Activity Of Secondary Alcohol Dehydrogenase Research Articles

Knockout of secondary alcohol dehydrogenase in Nocardia cholesterolicumNRRL 5767 by CRISPR/Cas9 genome editing technology.

Nocardia cholesterolicum NRRL 5767 is well-known for its ability to convert oleic acid to 10-hydroxystearic acid (~88%, w/w) and 10-ketostearic acid (~11%, w/w). Conversion of oleic acid to 10-hydroxystearic acid and then to 10-ketostearic acid has been proposed to be catalyzed by oleate hydratase and secondary alcohol dehydrogenase, respectively. Hydroxy fatty acids are value-added with many industrial applications. The objective of this study was to improve the Nocardia cholesterolicum NRRL5767 strain by CRISPR/Cas9 genome editing technology to knockout the secondary alcohol dehydrogenase gene, thus blocking the conversion of 10-hydroxystearic acid to 10-ketostearic acid. The improved strain would produce 10-hydroxystearic acid solely from oleic acid. Such improvement would enhance the production of 10-hydroxystearic acid by eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. Here, we report: (1) Molecular cloning and characterization of two functional recombinant oleate hydratase isozymes and a functional recombinant secondary alcohol dehydrogenase from Nocardia cholesterolicum NRRL5767. Existence of two oleate hydratase isozymes may explain the high conversion yield of 10-hydroxystearic acid from oleic acid. (2) Construction of a CRISPR/Cas9/sgRNA chimeric plasmid that specifically targeted the secondary alcohol dehydrogenase gene by Golden Gate Assembly. (3) Transformation of the chimeric plasmid into Nocardia cholesterolicum NRRL 5767 by electroporation and screening of secondary alcohol dehydrogenase knockout mutants. Two mutants were validated by their lack of secondary alcohol dehydrogenase activity at the protein level and mutation at the targeted 5’ coding region and the 5’ upstream at the DNA level. The knockout mutants offer improvements by converting added oleic acid to solely 10-hydroxystearic acid, thus eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. To the best of our knowledge, we report the first successful knockout of a target gene in the Nocardia species using CRISPR/Cas9/sgRNA-mediated genome editing technology.

Purification and characterization of a NAD+-dependent secondary alcohol dehydrogenase from propane-grown Rhodococcus rhodochrous PNKb1

NAD+-linked primary and secondary alcohol dehydrogenase activity was detected in cell-free extracts of propane-grown Rhodococcus rhodochrous PNKb1. One enzyme was purified to homogeneity using a two-step procedure involving DEAE-cellulose and NAD-agarose chromatography and this exhibited both primary and secondary NAD+-linked alcohol dehydrogenase activity. The Mr of the enzyme was approximately 86,000 with subunits of Mr 42,000. The enzyme exhibited broad substrate specificity, oxidizing a range of short-chain primary and secondary alcohols (C2−C8) and representative cyclic and aromatic alcohols. The pH optimum was 10. At pH 6.5, in the presence of NADH, the enzyme catalysed the reduction of ketones to alcohols. The K m values for propan-1-ol, propan-2-ol and NAD were 12 mM, 18 mM and 0.057 mM respectively. The enzyme was inhibited by metal-complexing agents and iodoacetate. The properties of this enzyme were compared with similar enzymes in the current literature, and were found to be significantly different from those thus far described. It is likely that this enzyme plays a major role in the assimilation of propane by R. rhodochrous PNKb1.

Oxidation of secondary alcohols to methyl ketones by immobilized yeast cells.

As HOU et al. (1, 2) mentioned, cell-free extract from methanol-grown yeasts catalyzes the oxidation reaction of secondary alcohols to methyl ketones. Recently, we found that glucose-grown yeasts also produce secondary alcohol dehydrogenase that is constitutively formed. Among several methanol-grown yeasts, Hansenula polymorpha P-5 was the most suitable enzyme source because of its thermal stability and broad substrate specificity. In addition to the secondary alcohols, primary alcohols and 1, 2-propanediol are also oxidized by the cell-free extract of H. polymorpha P-5. The oxidation of secondary alcohols to methyl ketones by immobilized yeast cells was examined and compared with the oxidation by the cell suspensions. Oxygen was essential for the conversion of 2-propanol to acetone. The secondary alcohol dehydrogenase activity of the immobilized cells was not affected by pH shock from 7.0 to 3.5, but with the cell suspension it was affected. The optimun pH for the cell suspension was about 8.5, but the immobilized yeast cells had a broad optimum pH range from 5.0 to 8.5. Only an 8% decrease in the enzyme activity of the immobilized yeast cells occurred when the pH was below 4.0. For the immobilized yeast cells, the tolerance concentration of the secondary alcohol dehydrogenase to acetone was up to 5% (v/v). However, with cell suspensions, the enzyme was not tolerant to acetone. The optimal enzyme activity of cell suspensions lasted 2 days; that of immobilized yeast cells lasted 3 days. The enzyme activity of immobilized cells could be restored with glucose medium.

Thermostable NAD-linked secondary alcohol dehydrogenase from propane-grown Pseudomonas fluorescens NRRL B-1244.

NAD-linked alcohol dehydrogenase activity was detected in cell-free crude extracts from various propane-grown bacteria. Two NAD-linked alcohol dehydrogenases, one which preferred primary alcohols (alcohol dehydrogenase I) and another which preferred secondary alcohols (alcohol dehydrogenase II), were found in propane-grown Pseudomonas fluorescens NRRL B-1244 and were separated from each other by DEAE-cellulose column chromatography. The properties of alcohol dehydrogenase I resembled those of well-known primary alcohol dehydrogenases. Alcohol dehydrogenase II was purified 46-fold; it was homogeneous as judged by acrylamide gel electrophoresis. The molecular weight of this secondary alcohol dehydrogenase is 144,500; it consisted of four subunits per molecule of enzyme protein. It oxidized secondary alcohols, notably, 2-propanol, 2-butanol, and 2-pentanol. Primary alcohols and diols were also oxidized, but at a lower rate. Alcohols with more than six carbon atoms were not oxidized. The pH and temperature optima for secondary alcohol dehydrogenase activity were 8 to 9 and 60 to 70 degrees C, respectively. The activation energy calculated from an Arrhenius plot was 8.2 kcal (ca. 34 kJ). The Km values at 25 degrees C, pH 7.0, were 8.2 X 10(-6) M for NAD and 8.5 X 10(-5) M for 2-propanol. The secondary alcohol dehydrogenase activity was inhibited by strong thiol reagents and strong metal-chelating agents such as 4-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), 5-nitro-8-hydroxyquinoline, and 1,10-phenanthroline. The enzyme oxidized the stereoisomers of 2-butanol at an equal rate. Alcohol dehydrogenase II had good thermal stability and the ability to catalyze reactions at high temperature (85 degrees C). It appears to have properties distinct from those of previously described primary and secondary alcohol dehydrogenases.

Production of Methyl Ketones from Secondary Alcohols by Cell Suspensions of C 2 to C 4 n -Alkane-Grown Bacteria

Nineteen new C(2) to C(4)n-alkane-grown cultures were isolated from lake water from Warinanco Park, Linden, N.J., and from lake and soil samples from Bayway Refinery, Linden, N.J. Fifteen known liquid alkane-utilizing cultures were also found to be able to grow on C(2) to C(4)n-alkanes. Cell suspensions of these C(2) to C(4)n-alkane-grown bacteria oxidized 2-alcohols (2-propanol, 2-butanol, 2-pentanol, and 2-hexanol) to their corresponding methyl ketones. The product methyl ketones accumulated extracellularly. Cells grown on 1-propanol or 2-propanol oxidized both primary and secondary alcohols. In addition, the activity for production of methyl ketones from secondary alcohols was found in cells grown on either alkanes, alcohols, or alkylamines, indicating that the enzyme(s) responsible for this reaction is constitutive. The optimum conditions for in vivo methyl ketone formation from secondary alcohols were compared among selected strains: Brevibacterium sp. strain CRL56, Nocardia paraffinica ATCC 21198, and Pseudomonas fluorescens NRRL B-1244. The rates for the oxidation of secondary alcohols were linear for the first 3 h of incubation. Among secondary alcohols, 2-propanol and 2-butanol were oxidized at the highest rate. A pH around 8.0 to 9.0 was found to be the optimum for acetone or 2-butanone formation from 2-alcohols. The temperature optimum for the production of acetone or 2-butanone from 2-propanol or 2-butanol was rather high at 60 degrees C, indicating that the enzyme involved in the reaction is relatively thermally stable. Metal-chelating agents inhibit the production of methyl ketones, suggesting the involvement of a metal(s) in the oxidation of secondary alcohols. Secondary alcohol dehydrogenase activity was found in the cell-free soluble fraction; this activity requires a cofactor, specifically NAD. Propane monooxygenase activity was also found in the cell-free soluble fraction. It is a nonspecific enzyme catalyzing both terminal and subterminal oxidation of n-alkanes.

Lipid and enzyme histochemistry in differentiation of the fetal adrenal cortex in the rat.

The development and differentiation of the fetal adrenal cortex was studied in the rat by lipid and enzyme histochemistry.The adrenal cortex began to have small amounts of lipid droplets at 171/2 days by oil red O stain and the amounts increased gradually. Cholesterol by Schultz reaction became clear at 201/2 days. At 151/2 days weak activity of glucose 6-phosphate dehydrogenase (G-6-PDH) was already present in the inner cortex, which was followed a day later by weakly positive reaction of 3β-hydroxysteroid dehydrogenase (3β-HSD). At 181/2 days positive activity of secondary alcohol dehydrogenase (SAD) was obvious in the inner cortex. Activity of alkaline phosphatase (Al-Pase) was observed in the subcapsular zone from day 181/2 on. Enzymatic activities of the cortex increased gradually after their appearance as development proceeded. These findings show that histochemical activities of such principal enzymes as G-6-PDH and 3β-HSD precede appearance of lipid droplets.As a conclusion, zona reticularis is not distinguishable from zona fasciculata during the fetal stage, zona fasciculo-reticularis differentiates histochemically in a 171/2 day old fetus, and finally differentiation of zona glomerulosa occurs at 201/2 days in gestation.

Regulation of Enzymes Associated with C-1 Metabolism in Three Facultative Methylotrophs

The levels of the oxidation enzyme methanol dehydrogenase and the serine pathway enzymes, hydroxypyruvate reductase, glycerate kinase, serine transhydroxymethylase, serine-glyoxylate aminotransferase, phosphoenolpyruvate carboxylase, and malyl-coenzyme A lyase, were studied in cells of the facultative methylotrophs Pseudomonas AM1, Pseudomonas 3A2 and Hyphomicrobium X grown on different substrates. Induction and dilution curves for these enzymes suggest they may be regulated coordinately in Hyphomicrobium X, but not in Pseudomonas AM1 or 3A2. Glyoxylate stimulated the serine transhydroxymethylase activity in methanol-grown cells of all three organisms. A secondary alcohol dehydrogenase activity was detected at low levels in Pseudomonas AM1 and Hyphomicrobium X, but not in Pseudomonas 3A2.

Microbial oxidation of gaseous hydrocarbons: production of methyl ketones from their corresponding secondary alcohols by methane- and methanol-grown microbes.

Cultures of methane- or methanol-utilizing microbes, including obligate (both types I and II) and facultative methylotrophic bacteria, obligate methanol utilizers, and methanol-grown yeasts were isolated from lake water of Warinanco Park, Linden, N.J., and lake and soil samples of Bayway Refinery, Linden, N.J. Resting-cell suspensions of these, and of other known C1-utilizing microbes, oxidized secondary alcohols to their corresponding methyl ketones. The product methyl ketones accumulated extracellularly. Succinate-grown cells of facultative methylotrophs did not oxidize secondary alcohols. Among the secondary alcohols, 2-butanol was oxidized at the highest rate. The optimal conditions for in vivo methyl ketone formation were compared among five different types of C1-utilizing microbes. Some enzymatic degradation of 2-butanone was observed. The product, 2-butanone, did not inhibit the oxidation of 2-butanol. The rate of the 2-butanone production was linear for the first 4 h of incubation for all five cultures tested. A yeast culture had the highest production rate. The optimum temperature for the production of 2-butanone was 35 degrees C for all the bacteria tested. The yeast culture had a higher temperature optimum (40 degrees C), and there was a reasonably high 2-butanone production rate even at 45 degrees C. Metal-chelating agents inhibit the production of 2-butanone, suggesting the involvement of metal(s) in the oxidation of secondary alcohols. Secondary alcohol dehydrogenase activity was found in the cell-free soluble extract of sonically disrupted cells. The cell-free system requires a cofactor, specifically nicotinamide adenine dinucleotide, for its activity. This is the first report of a nicotinamide adenine dinucleotide-dependent, secondary alcohol-specific enzyme.

Pathology of intracortical nodules in rat adrenal glands, especially on their fine-structure.

Intracortical nodules in the zona fasciculata of rat adrenal glands were studied electron microscopically and histochemically. The nodules were classified into the following three types. A) Hyperplastic small cell type having numerous elongated mitochondria with vesicular cristae. Alkaline phosphatase activity was increased in this type of nodule. B) Hypertrophic large cell type having well developed smooth‐surfaced endoplasmic reticulum, and occasionally forming a characteristic feature of fenestrated cisterna. Histochemical activity of steroid 3/3‐ol dehydrogenase was increased. These morphological appearances suggest that this type of nodule has a greater capacity for steroid synthesis. C) Lipid droplet type having many mitochondria containing lamellar cristae and intramltochondrial deposits often found near large lipid vacuoles. The activity of secondary alcohol dehydrogenase was generally increased.Structural and cellular relationships among these three types of nodule and the pathogenesis of cortical adenomatous nodule were also discussed.

Effect of x-irradiation on the endocrine function of the canine testis.

Observations were made in dogs to determine whether X-irradiation affects the secretion of androgens by the testes, and their responses to exogenous gonadotrophin. The secretory activity was, under pentobarbital anesthesia, determined by measuring 17-oxosteroids in spermatic vein blood. The radiation was applied with 200 or 2, 000 R only to the testes, but it failed to stimulate or significantly alter the secretion of 17-oxosteroids in a period of up to 4 hr after exposure. However, at 200 R an increased secretion of testicular 17-oxosteroids into the spermatic vein became evident within 1-6 days, whereas after 2, 000 R it was observed within 13 days only in a few cases. During the post-irradiation period of 30 days, irradiated testes responded to pregnant mare serum in doses of 40iu/kg by producing larger amounts of 17-oxosteroids than normal, the maximum response occurring within a few days following irradiation. In connection with these experiments, enzyme histochemical studies were carried out on the activity of secondary alcohol dehydrogenase in the testicular tissue. At 200 R, the enzyme activity in the Leydig cell areas was increased considerably and attained to the maximum in a faw days after irradiation, and was then progressively reduced.

HISTOCHEMICAL OBSERVATIONS ON NODULAR PORTIONS IN ADRENAL CORTEX OF AGING RATS

Nodular portions of the adrenal cortex in the aging rats were examined histochemically.From the observations on lipid distribution and histological feature, the cortical nodules were classified into the following three types:1) Type I consists of the compact cells containing no or scanty fine granules of lipid.2) Type II consists of the large cells containing moderate amounts of small of middle sized granules of lipid.3) Type III consists of the cells with vacuolated cytoplasm and abundant lipid droplets.In the nodule of Type III, increased activity of secondary alcohol dehydrogenase was usually consistent. On the other hand, histochemical activities of the enzyme tended to more variable in Type I and Type II.The possible relations of these nodules including few transitional forms were considered.

Enzyme and lipid histochemistry of the rat adrenal cortex stimulated by SU-4885.

The effects of SU-4885 on the rat adrenal cortex by oral administration of 50mg/kg, single or daily for 10 days, were morphologically studied particularly with histochemical methods for the enzyme and lipid. Lipid depletion with fragmentation of droplets throughout the zona fasciculata in rats with single administration conformed to the pattern of adreno-cortical response to an acute moderate stimulus. Reduced activity of secondary alcohol dehydrogenase, fragmentation of lipid droplets, and acidophilic and trabecular changes in the cell cords were observed in the inner fasciculata following a single or daily administration for 10 days. The medication apparently brought about hypersecretion of adrenocortical steroids, which was similar to the effect of ACTH administration. Reduced activity of secondary alcohol dehydrogenase, which reflected the transformation of cholesterol in the steroid biosynthesis, was always observed in the zona fasciculata following the administration of SU-4885 or ACTH. The result might indicate that the administration inhibits some decrease in the steroid biosynthesis from cholesterol possibly through the ‘short route’.

HISTOCHEMICAL DISTRIBUTION OF THE SECONDARY ALCOHOL DEHYDROGENASE IN THE RAT, ESPECIALLY IN THE ADRENAL CORTEX

In the site of steroid production, the histochemical activity of secondary alcohol dehydrogenase on isopropanol as its substrate with NAD as its coenzyme will reflect the inversion of 20ƒ?-22R-dihydroxycholesterol in the biosynthesis of steroid hormone, We applied this method on organs from all parts of the body of Wistar strain rats and observed positive reaction in the adrenal cortex, ovary and testis. In the adrenal cortex of mature rats, the zona glomerulosa exhibited a weakly positive activity, the outer to middle portions of the zona fasciculata an intensely positive activity, the inner portion of the zona fasciculata a weakly positive or negative activity, and the zona reticularis a positive activity. A proportional relationship between secondary alcohol dehydrogenase activity and the amount of lipid was present in the adrenal cortex of mature rats. In addition, the specific distribution of young and old rats was described. The secondary alcohol dehydrogenase is the enzyme of dehydration on the secondary alcohol. In 1965, Hardonkl' noted the similarity between the enzymes participating in the oxidative cleavage of the side chain of cholesterol as a precursor of steroid and secondary alcohol dehydrogenase. Using various secondary alcohols, the distribution of activity of the dehydrogenase in the human body was studied. Among them an enzyme acting on isopropanol as its substrate with nicotinamideadenine dinucleotide (NAD) as its coenzyme had a distribution of activity in the human body in agreement with the site of steroid production such as the adrenal cortex, ovary and testis, according to the description of Hardonkl'. The histochemical detection of various enzymes participating in the biosynthesis and metabolism of steroid hormones in the adrenal cortex and other tissues has been carried out on steroid-3/9-o1 dehydrogenase6', glucose-6-phosphate dehydrogenase and other enzymes2>''4). However, when no specific significance has yet been established, the secondary alcohol dehydrogenase activity will be important, too. We applied this method on organs from all parts of the body, particulary the site of steroid production of male and female Wistar rats, considering the relationship between this enzyme activity and lipid stain.