Enhancement of the productivity of xylanase and beta-xy-losidase of Aspergillus ochraceus was investigated by multistep mutagenesis. The spores of the wild strain were subjected to UV and N-methyl-N-nitro-N-nitro-soguanidine (NTG). The hyperxylanolytic mutant (NG-13), which showed good clearing on the surface of the xylan-agar plate, secretes xylanase and beta-xylosidase at high levels during growth on commercial xylan and on agricultural wastes. Both liquid and solid state cultures were employed in the study for enzyme production. The xylanase from NG-13 was purified to homogeneity by ammonium sulfate precipitation and gel filtration. This purified enzyme showed a pH optimum of 6.0 and was stable in the range of pH 5 to 10. Prolonged stability of the enzyme was observed at 45 degrees C though its activity was maximal at 50 degrees C. The molecular weight of the enzyme was estimated to be 4.3 x 10(4) by SDS-polyacrylamide gel electrophoresis and 5 x 10(4) by gel filtration on Sephadex G-75. The kinetic data showed that the K(m) and V(max) values for xylan were 1 x 10(-3)M and 19.6 mumol/ min/mg protein, respectively. The enzyme was both more active and thermostable in the presence of K(+)and was inactivated by thiol reagents such as Hg(2+), p-hydroxymercuribenzoate (PHMB), 3', 5'-dithiobis (2'-nitrobenzoic acid) (DTNB), and N-ethylmaleimide (NEM).
Transposon insertion mutagenesis was used to isolate mutants of Bosea thiooxidans which are impaired in thiosulfate oxidation. Suicide plasmid pSUP5011 was used to introduce the transposon Tn5 into B. thiooxidans via Escherichia coli S17.1-mediated conjugation. Neomycin-resistant transconjugants occurred at a frequency of 2.2 ؋ 10 ؊4 per donor. Transconjugants defective in thiosulfate oxidation were categorized into three classes on the basis of growth response, enzyme activities, and cytochrome patterns. Class I mutants were deficient in cytochrome c, and no thiosulfate oxidase activity was detected. Class II mutants retained the activities of key enzymes of thiosulfate metabolism, although at reduced levels. Mutants of this class grown on mixed-substrate agar plates deposited elemental sulfur on the colony surfaces. Class III mutants were unable to utilize thiosulfate, though they had normal levels of cytochrome c. The transposon insertions occurred at different chromosomal positions, as confirmed by Southern blotting of chromosomal DNA of mutants deficient in thiosulfate oxidation, a deficiency which resulted from single insertions of Tn5.Little is known about the structure, expression, and regulation of the genes associated with the oxidation of inorganic sulfur compounds of the obligately (17, 44) or facultatively (13,31,43) . These heterotrophs may benefit from sulfur oxidation in the detoxification of hydrogen peroxide (18,29). To date, preliminary work has been done to understand the genetical basis of sulfur metabolism by obligate chemolithoautotrophic (Thiobacillus ferrooxidans) (30) and facultatively chemolithoautotrophic (Thiobacillus versutus and Thiobacillus novellus) bacteria (9). However, little is known at the genetic level, and no genes involved in oxidation of sulfur have been identified or cloned (47-49). Until recently, no DNA probes for reduced inorganic sulfur oxidation genes existed (30). In addition, there is no defined mutant deficient in sulfur oxidation and an only rudimentary genetic system has been discussed for Thiobacillus novellus, Thiobacillus versutus (9), Paracoccus denitrificans GB17 (51), Thiosphaera pantotropha (5, 28), and a bacterial endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila (19). Genetic systems to study bacterial species capable of oxidizing inorganic sulfur compounds are only now being developed. Transposon mutagenesis of Bosea thiooxidans (8) was used to generate mutants impaired in thiosulfate oxidation.Transposon insertion mutagenesis has been applied to a wide range of gram-negative bacteria (5,16,34,35,45) for both strain construction and the analysis of mutant phenotypes, but this approach has not been exploited to study the mechanism of inorganic sulfur oxidation for chemolithoheterotrophs.B. thiooxidans BI-50 was selected for genetic studies of thiosulfate-oxidizing activity by transposon Tn5 insertion mutagenesis because (i) its level of spontaneous neomycin resistance above its base sensitivity level is very low; (ii) its generation ti...
Attempts to find streptomycin-dependent (str-d) mutations in the sensitive SIII-1 strain of Diplococcus pneumoniae have been unsuccessful, for, if they occur at all, such mutations must arise with a frequency of <10-"1 (A. W. Ravin and
Alteration of carbon sources significantly altered the analogue sensitivity of Bacillus megaterium B71. DL‐Ethionine (ETN) was highly inhibitory with glucose, mannitol, sucrose, citrate, glycerol and arabinose. DL‐Norleucine, L‐homoserine and S(2′‐aminoethyl)‐L‐cysteine were either highly inhibitory, slightly inhibitory or non‐inhibitory depending on the carbon sources used. Maltose markedly overcame the inhibitory effect of ETN in liquid culture. Uninhibited growth was poor on citrate and arabinose when compared with other carbon sources. Six carbon sources showing comparable growth were used to determine minimum inhibitory concentrations (MIC) of the analogues. The MIC of ETN was highest (450 μg/ml) with maltose and lowest (4 μg/ml) with mannitol. ETN sensitivity was inversely related to the endogenous L‐methionine pool size, and was relatively low with mannitol which was used to isolate ETN resistant mutants of B. megaterium B71. The best mutant BUE‐118 produced 435 μg/ml of L‐methionine.
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