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1.
δ1-Pyrroline-5-carboxylate (P5C) dehydrogenase (EC 1.5.1.12) activity was measured in extracts from cultured tobacco (Nicotiana plumbaginifolia Viviani) cells. Two putative isozymes were resolved by anion-exchange fast protein liquid chromatography. These enzyme forms
showed different patterns of expression during the culture growth cycle: activity-I increased in exponentially growing cells
and declined rapidly in late logarithmic phase, while activity-II was found at substantial level only in cells which were
entering the stationary phase. Both P5C dehydrogenases were partially purified and characterized with respect to kinetic and
biochemical properties. They showed similar molecular masses as judged from retention patterns upon gel-filtration chromatography.
The in vitro activity of both enzymes had a broad maximum around pH 7.4, and was progressively inhibited by Cl− at concentrations ranging from 0.1 to 1 M. A pronounced difference was found between their apparent K
m values for the two substrates, P5C and NAD+, the higher affinities being shown by activity-I. Regulation of P5C dehydrogenase during salt-stress-induced proline accumulation
was investigated. Following the addition of 175 mM NaCl to the culture medium the level of activity-I was substantially unaffected,
while the specific activity of the other isozyme failed to increase even after the onset of the stationary phase of growth.
Possible roles for P5C dehydrogenase isozymes in proline and arginine metabolism are discussed.
Received: 23 May 1996 / Accepted: 18 December 1996 相似文献
2.
M. A. Ferrero G. R. Castro C. M. Abate M. D. Baigorí F. Siñeriz 《Applied microbiology and biotechnology》1996,45(3):327-332
Bacillus licheniformis MIR 29 has been isolated and produces extracellular proteases. It is able to grow at temperatures up to 60 °C and at pH values
up to 9.0. Casein was the best carbon source for production of a thermostable protease activity which, in some conditions,
is 90% extracellular. The synthesis of alkaline protease is not constitutive; different levels of production were found with
different carbon and nitrogen sources. Casein was thought to be an inducer of enzyme synthesis. The optimal pH and temperature
of the enzyme activity were 12 °C and 60 °C, respectively. The enzyme was stable up to 60 °C in the absence of stabilizers.
The protease activity was inhibited with phenylmethylsulphonyl fluoride, indicating a serine-protease activity. The proteolytic
activity was lowered by molecules present in the culture supernatant, which include amino acids and peptides, indicating end-product
inhibition. Electrophoresis assay on denaturating gels showed two bands with alkaline protease activity, in the 25 to 40-kDa
molecular mass range.
Received: 7 June 1995/Received revision: 14 September 1995/Accepted: 20 September 1995 相似文献
3.
The influence of low temperature (5–29 °C) on the methanogenic activity of non-adapted digested sewage sludge and on temperature/leachate-adapted
biomass was assayed by using municipal landfill leachate, intermediates of anaerobic degradation (propionate) and methane
precursors (acetate, H2/CO2) as substrates. The temperature dependence of methanogenic activity could be described by Arrhenius-derived models. However,
both substrate and adaptation affected the temperature dependence. The adaptation of biomass in a leachate-fed upflow anaerobic
sludge-blanket reactor at approximately 20 °C for 4 months resulted in a sevenfold and fivefold increase of methanogenic activity
at 11 °C and 22 °C respectively. Both acetate and H2/CO2 were methanized even at 5 °C. At 22 °C, methanogenic activities (acetate 4.8–84 mM) were 1.6–5.2 times higher than those
at 11 °C. The half-velocity constant (K
s) of acetate utilization at 11 °C was one-third of that at 22 °C while a similar K
i was obtained at both temperatures. With propionate (1.1–5.5 mM) as substrate, meth‐anogenic activities at 11 °C were half
those at 22 °C. Furthermore, the residual concentration of the substrates was not dependent on temperature. The results suggest
that the adaptation of biomass enables the achievement of a high treatment capacity in the anaerobic process even under psychrophilic
conditions.
Received: 23 December 1996 / Received last revision: 18 June 1997 / Accepted: 23 June 1997 相似文献
4.
Y. Morita Q. Hasan T. Sakaguchi Y. Murakami K. Yokoyama E. Tamiya 《Applied microbiology and biotechnology》1998,50(6):669-675
Protease activity was detected in the culture medium of Flavobacterium balustinum P104 grown at 10 °C, which was isolated from salmon (Oncorhynchus keta) intestine. The enzyme, designated as CP-70 protease, was purified to homogeneity from the culture broth by ion exchange
and gel filtration chromatographyies. The molecular mass of the protease was 70 kDa, and its isoelectric point was close to
3.5. Maximal activity toward azocasein was observed at 40 °C and from pH 7.0 to 9.0. The activity was strongly inhibited by
phenylmethylsulfonyl fluoride, suggesting that the enzyme is a serine protease. The n-terminal amino acid sequence was Asp-Thr-Arg-Gln-Leu-Leu-Asn-Ala-Asn-Ser-Asp-Leu-Leu-Asn-Thr-Thr-Gly-Asn-Val-Thr-Gly-Leu-Thr-Gly-Ala-Phe-Asn-Gly-Glu-Asn.
A search through the database for sequence homology yielded no significant match. The initial cleavage sites for oxidized
insulin B-chain were found to be the Glu13-Ala14 and Phe24-Phe25 bonds. The result of the cleavage pattern of oxidized insulin
B-chain suggests that CP-70 protease has a broader specificity than the other cold-active proteases against the peptide substrate.
Received: 17 April 1998 / Received last revision: 17 June 1998 / Accepted: 10 July 1998 相似文献
5.
E. V. Eneyskaya A. A. Kulminskaya A. N. Savel'ev N. V. Savel'eva K. A. Shabalin K. N. Neustroev 《Applied microbiology and biotechnology》1999,52(2):226-231
Mechanisms regulating post-secretory limited proteolysis, carried out by the acid protease from Trichoderma reesei, were studied by following the release of α-galactosidase and multiple forms of cellobiohydrolase from this species. Both
the rate of the proteolysis and the mode of action of the protease were affected by the pH of the culture medium, and only
weakly depended on the amount of the enzyme. At pH between 2.7 and 3.5 the proteolytic reaction was limited, while at lower
pH proteins were completely digested. Proteolysis depended on the degree of glycosylation of secreted enzymes. Inhibition
of post-secretory deglycosylation decreased the rate of limited proteolysis in the culture medium in the course of fungal
growth. Glucose and cellobiose, the main products of cellulose degradation carried out by the fungal cellulolytic complex,
inhibited the proteolysis of the cellobiohydrolase in a concentration-dependent manner. A 32-kDa aspartic protease (EC 3.4.23.18)
secreted by T. reesei was purified to homogeneity. The acid protease cleaved α-galactosidase and cellobiohydrolase into the same proteolytic fragments
that had been isolated from the culture medium.
Received: 4 December 1998 / Received revision: 22 February 1999 / Accepted: 5 March 1999 相似文献
6.
B. L. García A. S. Ball J. Rodríguez M. I. Pérez-Leblic M. E. Arias J. L. Copa-Patiño 《Applied microbiology and biotechnology》1998,50(2):213-218
Streptomyces avermitilis CECT 3339 produces extracellular ferulic acid esterase (FAE) activity during growth on a range of lignocellulose substrates.
Maximal levels of FAE activity were detected in culture filtrates from S. avermitilis CECT 3339 grown in media containing wheat bran and yeast extract as carbon and nitrogen sources respectively. Biochemical
characterization of this enzyme activity revealed that it was 100-fold higher when wheat bran was pretreated with Celluclast
(a mix of hydrolytic enzymes). FAE was found to be end-product-inhibited. Characterization of the properties of the enzyme
showed that FAE exhibited an activity optimum pH at 6 with pH stability between pH 6 and 8. The optimum temperature was 50 °C
while the temperature stability was between 30 °C and 40 °C, with rapid inactivation at 60 °C and above. The characteristics
and stability of FAE from S. avermitilis CECT 3339 suggest a potential role for this enzyme in combination with endoxylanases for the upgrading of plant-residue silage
and for biopulping.
Received: 17 November 1997 / Received revision: 13 March 1998 / Accepted: 13 April 1998 相似文献
7.
A number of nutritional factors influencing growth and glucose oxidase (EC 1.1.3.4) production by a newly isolated strain
of Penicillium pinophilum were investigated. The most important factors for glucose oxidase production were the use of sucrose as the carbon source,
and growth of the fungus at non-optimal pH 6.5. The enzyme was purified to apparent homogeneity with a yield of 74%, including
an efficient extraction step of the mycelium mass at pH 3.0, cation-exchange chromatography and gel filtration. The relative
molecular mass (M
r) of native glucose oxidase was determined to be 154 700 ± 4970, and 77 700 for the denatured subunit. Electron-microscopic
examinations revealed a sandwich-shaped dimeric molecule with subunit dimensions of 5.0 × 8.0 nm. Glucose oxidase is a glycoprotein
that contains tightly bound FAD with an estimated stoichiometry of 1.76 mol/mol enzyme. The enzyme is specific for d-glucose, for which a K
m value of 6.2 mM was determined. The pH optimum was determined in the range pH 4.0–6.0. Glucose oxidase showed high stability
on storage in sodium citrate (pH 5.0) and in potassium phosphate (pH 6.0), each 100 mM. The half-life of the activity was
considerably more than 305 days at 4 °C and 30 °C, and 213 days at 40 °C. The enzyme was unstable at temperatures above 40 °C
in the range pH 2.0–4.0 and at a pH above 7.0.
Received: 18 November 1996 / Received revision: 3 March 1997 / Accepted: 7 March 1997 相似文献
8.
A. Blanco P. Díaz J. Martínez T. Vidal A. L. Torres F. I. J. Pastor 《Applied microbiology and biotechnology》1998,50(1):48-54
The gene celA, encoding an endoglucanase from the strain Bacillus sp. BP-23, was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1867-bp DNA fragment containing the celA gene was determined, revealing an open reading frame of 1200 nucleotides that encodes a protein of 44 803 Da. The deduced
amino acid sequence of the encoded enzyme shows high homology to those of enzymes belonging to subtype 4 of the family-A cellulases.
The celA gene product synthesized in E. coli showed activity on carboxymethylcellulose and lichenan but no activity was found on Avicel. Activity was enhanced in the
presence of 10 mM Mg2+ and Ca2+ and showed its maximum at 40 °C and pH 4.0. Study of the performance of CelA on paper manufacture from agricultural fibres
showed that treatment with the enzyme improved the properties of the pulp and the quality of paper. CelA treatment enhanced
the physical properties (stretch and tensile index) of paper from wheat straw, while dewatering properties were slightly diminished.
Electron-microscope analysis showed that the surface of straw fibres was modified by CelA.
Received: 11 February 1998 / Received revision: 20 March 1998 / Accepted: 20 March 1998 相似文献
9.
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
10.
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
11.
Production of high yields of arachidonic acid in a fed-batch system by Mortierella alpina ATCC 32222
Of six strains of Mortierella tested, Mortierella alpina ATCC 32222 produced the highest yields of arachidonic acid. Supplementation of soy flour (1% w/v) and vegetable oils (1%
v/v) significantly increased the biomass, lipid content and arachidonic acid level. Replacement of NaNO3 with corn steep liquor (1% w/v) also improved arachidonic acid production. A fed-batch culture system at 25 °C, producing
a high biomass (52.4 g/l) and arachidonic acid content (9.1 g/l) in 8␣days, was developed. A fed-batch system at low temperature
(15 °C) gave even higher arachidonic acid levels (11.1 g/l) in 11 days.
Received: 28 October 1996 / Received revision: 3 March 1997 / Accepted: 7 March 1997 相似文献
12.
A. De Smul J. Dries L. Goethals H. Grootaerd W. Verstraete 《Applied microbiology and biotechnology》1997,48(3):297-303
In a mesophilic (30–35 °C), sulphidogenic, ethanol-fed expanded-granular-sludge-blanket reactor, sulphate, at loading rates
of up to 10.0–12.0 g Sl−1␣day−1, was removed with an average efficiency of more than 80%. The pH was between 7.7 and 8.3 and the maximal total dissolved
sulphide concentration was up to 20 mM S (650 mg S/l). The alkaline pH was maintained by either a pH-control unit with sodium
hydroxide or by stripping part of the sulphide and CO2 from the recycle with nitrogen gas. The superficial upstream liquid velocity (v
up) was 3.0–4.5 m/h. The ratio of ethanol to sulphur was near stoichiometry. At alkaline pH, the activity of the acetotrophic
sulphate-reducing bacteria, growing on acetate, was strongly enhanced, whereas at pH below 7.7 the acetotrophic sulphate-reducing
bacteria were inhibited by aqueous H2S. With regard to the removal efficiency and operational stability, external stripping with N2 and pH control were equally successful.
Received: 2 December 1996 / Received revision: 13 March 1997 / Accepted: 15 March 1997 相似文献
13.
An extremely halotolerant mannan-degrading bacterium (strain NN) was isolated from the Great Salt Lake, Utah, USA. Strain
NN grew at salinities from 0 to 20% NaCl with optimal growth at 0% NaCl. When grown on 0.2% (w/v) locust bean gum as the carbon
source at 10% NaCl, both β-mannanase and β-mannosidase activities were produced. β-Mannosidase activity was shown to be cell-associated,
while at least 23% of the total β-mannanase activity was extracellular. The optimum temperature and pH for β-mannanase activity
were 70 °C and 7.6, and for β-mannosidase 25 °C and 7.0. The β-mannanase system retained full activity after 24 h of incubation
at 60 °C and 10% NaCl. β-Mannanase activity was maximal at 1% NaCl and β-mannosidase activity at 0.5% NaCl. Despite these
low salinity optima, 50% and 100% respectively of the initial β-mannanase and β-mannosidase activities remained after 48 h
of incubation at 20% NaCl, indicating a high degree of halostability. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis
revealed the presence of at least eight different mannan-degrading proteins in the cell-free culture supernatant of cultures
grown on locust bean gum.
Received: 19 March 1998 / Received revision: 8 June 1998 / Accepted: 14 June 1998 相似文献
14.
M. Graf A. Brunella M. Kittelmann K. Laumen O. Ghisalba 《Applied microbiology and biotechnology》1997,47(6):650-657
A new amidohydrolase deacetylating several N-acetyl-1-phenylethylamine derivatives (R)-specifically was found in Arthrobacter aurescens AcR5b. The strain was isolated from a wet haystack by enrichment culture with (R)-N-acetyl-1-phenylethylamine as the sole carbon source. (R) and (S )-N-acetyl-1-phenylethylamine do not serve as inducers for acylase formation. By improving the growth conditions the enzyme production
was increased 47-fold. The amidohydrolase was purified to homogeneity leading to a 5.2-fold increase of the specific activity
with a recovery of 67%. A molecular mass of 220 kDa was estimated by gel filtration. Sodium dodecyl sulfate/polyacrylamide
gel electrophorosis shows two subunits with molecular masses of 16 kDa and 89 kDa. The optimum pH and temperature were pH 8
and 50 °C, respectively. The enzyme was stable in the range of pH 7–9 and at temperatures up to 30 °C. The enzyme activity
was inhibited by Cu2+, Co2+, Ni2+, and Zn2+, and this inhibition was reversed by EDTA.M
Received: 20 September 1996 / Received version: 23 December 1996 / Accepted: 30 December 1996 相似文献
15.
J. Blanco J. J. R. Coque J. Velasco J. F. Martín 《Applied microbiology and biotechnology》1997,48(2):208-217
Several thermophilic actinomycetes were isolated from urban solid waste. One of them, Thermomonospora alba ULJB1, showed a broad degradative activity on xylan, cellulose, starch and other polymers. Xylanase and cellulase activities
were quantified and compared with those of Thermomonospora fusca. Genes encoding two different endo-β-1,4-xylanases were cloned from T.␣alba ULJB1. One of them, xylA, was sequenced, subcloned and overexpressed in Streptomyces lividans. It encodes a protein of 482 amino acids with a deduced molecular mass of 48 456 Da. The protein contains a 38-amino-acid
leader peptide with six Arg+ residues in its amino-terminal end, a catalytic domain and a cellulose-binding domain connected by a linker region rich in
proline and glycine. The XylA protein was purified to near homogeneity from S. lividans/xylA cultures. Two forms of the extracellular xylanase, of 48 kDa and 38 kDa, were produced that differed in their cellulose-binding
ability. The 48-kDa protein showed a strong binding to cellulose whereas the 38-kDa form did not bind to this polymer, apparently
because of the removal during processing of the cellulose-binding domain. Both forms were able to degrade xylans form different
origins but not lichenam or carboxymethylcellulose. The major degradation product was xylobiose with traces of xylose. The
xylanase activity was thermostable, showing a good activity up to 95 °C, and had broad pH stability in the range from pH 4.0
to pH 10.0.
Received: 9 January 1997 / Received revision: 27 March 1997 / Accepted: 13 April 1997 相似文献
16.
A highly thermostable endo-(1,4)-β-mannanase from the marine bacterium Rhodothermus marinus 总被引:2,自引:0,他引:2
Rhodothermus marinus ATCC 43812, a thermophilic bacterium isolated from marine hot springs, possesses hydrolytic activities for depolymerising
substrates such as carob-galactomannan. Screening of expression libraries identified mannanase-positive clones. Subsequently,
the corresponding DNA sequences were determined, eventually identifying a coding sequence specifying a 997 amino acid residue
protein of 113 kDa. Analyses revealed an N-terminal domain of unknown function and a C-terminal mannanase domain of 550 amino
acid residues with homology to known mannanases of glycosidase family 26. Action pattern analysis categorised the R. marinus mannanase as an endo-acting enzyme with a requirement for at least five sugar moieties for effective catalytic activity.
When expressed in Escherichia coli, purified gene product with catalytic activity was mainly found as two protein fragments of 45 kDa and 50 kDa. The full-length
protein of 113 kDa was only detected in crude extracts of R. marinus, while truncated protein-containing fractions of the original source resulted in a major active protein of 60 kDa. Biochemical
analysis of the mannanase revealed a temperature and pH optimum of 85 °C and pH 5.4, respectively. Purified, E. coli-produced protein fragments showed high heat stability, retaining more than 70% and 25% of the initial activity after 1 h
incubation at 70 °C and 90 °C, respectively. In contrast, R. marinus-derived protein retained 87% activity after 1 h at 90 °C. The enzyme hydrolysed carob-galactomannan (locust bean gum) effectively
and to a smaller extent guar gum, but not yeast mannan.
Received: 5 November 1999 / Received revision: 19 January 2000 / Accepted: 23 January 2000 相似文献
17.
A. Brunella M. Graf M. Kittelmann K. Laumen O. Ghisalba 《Applied microbiology and biotechnology》1997,47(5):515-520
Rhodococcus equi Ac6 was found to express an inducible (S )-specific N-acetyl-1-phenylethylamine amidohydrolase. Optimal bacterial growth and amidohydrolase expression were both observed around
pH 6.5. Purification of the enzyme to a single band in a Coomassie-blue-stained sodium dodecyl sulfate/polyacrylamide gel
(SDS-PAGE) was achieved by ammonium sulfate precipitation of R. equi Ac6 crude extract and column chromatographies on Fractogel TSK Butyl-650(S) and Superose 12HR. At pH 7.0 and 30 °C the amidohydrolase
had a half-life of around 350 days; at 44 °C it was only 10 min. Except for Ni2+ and, to some extent, Zn2+ and Co2+, the enzyme was neither strongly influenced by metal cations nor by chelating agents, but was inhibited by 95% at 0.1 mM
phenylmethylsulfonyl fluoride. The molecular mass of the native enzyme was estimated to be 94 kDa by gel filtration and 50 kDa
by SDS-PAGE, suggesting a dimeric structure. Specificity experiments revealed a spectrum of related N-acetylated compounds being hydrolyzed with variable enantiomeric selectivities.
Received: 20 September 1996 / Received revision: 23 December 1996 / Accepted: 30 December 1996 相似文献
18.
Growth hormone (GH) enhances the growth rate of aquacultured fish and shellfish, but it is difficult to extract native GH
from fish pituitary glands. However, fish recombinant GH (rGH) can be efficiently synthesized by Escherichia coli cells, although it exists in denatured form in inclusion bodies (IB). We studied the solubilization of IB and the renaturation
of rGH to help facilitate the production of a large amount of biologically active rGH. A 100-ml sample of rGH-producing E. coli produced 73.43 ± 5.47 mg IB (dry weight, n = 3) after 20 h induction by 1 mM isopropyl β-o-thiogalactopyranoside. Interestingly, if the bacteria were induced by 0.1 mM β-lactose, 95.3 ± 3.43 mg of IB was obtained.
The optimal conditions for denaturation and renaturation of rGH were when IB were solubilized in 6 M guanidine hydrochloride
and then dialysed against pH 10 dialysis buffer (50 mM ammonium bicarbonate and 2 mM EDTA) containing 100 mM l-arginine, 2 mM oxidized glutathione and 2 mM reduced glutathione for 24 h at 4 °C in a volume ratio of 3 to 500. At least
20% of the denaturated rGH in IB was renatured. Juvenile black sea bream injected with 0.05 μg/g resultant rGH once every
2 weeks exhibited significant increases (P < 0.05) in weight gain (84%) relative to fish in the control group over a 16-week period. This process is an economical and
effective way to obtain an active form of rGH biosynthesized by a prokaryotic system.
Received: 18 November 1996 / Received revision: 5 March 1997 / Accepted: 7 March 1997 相似文献
19.
The fermentability of commercial xylans and municipal waste hemicelluloses in the presence of Clostridium sp. (C.SAIV; ATCC 700188) has been evaluated. Teak, deal wood, banana stalk and bagasse of the municipal waste contained
significant amounts (approx. 12 %–23 %) of hemicellulose. Under optimized growth conditions, the growth rate of C.SAIV was
improved as indicated by an increase in the concentration of ethanol in the culture broth. Commercial xylans were utilized
fairly efficiently and ethanol formed from larch wood xylan and bagasse hemicellulose was at least 64 mM. The amount of ethanol
formed from the bagasse hemicellulose was at least three times higher than any other reported value. The current study also
indicated that the source and composition of hemicellulose played an important role in determining the fermentability of the
substrate for some microorganisms.
Received: 19 June 1996 / Received revision: 22 October 1996 / Accepted: 25 October 1996 相似文献
20.
Characteristics of glycosylated streptokinase secreted from Pichia pastoris: enhanced resistance of SK to proteolysis by glycosylation 总被引:1,自引:0,他引:1
Degradation of streptokinase (SK) has been frequently observed during large-scale protein production. An enhanced susceptibility
of SK to degradation has been correlated with its existence in a partially unfolded state. The influence of the carbohydrate
moiety on the stability and functional characteristics of SK has been examined by obtaining the glycoform of SK following
its secretion through the methylotrophic yeast Pichia pastoris. Secretion of the protein product was achieved by replacing the native secretion signal codons of SK with those from α-factor
leader peptide and expressing the fusion construct under the control of the methanol-inducible alcohol oxidase (ox) promoter of P. pastoris after its integration into the host chromosome. Western blot and zymographic analysis of proteins secreted from the recombinant
P. pastoris indicated that SK was glycosylated by the host cells, which resulted in the appearance of a SK species migrating slowly,
corresponding to a 55-kDa protein product as compared to the 47-kDa native SK. The glycosylated SK retained a plasminogen
activation capability identical to that of its unglycosylated counterpart. Glycoform SK exhibited an enhanced stability profile
at 25 °C and 37 °C and improved resistance towards protease treatment compared to unglycosylated SK secreted through P. pastoris after tunicamycin treatment or that secreted from the recombinant Escherichia coli. The results presented thus illustrate that N-linked glycosylation of SK results in 30–40% enhancement of the protein stability
and resistance towards degradation but does not interfere with its fibrinolytic function.
Received: 1 March 1999 / Received last revision: 5 October 1999 / Accepted: 10 October 1999 相似文献