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1.
The extracellular metalloprotease (SMP 6.1) produced by a soil isolate of Serratia marcescens NRRL B-23112 was purified and characterized. SMP 6.1 was purified from the culture supernatant by ammonium sulfate precipitation,
acetone fractional precipitation, and preparative isoelectric focusing. SMP 6.1 has a molecular mass of approximately 50 900 Da
by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The following substrates were hydrolyzed: casein,
bovine serum albumin, and hide powder. SMP 6.1 has the characteristics of a metalloprotease, a pH optimum of 10.0, and a temperature
optimum of 42° C. The isoelectric point of the protease is 6.1. Restoration of proteolytic activity by in-gel renaturation
after SDS-PAGE indicates a single polypeptide chain. SMP 6.1 is inhibited by EDTA (9 μg/ml) and not inhibited by antipain
dihydrochloride (120 μg/ml), aprotinin (4 μg/ml), bestatin (80 μg/ml), chymostatin (50 μg/ml), E-64 (20 μg/ml), leupeptin
(4 μg/ml), Pefabloc SC (2000 μg/ml), pepstatin (4 μg/ml), phosphoramidon (660 μg/ml), or phenylmethylsulfonyl fluoride (400
μg/ml). SMP 6.1 retains full activity in the presence of SDS (1% w/v), Tween-20 (1% w/v), Triton X-100 (1% w/v), ethanol (5%
v/v), and 2-mercaptoethanol (0.5% v/v). The extracellular metalloprotease SMP 6.1 differs from the serratiopeptidase (Sigma)
produced by S. marcescens ATCC 27117 in the following characteristics: isoelectric point, peptide mapping and nematolytic properties.
Received: 22 November 1996 / Received revision: 27 February 1997 / Accepted: 7 March 1997 相似文献
2.
M. B. Cassidy K. W. Shaw H. Lee J. T. Trevors 《Applied microbiology and biotechnology》1997,47(2):108-113
A pentachlorophenol(PCP)-degrading Pseudomonas sp. strain UG30 was encapsulated in κ-carrageenan for use in PCP degradation. Free and encapsulated cells were compared for
their ability to dechlorinate and mineralize 100–800 μg/ml sodium pentachlorophenate in broth. Dechlorination was measured
with a chloride ion electrode, and mineralization was measured by 14CO2 evolution from radiolabelled [U-14C]PCP. Free and encapsulated Pseudomonas sp. UG30 cells mineralized up to 200 μg/ml and 600 μg/ml PCP, respectively, after 21 days. Encapsulation of UG30 cells provided
a protective effect, allowing dechlorination and mineralization of high levels of PCP to occur.
Received: 3 May 1996 / Received revision: 4 September 1996 / Accepted: 13 September 1996 相似文献
3.
Isochorismate hydroxymutase (i.e. isochorismate synthase, EC 5.4.99.6) was purified from an anthraquinone-producing cell-suspension
culture of Galium mollugo L. Although attempts to stabilize the labile enzyme met with little success, a substantial increase in enzyme activity was
observed in the presence of glycine betaine (500 mM). Column chromatography on solid supports other than diethylaminoethyl
(DEAE)-Sephacel, Phenylsepharose Cl-4B or Cibacron Blue 3G-A did not give active enzyme preparations. In spite of these drawbacks
the enzyme was purified 573-fold. Enzyme activity depended strictly on the presence of Mg2+. Kinetic data for chorismate in the forward reaction (K
m = 807 μM, V
max = 6.2 pkat · mg−1) and for isochorismate in the reverse reaction (K
m = 675 μM, V
max = 5.9 pkat · mg−1) were determined.
Received: 18 November 1996 / Accepted: 28 December 1996 相似文献
4.
The effect of extracellular pH and dissolved oxygen on regulation of the pcbAB gene in P.␣chrysogenum was examined, using Northern analysis and a reporter gene fusion. It was found that ambient pH markedly affected levels of
pcbAB mRNA whereas maintenance of dissolved oxygen concentration above 10 % had no detectable effect. The presence of a DNA-binding
protein, which binds upstream of the pcbAB translational start codon, was also related to ambient pH. In all fermentations, pcbAB mRNA was most abundant at around the late exponential/early stationary phase of a culture.
Received: 10 May 1996 / Received revision: 14 October 1996 / Accepted: 25 October 1996 相似文献
5.
Glycine-rich peptide toxin of cyanobacterium Scytonema MKU 106 was purified. UV spectral analysis showed an absorption maximum at 228 nm and the molecular mass was less than 12
kDa. The mortality rate of American boll worms (Helicoverpa armigera) was about 80% and 40% 84 h after treatment with 0.001% crude and purified peptide toxins respectively; 100% mortality was
observed after 108 h treatment with both purified and crude peptide toxins. The LC50 (lethal concentration to 50% of the population) for Heliothis larvae after 96 h was 8.3 μg/ml purified peptide toxin and 6.2 μg/ml crude peptide toxin. Observations also show that the
peptide toxin at 0.01% concentration acts as a biopesticide and at high (0.1%) concentrations it will act as an anti-feeding
compound for Stylepta derogata (leaf-roller) larvae of the cotton crop.
Received: 22 May 1996 / Accepted: 8 July 1996 相似文献
6.
K. Kirimura T. Sato N. Nakanishi M. Terada S. Usami 《Applied microbiology and biotechnology》1997,47(2):127-131
Interspecific protoplast fusion between␣Aspergillus terreus, an itaconic acid producer, and A.␣usamii, a glucoamylase producer, was done to breed new koji molds producing itaconic acid from starch. Protoplast fusion between auxotrophic mutant strains by poly(ethylene glycol) treatment
produced prototrophic fusants with a fusion frequency of 10−5−10−4. The stabilities of some fusants obtained were confirmed by successive subcultures. Conidial analyses of DNA contents and
the number of nuclei indicated that the fusants obtained were haploids like the parental strains. One of the stable fusants,
F-112, morphologically resembled A. terreus, and produced maximally 35.9 mg/ml itaconic acid from soluble starch (120 mg/ml) at day 6 of cultivation. This productivity
from soluble starch was five times as high as that of A. terreus and 70 % of that of A. terreus from glucose (120 mg/ml).
Received: 28 June 1996 / Received revision: 3 September 1996 / Accepted: 29 September 1996 相似文献
7.
R. W. Herzog N. K. Singh D. W. Urry H. Daniell 《Applied microbiology and biotechnology》1997,47(4):368-372
A gene for a synthetic protein-based polymer, G-(VPGVG)119-VPGV, coding for the EG-120mer (elastomer), was cloned into a fungal expression vector to allow constitutive expression of
the polymer controlled by the gpdA (glyceraldehyde-3-phosphate dehydrogenase) promoter sequence of Aspergillus nidulans. Stable transformants of A. nidulans showed plasmid integration with varying copy number when analyzed by Southern-blot hybridization. Expression of the synthetic
gene was demonstrated by Northern-blot hybridization. However, the translational efficiency for production of the polymer
polypeptide was low, presumably because of certain codons in the polymer gene (CCG and GUA) that are rarely used by A. nidulans. Partial purification by reversible phase transition followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis
revealed the presence of polymer protein in a transformant that contained multiple copies of the polymer gene. This study
represents the first attempt to express a synthetic gene (with no natural analog) in a fungus.
Received: 23 July 1996 / Received revision: 19 November 1996 / Accepted: 23 November 1996 相似文献
8.
Xanthomonas campestris pv. campestris, the causal agent of black-rot disease of cruciferous plants, and an important industrial microbe, was able to express the
Escherichia coliβ-glucuronidase reporter gene (uidA) when fused to the E. coli lactose operon promoter on a wide-host-range plasmid vector. The gene fusion is expressed constitutively at high levels in
both complex and defined media using a wide range of carbon sources, and is not repressible by glucose or inducible by the
gratuitous lac inducer isopropyl β-d-thiogalactoside. An X. campestris campestris strain with a lesion in the clp (catabolite-repressor-like protein) locus, and containing the plac/uidA fusion, was tested for β-glucuronidase activity. We found that the expression of the plac/uidA fusion gene is dependent on the presence of catabolite-repressor-like protein, with an approximately 75% reduction of expression
in the clp -deficient mutant.
Received: 1 April 1996 / Received revision: 21 June 1996 / Accepted: 15 July 1996 相似文献
9.
M. S. Umikalsom A. B. Ariff Z. H. Shamsuddin C. C. Tong M. A. Hassan M. I. A. Karim 《Applied microbiology and biotechnology》1997,47(5):590-595
Studies on the feasibility of using delignified oil palm empty-fruit-bunch (OPEFB) fibres as a substrate for cellulase production
by Chaetomium globosum strain 414 were carried out in shake-flask cultures containing different types and concentrations of nitrogen source. Peptone,
as nitrogen source, gave maximum production of all the three main components of the cellulase complex (endoglucanase or carboxymethylcellulase,
cellobiohydrolase or filter-paper-hydrolysing enzyme and β-glucosidase), followed by yeast extract, urea, KNO3 and (NH4)2SO4. The maximum specific growth rate (μmax) of C. globosum strain 414 grown in medium containing OPEFB and peptone was 0.038 h−1. In all the fermentations, the fungus was able to produce all the three cellulases with significant amounts of β-glucosidase,
except when using (NH4)2SO4 as nitrogen source, where β-glucosidase was not produced. With 6 g/l peptone and 10 g/l delignified OPEFB fibres, the fungus
produced maximum concentrations of FPase, carboxymethylcellulase and β-glucosidase: 1.4, 30.8 and 9.8 U/ml, giving productivities
of 10, 214 and 24 U l−1h−1, respectively. The cellulase mixture, partially purified by ammonium sulphate precipitation, was able to hydrolyse delignified
OPEFB fibres, converting about 68 % of the cellulosics to reducing sugars after 5 days.
Received: 17 June 1996 / Received revision: 18 November 1996 / Accepted: 23 November 1996 相似文献
10.
N. Christiansen S. R. Christensen E. Arvin B. K. Ahring 《Applied microbiology and biotechnology》1997,47(1):91-94
Reductive dechlorination of tetrachloroethene was studied in a mesophilic upflow anaerobic sludge blanket reactor. Operating
the reactor in batch mode the dynamic transformation of tetrachloroethene, trichloroethene and dichloroethene (DCE) was monitored.
Tetrachloroethene was reductively dechlorinated to trichloroethene, which again was dechlorinated at the same rate as DCE
was produced. DCE showed a lag period of 40 h before transformation was observed. During normal reactor operation trans-1,2-DCE was the major DCE isomer, followed by cis-1,2-DCE. Small amounts of 1,1-DCE but no vinyl chloride were detected. When the influent tetrachloroethene concentration
was increased from 4.6 μM to 27 μM, the transformation rate increased, indicating that the system was not saturated with tetrachloroethene.
The main organic component in the effluent was acetate, indicating that the aceticlastic methane-producing bacteria were inhibited
by the chlorinated ethenes.
Received: 29 July 1996 / Received revision: 13 September 1996 / Accepted: 13 September 1996 相似文献
11.
Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. We have developed a versatile
monitoring technique for detecting the amount of OPH during the expression and purification steps. This involves fusion of
the gene for green fluorescent protein (GFP) to the 5′ end of the OPH gene and subsequent expression in Escherichia coli. The synthesized fusion protein was directly visualized due to the optical properties of GFP. Western blot analyses showed
that the correct fusion protein was expressed after IPTG-induction. Also, the in vivo GFP fluorescence intensity was proportional
to the OPH enzyme activity. Moreover, the OPH, which forms a dimer in its active state, retained activity while fused to GFP.
Enterokinase digestion experiments showed that OPH was separated from the GFP reporter after purification via immobilized
metal affinity chromatography, which in turn was monitored by fluorescence. The strategy of linking GFP to OPH has enormous
potential for improving enzyme production efficiency, as well as enhancing field use, as it can be monitored at low concentrations
with inexpensive instrumentation based on detecting green fluorescence.
Received: 27 April 1999 / Received last revision: 18 October 1999 / Accepted: 1 November 1999 相似文献
12.
Sari Tähtiharju Veena Sangwan Antonio F. Monroy Rajinder S. Dhindsa Marianne Borg 《Planta》1997,203(4):442-447
The involvement of calcium signaling during cold-induction of the kin genes of Arabidopsis thaliana (L.) Heynh. was examined. Treatments with chemicals which either chelate extracellular calcium (EGTA) or block the plasma-membrane
calcium channels (La3+, Gd3+) inhibited cold acclimation as well as kin gene expression. Ruthenium red, an inhibitor of calcium release from intracellular stores partially inhibited kin gene expression and development of freezing tolerance. An inhibitor of calcium-dependent protein kinases (CDPKs) and calmodulin
prevented cold acclimation as well as the cold induction of kin genes. Using restriction fragment length polymorphism-coupled domain-directed differential display, five CDPK clones were
identified which showed differential regulation by cold. The amplified fragments showed homology to known plant CDPKs. The
involvement of calcium and calcium-binding proteins in cold acclimation of A. thaliana is discussed.
Received: 28 November 1996 / Accepted: 5 May 1997 相似文献
13.
Degradation of styrene by white-rot fungi 总被引:2,自引:0,他引:2
A. Braun-Lüllemann A. Majcherczyk A. Hüttermann 《Applied microbiology and biotechnology》1997,47(2):150-155
Degradation of styrene in the gaseous phase was investigated for white-rot fungi Pleurotus ostreatus (two strains), Trametes versicolor, Bjerkandera adusta and Phanerochaete chrysosporium. Fungi were grown in liquid culture and the gas/mycelium contact surface was enhanced with the help of perlite. The influence
of various inducers on styrene degradation was studied. The best inducers for styrene degradation were lignosulphonate for
P. ostreatus and T. versicolor and wood meal for B. adusta and P. chrysosoporium. Under these conditions all fungi were able to degrade styrene almost completely in 48 h at a concentration of 44 μmol/250 ml
total culture volume; one strain of P. ostreatus was able to remove 88 μmol styrene under these conditions. Three transformation products of [14C]styrene in cultures of P. ostreatus were identified: phenyl-1,2-ethanediol, 2-phenylethanol and benzoic acid; 4% of the styrene was metabolised to CO2 in 24 h and no other volatile products were found.
Received: 16 July 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996 相似文献
14.
Poly[(R)-3-hydroxybutyric acid] (PHB) was produced at 37 °C by a recombinant Escherichia coli harboring the Alcaligenes eutrophus biosynthesis phbCAB genes in Luria-Bertani media containing glucose at 10–30 g/l at different pH values and the time-dependent changes in the
molecular mass of PHB were studied. PHB polymers accumulated within cells while glucose was present in the medium. The number-average
molecular mass of PHB decreased with time during the course of PHB accumulation, and the values for PHB were markedly dependent
on the cultivation conditions of the E. coli, ranging from 0.5 MDa to 20 MDa. Under specific conditions (pH 6.0), E. coli produced PHB with an extremely high molecular mass (20 MDa). It has been suggested that a chain-transfer agent is generated
in E. coli cells during the accumulation of PHB.
Received: 18 July 1996 / Received revision: 4 November 1996 / Accepted: 4 November 1996 相似文献
15.
M. Kataoka K. Yamamoto H. Kawabata M. Wada K. Kita H. Yanase S. Shimizu 《Applied microbiology and biotechnology》1999,51(4):486-490
The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate [(R)-CHBE] using Escherichia coli cells, which coexpress both the aldehyde reductase gene from Sporobolomyces salmonicolor and the glucose dehydrogenase (GDH) gene from Bacillus megaterium as a catalyst was investigated. In an organic solvent-water two-phase system, (R)-CHBE formed in the organic phase amounted to 1610 mM (268 mg/ml), with a molar yield of 94.1% and an optical purity of 91.7%
enantiomeric excess. The calculated turnover number of NADP+ to CHBE formed was 13 500 mol/mol. Since the use of E. coli JM109 cells harboring pKAR and pACGD as a catalyst is simple, and does not require the addition of GDH or the isolation of
the enzymes, it is highly advantageous for the practical synthesis of (R)-CHBE.
Received: 5 October 1998 / Received revision: 16 November 1998 / Accepted: 5 December 1998 相似文献
16.
N. Sriubolmas W. Panbangred S. Sriurairatana V. Meevootisom 《Applied microbiology and biotechnology》1997,47(4):373-378
Various concentrations of isopropyl β-d-thiogalactopyranoside (IPTG) were used to induce production of the enzyme penicillin G acylase by recom binant Escherichia coli harboring plasmid pQEA11. The plasmid pQEA11 carries a wild-type pga gene, which is under the control of the tac promoter and lacI q. At low IPTG concentrations (0.025 – 0.1 mM), enzyme activity increased with increasing IPTG concentrations. At higher IPTG
concentrations (0.2 and 0.5 mM), enzyme activity declined progressively. Examination of induced recombinant E. coli cells by transmission electron microscopy showed the presence of only periplasmic inclusion bodies at low IPTG concentrations
(up to 0.1 mM) and both periplasmic and cytoplasmic inclusion bodies at high IPTG concentrations (0.2 mM and 0.5 mM). Results
from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblots of whole-cell proteins, membrane proteins and
inclusion body proteins in these cells indicated that cytoplasmic inclusion bodies constituted an accumulation of preproenzyme
(i.e., precursor polypeptide containing a signal peptide) and that periplasmic inclusion bodies constituted an accumulation
of proenzyme (i.e., precursor polypeptide lacking a signal peptide).
Received: 27 March 1996 / Received revision: 2 July 1996 / Accepted: 10 November 1996 相似文献
17.
K. Sangthongpitag R. J. Penfold S. F. Delaney P. L. Rogers 《Applied microbiology and biotechnology》1997,47(4):379-384
Genes encoding the mosquitocidal binary toxin of Bacillus sphaericus 2362 were introduced into Synechococcus PCC6301, a cyanobacterium that can tolerate a number of potential variations in the mosquito breeding environment, and can
serve as a food source for mosquito larvae. The toxin genes, preceded by a Synechococcus rbcL promoter, were located on a mobilizable Escherichia coli Synechococcus shuttle vector, which was introduced into Synechococcus PCC6301 at frequencies of 10−5–10−7 exconjugants/recipient, depending on the selective conditions used. Recombinant Synechococcus exhibited significant toxicity against 2-day-old and 6-day-old Culex quinquefasciatus larvae, the concentration required to kill 50 % of larvae (LC50) being 2.1 × 105 and 1.3 × 105 cells/ml respectively. Mosquitocidal activity decreased tenfold after 20 generations of non-selective growth.
Received: 23 July 1996 / Received revision: 11 November 1996 / Accepted: 15 November 1996 相似文献
18.
19.
A xylose reductase gene (xyl1) of Candida guilliermondii ATCC 20118 was cloned and characterized. The open reading frame of xyl1 contained 954 nucleotides encoding a protein of 317 amino acids with a predicted molecular mass of 36 kDa. The derived amino
acid sequence of C. guilliermondii xylose reductase was 70.4% homologous to that of Pichia stipitis. The gene was placed under the control of an alcohol oxidase promoter (AOX1) and integrated into the genome of a methylotrophic yeast, Pichia pastoris. Methanol induced the expression of the 36-kDa xylose reductase in both intracellular and secreted expression systems. The
expressed enzyme preferentially utilized NADPH as a cofactor and was functional both in vitro and in vivo. The different cofactor
specificity between P. pastoris and C. guilliermondii xylose reductases might be due to the difference in the numbers of histidine residues and their locations between the two
proteins. The recombinant was able to ferment xylose, and the maximum xylitol accumulation (7.8 g/l) was observed when the
organism was grown under aerobic conditions.
Received: 26 August 1997 / Received revision: 6 November 1997 / Accepted: 21 November 1997 相似文献
20.
J. M. Crous I. S. Pretorius W. H. van Zyl 《Applied microbiology and biotechnology》1996,46(3):256-260
First-strand cDNA was prepared from mRNA of Aspergillus niger MRC11624 induced on oat spelts xylan. Using the cDNA as a template, the α-L-arabinofuranosidase gene (abf B) was amplified with the polymerase chain reaction technique. The abf B DNA fragment was inserted between the yeast phosphoglycerate kinase I gene promoter (PGK1
P
) and terminator (PGK1
T
) sequences on a multicopy episomal plasmid. The resulting construct PGK1
P
-abf B-PGK1
T
was designated ABF2. The ABF2 gene was expressed successfully in Saccharomyces cerevisiae and functional α-L-arabinofuranosidase was secreted from the yeast cells. The ABF2 nucleotide sequence was determined and verified to encode a 449-amino-acid protein (Abf 2) that is 94% identical to the α-L-arabinofuranosidase B of A. niger N400. Maximum α-L-arabinofuranosidase activities of 0.020 U/ml and 1.40 U/ml were obtained with autoselective recombinant S. cerevisiae strains when grown for 48 h in synthetic and complex medium respectively.
Received: 29 January 1996/Received revision: 3 May 1996/Accepted: 9 May 1996 相似文献