturation and activity. The gene was cloned from a genomic library by complementation
of a spe-2 mutant. Like other AdoMetDCs, that of Neurospora is derived by cleavage of a proenzyme. The deduced sequence of the Neurospora proenzyme (503 codons) is over 100 codons longer than any other AdoMetDC sequence available in genomic databases. The additional
amino acids are found only in the AdoMetDC of another fungus, Aspergillus nidulans, a cDNA for which we also sequenced. Despite the conserved processing site and four acidic residues required for putrescine
stimulation of human proenzyme processing, putrescine has no effect on the rate (t
0.5∼10 min) of processing of the Neurospora gene product. However, putrescine is absolutely required for activity of the Neurospora enzyme (K
0.5∼100 μM). The abundance of spe-2 mRNA and enzyme activity is regulated 2- to 4-fold by spermidine.
Received: 4 August 1999 / Accepted: 14 February 2000 相似文献
12.
Manish Kumar Tiwari Hee-Jung Moon Marimuthu Jeya Jung-Kul Lee 《Applied microbiology and biotechnology》2010,87(2):571-581
An NAD+-dependent xylitol dehydrogenase from Rhizobium etli CFN42 (ReXDH) was cloned and overexpressed in Escherichia coli. The DNA sequence analysis revealed an open reading frame of 1,044 bp, capable of encoding a polypeptide of 347 amino acid
residues with a calculated molecular mass of 35,858 Da. The ReXDH protein was purified as an active soluble form using GST
affinity chromatography. The molecular mass of the purified enzyme was estimated to be ∼34 kDa by sodium dodecyl sulfate–polyacrylamide
gel and ∼135 kDa with gel filtration chromatography, suggesting that the enzyme is a homotetramer. Among various polyols,
xylitol was the preferred substrate of ReXDH with a K
m = 17.9 mM and kcat
/K
m = 0.5 mM−1 s−1 for xylitol. The enzyme had an optimal pH and temperature of 9.5 and 70 °C, respectively. Heat inactivation studies revealed
a half life of the ReXDH at 40 °C of 120 min and a half denaturation temperature (T
1/2) of 53.1 °C. ReXDH showed the highest optimum temperature and thermal stability among the known XDHs. Homology modeling and
sequence analysis of ReXDH shed light on the factors contributing to the high thermostability of ReXDH. Although XDHs have
been characterized from several other sources, ReXDH is distinguished from other XDHs by its high thermostability. 相似文献
13.
A. Arai S. Masuda A. Matsuyama S. Murakami M. Nakajima 《Applied microbiology and biotechnology》1998,49(3):272-276
The gene for the thermostable pyruvate kinase of Microbispora thermodiastatica IFO 14046, a moderate thermophilic actinomycete, was cloned in Escherichia coli. This gene consists of an open reading frame of 1422 nucleotides and encodes a protein of 474 amino acids with molecular
mass of 50 805 Da. The open reading frame was confirmed as the pyruvate kinase gene by comparison with the N-terminal amino
acid sequence of the purified pyruvate kinase from M. thermodiastatica.
Received: 19 May 1997 / Received last revision: 22 September 1997 / Accepted: 14 October 1997 相似文献
14.
Hee-Jung Moon Manish Tiwari Marimuthu Jeya Jung-Kul Lee 《Applied microbiology and biotechnology》2010,87(1):205-214
Ribitol dehydrogenase (RDH) catalyzes the conversion of ribitol to d-ribulose. A novel RDH gene was cloned from Zymomonas mobilis subsp. mobilis ZM4 and overexpressed in Escherichia coli BL21(DE3). DNA sequence analysis revealed an open reading frame of 795 bp, capable of encoding a polypeptide of 266 amino
acid residues with a calculated molecular mass of 28,426 Da. The gene was overexpressed in E. coli BL21(DE3) and the protein was purified as an active soluble form using glutathione S-transferase affinity chromatography. The molecular mass of the purified enzyme was estimated to be ∼28 kDa by sodium dodecyl
sulfate-polyacrylamide gel and ∼58 KDa with gel filtration chromatography, suggesting that the enzyme is a homodimer. The
enzyme had an optimal pH and temperature of 9.5 and 65°C, respectively. Unlike previously characterized RDHs, Z. mobilis RDH (ZmRDH) showed an unusual dual coenzyme specificity, with a k
cat of 4.83 s−1 for NADH (k
cat/K
m = 27.3 s−1 mM−1) and k
cat of 2.79 s−1 for NADPH (k
cat/K
m = 10.8 s−1 mM−1). Homology modeling and docking studies of NAD+ and NADP+ into the active site of ZmRDH shed light on the dual coenzyme specificity of ZmRDH. 相似文献
15.
The gene coding for cyanidase, which catalyzes the hydrolysis of cyanide to formate and ammonia, was cloned from chromosomal
DNA of Pseudomonas stutzeri AK61 into Escherichia coli. The cyanidase gene consisted of an open reading frame of 1004 bp, and it was predicted that cyanidase was composed of 334
amino acids with a calculated molecular mass of 37 518 Da. The amino acid sequence of cyanidase showed a 35.1% and 26.4% homology
to aliphatic nitrilase from Rhodococcus rhodochrous K22 and cyanide hydratase from Fusarium lateritium, respectively. A unique cysteine residue of aliphatic nitrilase, which was suggested to play an essential role in the catalytic
activity, was conserved in cyanidase. The active form of cyanidase was successfully expressed by a DNA clone containing the
cyanidase gene in E.coli. Its productivity was approximately 230 times larger than that of P. stutzeri AK61. The characteristics of the expressed cyanidase, including optimum pH, optimum temperature, Michaelis constant (K
m) for cyanide and specific activity, were similar to those of the native enzyme from P. stutzeri AK61.
Received: 24 October 1997 / Received last revision: 17 March 1998 / Accepted: 20 March 1998 相似文献
16.
A. Phongdara A. Merckelbach P. Keup G. Gellissen C. P. Hollenberg 《Applied microbiology and biotechnology》1998,50(1):77-84
A cloned cDNA, generated from mRNA isolates of phosphate-derepressed H. polymorpha cells, was identified to harbour an incomplete sequence of the coding region for a repressible acid phosphatase. The cDNA
fragment served as a probe to screen a plasmid library of H. polymorpha genomic DNA. A particular clone, p606, of a 1.9-kb insert contained a complete copy of the PHO1 gene. Sequencing revealed the presence of a 1329-nucleotide open reading frame encoding a protein of 442 amino acids with
a calculated M
r of 49400. The␣encoded protein has an N-terminal 17-amino-acid secretory leader sequence and seven potential N-glycosylation sites. The leader cleavage site was confirmed by N-terminal sequencing of the purified enzyme. The nucleotide
sequence is 48.9% homologous, the derived amino acid sequence 36% homologous to its Saccharomyces cerevisiae counterpart. The derived amino acid sequence harbours a consensus sequence RHGXRXP, previously identified as a sequence involved
in active-site formation of acid phosphatases. The PHO1 promoter and the secretion leader sequence present promising new tools for heterologous gene expression.
Received: 15 January 1998 / Received revision: 2 March 1998 / Accepted: 4 March 1998 相似文献
17.
Cloning,sequence analysis,and expression of a gene encoding <Emphasis Type="Italic">Chromobacterium</Emphasis> sp. DS-1 cholesterol oxidase 总被引:1,自引:1,他引:0
Noriyuki Doukyu Kanpei Shibata Hiroyasu Ogino Martin Sagermann 《Applied microbiology and biotechnology》2009,82(3):479-490
Chromobacterium sp. strain DS-1 produces an extracellular cholesterol oxidase that is very stable at high temperatures and in the presence
of organic solvents and detergents. In this study, we cloned and sequenced the structural gene encoding the cholesterol oxidase.
The primary translation product was predicted to be 584 amino acid residues. The mature product is composed of 540 amino acid
residues. The amino acid sequence of the product showed significant similarity (53–62%) to the cholesterol oxidases from Burkholderia spp. and Pseudomonas aeruginosa. The DNA fragment corresponding to the mature enzyme was subcloned in the pET-21d(+) expression vector and expressed as an
active product in Escherichia coli. The cholesterol oxidase produced from the recombinant E. coli was purified to homogeneity. The physicochemical properties were similar to those of native enzyme purified from strain DS-1.
K
m and V
max values of the cholesterol oxidase were estimated from Lineweaver–Burk plots. The V
max/K
m ratio of the enzyme was higher than those of commercially available cholesterol oxidases. The circular dichroism spectral
analysis of the recombinant DS-1 enzyme and Burkholderia cepacia ST-200 cholesterol oxidase showed that the conformational stability of the DS-1 enzyme was higher than that of B. cepacia ST-200 enzyme at higher temperatures. 相似文献
18.
Liu JQ Odani M Yasuoka T Dairi T Itoh N Kataoka M Shimizu S Yamada H 《Applied microbiology and biotechnology》2000,54(1):44-51
The dtaAX gene encoding a pyridoxal 5′-phosphate (pyridoxal-P)-dependent low-specificity d-threonine aldolase was cloned from the chromosomal DNA of Alcaligenes xylosoxidans IFO 12669. It contains an open reading frame consisting of 1,134 nucleotides corresponding to 377 amino acid residues. The
predicted amino acid sequence displayed 54% identity with that of d-threonine aldolase from gram-positive bacteria Arthrobacter sp. DK-38, but showed no significant similarity with those of other known pyridoxal-P enzymes. This gram-negative bacterial enzyme was highly overproduced in recombinant Escherichia coli cells, and the specific activity of the enzyme in the cell extract was as high as 18 U/mg (purified enzyme 38.6 U/mg), which
was 6,000 times higher than that from the wild-type Alcaligenes cell extract. The recombinant enzyme was thus feasibly purified to homogeneity by ammonium sulfate fractionation and DEAE-Toyopearl chromatography
steps. The recombinant low-specificity d-threonine aldolase was shown to be an efficient biocatalyst for resolution of l-β-3,4-methylenedioxyphenylserine, an intermediate for production of a therapeutic drug for Parkinson's disease.
Received: 9 September 1999 / Received revision: 1 November 1999 / Accepted: 12 November 1999 相似文献
19.
Glucose oxidase from Penicillium amagasakiense was purified to homogeneity by ion-exchange chromatography and deglycosylated with endoglycosidase H. On the basis of gas
chromatography and sodium dodecyl sulphate/polyacrylamide gel electrophoretic (SDS-PAGE) analyses, the protein-bound high-mannose-type
carbohydrate moiety corresponded to 13% of the molecular mass of glycosylated glucose oxidase. A total of six N-glycosylation sites per dimer were determined from the N-acetylglucosamine content. The enzymatically deglycosylated enzyme contained less than 5% of the original carbohydrate moiety.
A molecular mass of 130 kDa (gel filtration) and 133 kDa (native PAGE) was determined for the dimer and 67 kDa (SDS-PAGE)
for the monomer of the deglycosylated enzyme. The N-terminal sequence, which has not been published for glucose oxidase from
P. amagasakiense to date and which showed less than 50% homology to the N terminus of glucose oxidase from Aspergillus niger, and the amino acid composition were not altered by the deglycosylation. Deglycosylation also did not affect the kinetics
of glucose oxidation or the pH and temperature optima. It also did not increase the susceptibility of the enzyme to proteolytic
degradation. However, deglycosylated glucose oxidase exhibited decreased pH and thermal stability. The thermal stability of
both enzymes was shown to be dependent on the buffer concentration and was enhanced by certain additives, particularly 1 M
(NH4)2SO4, which stabilised glucose oxidase 100- to 300-fold at 50 °C and pH 7–8, and 2 M KF, which stabilised the enzyme up to 36-fold
at 60 °C and pH 6. In sodium acetate buffer, changes in pH (4–6) affected the affinity for glucose but had no effect on the
V
max of the reaction. In contrast, in TRIS buffer, pH 8, a 10-fold decrease in V
max and a 2-fold decrease in K
m were observed.
Received: 8 October 1996 / Received revision: 14 January 1997 / Accepted: 17 January 1997 相似文献
20.
A new cytochrome P450 monooxygenase, FcpC, from Streptomyces virginiae IBL-14 has been identified. This enzyme is found to be responsible for the bioconversion of a pyrano-spiro steroid (diosgenone) to a rare nuatigenin-type spiro steroid (isonuatigenone), which is a novel C-25-hydroxylated diosgenone derivative. A whole-cell P450 system was developed for the production of isonuatigenone via the expression of the complete three-component electron transfer chain in an Escherichia coli strain.Nuatigenin-type steroids, such as nuatigenin and isonuatigenin (9, 13, 22), are rare natural steroidal sapogenins that are important pharmacological compounds. They are found in several healthy foods and traditional medicinal herbs. These compounds have been shown to have potential anticancer effects, antagonistic effects on rheumatoid arthritis, beneficial cardiovascular activities, and antimalarial activities. Examples include ophiofurospiside in Ophiopogon japonicus (28), nuatigenosido in Solanum sisymbriifolium (13), avenacoside in oat (20), and glycosides in Paris polyphylla SM (7). Since the majority of these nuatigenin-type steroids are very rare, strategies for their isolation can lead to very high production costs. As a result, with a more economical production process in mind, it would be worthwhile to search for a suitable reagent capable of converting the abundant amounts of pyrano-spirostanol sapogenins found in nature, such as diosgenone, to rare nuatigenin-type steroids. At this time, we plan to focus on microbial transformation systems.A previous article (25) described an actinomycete strain named Streptomyces virginiae IBL-14, isolated from soil, that can transform diosgenone to isonuatigenone by introducing a hydroxyl group to the tertiary C-25 atom of the F-ring (Fig. (Fig.1).1). To our knowledge, this was the first report of producing a rare nuatigenin-type spiro steroid from diosgenone by microbial biotransformation. The present study was conducted in order to identify the determinant enzyme from S. virginiae IBL-14 that catalyzes the biotransformation and to design a whole-cell cytochrome P450 system to produce isonuatigenone by using Escherichia coli.Open in a separate windowFIG. 1.(Bio)synthetic conversion of diosgenone (1) to isonuatigenone (2) and nuatigenone (3). Diosgenone can be transformed into isonuatigenone by cytochrome P450 FcpC from S. virginiae IBL-14. Nuatigenone is the rearrangement product of isonuatigenone during acidic work-up (8). 相似文献