Purification and characterization of a novel alcohol oxidase from Paenibacillus sp. AIU 311

An oxidase catalyzing the conversion of glycolaldehyde to glyoxal was purified to the homogeneous state from Paenibacillus sp. AIU 311, and its properties were revealed. This enzyme was specific to glycolaldehyde and glyceraldehyde, and the reaction rates to other alcohols and aldehydes were less than 6% of that of glycolaldehyde. The Km values for glycolaldehyde and glyceraldehyde were estimated to be 13.2 and 7.5 mM, respectively. The glycolaldehyde oxidation was optimum at pH 6.5 and 50 degrees C. The molecular mass of this enzyme was 49 kDa, and it consisted of two identical subunits of 24 kDa. The NH2-terminal sequence was not homologous to those of alcohol oxidases. This is the first report of an oxidase exhibiting high specificity to a hydroxy group of aldehyde alcohols.     

Cloning and expression of a NAD+-dependent xylitol dehydrogenase gene (xdhA) of Aspergillus oryzae

XdhA, which encodes a xylitol dehydrogenase gene, was cloned from Aspergillus oryzae genomic DNA. It consists of 1214 bp structural region, which is interrupted by two introns, and encodes 358-amino-acid protein (38,197 Da). It is similar to the known NAD(+)-dependent xylitol dehydrogenase (EC 1.1.1.9). The gene was expressed in Escherichia coli BL21-AI using a T7 promoter. The cell-free extract of the transformant showed a 36.5 kDa band upon SDS-PAGE and NAD(+)-dependent xylitol dehydrogenase activity.     

Superoxide dismutases exhibit oxidase activity on aldehyde alcohols similar to alcohol oxidase from Paenibacillus sp. AIU 311

The relations between oxidase activity on aldehyde alcohols and superoxide dismutase (SOD) were investigated, since the amino terminal amino acid sequence of alcohol oxidase (AOD) from Paenibacillus sp. AIU 311, which was specific to aldehyde alcohols, exhibited high similarity to those of SODs containing manganese (Mn(2+)-SOD). Paenibacillus AOD had high SOD activity. The SODs containing manganese, iron, or copper and zinc also exhibited oxidase activities on aldehyde alcohols, and the relative values of oxidase activities on aldehyde alcohols to SOD activity of Mn(2+)-SOD were closer to those of Paenibacillus AOD compared with those of the other SODs. Thus, SODs had AOD activity on aldehyde alcohols as another enzyme activity, and the Paenibacillus AOD and Mn(2+)-SOD were classified into a similar group.     

Cloning and expression of NAD+-dependent L-arabinitol 4-dehydrogenase gene (ladA) of Aspergillus oryzae

A gene of Aspergillus oryzae, ladA, which encodes L-arabinitol 4-dehydrogenase (EC 1.1.1.12), and its cDNA were cloned in Escherichia coli. The gene consisted of a 1209-bp coding region, interrupted by a 59-bp intron, which encoded a 382-amino-acid polypeptide (40,812 Da). The protein showed 67% identity to a well-studied L-arabinitol 4-dehydrogenase (Lad1) of Hypocrea jecorina. The cell-free extract of E. coli, which expressed ladA cDNA, showed L-arabinitol dehydrogenase activity with NAD+. It was also reactive for ribitol and xylitol.     

Effect of NAD+-dependent isocitrate dehydrogenase gene (IDH1, IDH2) disruption of sake yeast on organic acid composition in sake mash

The ratio of organic acids in sake mash is a very important factor affecting the taste of alcoholic beverages. To alter the organic acid composition in sake and investigate the mechanism of producing organic acids in sake mash, we examined the effect of NAD+-dependent isocitrate dehydrogenase (IDH) activity deficiency in sake yeast by disrupting the IDH1 or IDH2 gene. Two haploid strains (MATa or MATa genotype) isolated from sake yeast Kyokai no. 701 (K701) were disrupted using the aureobasidin A resistant gene (AUR1-C) as a selection marker. These disruptants were defective in the activity of IDH and failed to grow on medium containing glycerol as a sole carbon source. Sake meter, alcohol concentration, and glucose consumption in sake brewed with the disruptants were reduced in comparison with those of the parental strains. The production of citrate (including isocitrate), malate, and acetate by the disruptants was increased, but succinate production was reduced to approximately half in comparison with the parental strains. These results indicate that approximately half the amount of succinate in sake mash is produced via the oxidative pathway of the TCA cycle in sake yeast. While the diploid strain constructed by mating haploid disruptants for the IDH gene exhibited stronger fermentation ability than the haploid disruptants, almost similar profiles of components in sake were obtained for both strains.     

Purification and characterization of a dehydrogenase catalyzing conversion of N alpha-benzyloxycarbonyl-L-aminoadipic-delta-semialdehyde to N alpha-benzyloxycarbonyl-L-aminoadipic acid from rhodococcus sp. AIU Z-35-1

The enzyme catalyzing conversion of N alpha-benzyloxycarbonyl-L-aminoadipic-delta-semialdehyde (N alpha-Z-L-AASA) to N alpha-benzyloxycarbonyl-L-aminoadipic acid (N alpha-Z-L-AAA) in Rhodococcus sp. AIU Z-35-1 was identified, and its characteristics were revealed. This reaction was catalyzed by a dehydrogenase with a molecular mass of 59 kDa. The dehydrogenase exhibited enzyme activity on not only N alpha-Z-L-AASA but also N alpha-Z-D-AASA and short chain aliphatic aldehydes, but not on aromatic aldehydes and alcohols. The apparent Km values for N alpha-Z-L-AASA, N alpha-Z-D-AASA and NAD+ were estimated to be 3.8 mM, 14.1 mM and 0.16 mM, respectively. The NH2 terminal amino acid sequence of this enzyme exhibited a similarity to those of a piperidein-6-carboxylate dehydrogenase from Streptomyces clavuligerus and a putative dehydrogenase from Rhodococcus sp. RHA 1, but not to those of other microbial aldehyde dehydrogenases.     

Purification and characterization of the alcohol dehydrogenase with a broad substrate specificity originated from 2-phenylethanol-assimilating Brevibacterium sp. KU 1309

A novel 2-phenylethanol dehydrogenase has been purified from a soil bacterium Brevibacterium sp. KU 1309. The enzyme was purified about 1400-fold to homogeneity, and found to be a monomeric enzyme of apparent 39 kDa. The enzyme had broad substrate specificity and catalyzes a reversible oxidation of various primary alcohols to aldehydes. The enzyme required NAD+, but not NADP+ as a cofactor. Thus, the enzyme was classified into a group of NAD+-dependent primary alcohol dehydrogenase. The activity was inhibited by Cu2+, Ni2+, Ba2+, Hg2+ and p-chloromercuribenzoate. The enzyme is expected to be applicable as an effective biocatalyst in the oxidation of various alcohols.     

Biochemical and genetic characterization of a D(-)-3-hydroxybutyrate dehydrogenase from Acidovorax sp. strain SA1

D(-)-3-hydroxybutyrate dehydrogenase (BDH; EC 1.1.1.30) from a poly(D(-)-3-hydroxybutyrate) (PHB) degrading bacterium, Acidovorax sp. SA1, was purified using Toyopearl DEAE-650M, red-Sepharose CL-4B, and Q Sepharose FF. The molecular mass of the enzyme was estimated as 27 kDa by SDS-PAGE and 110 kDa by gel filtration. The gene encoding BDH was cloned and sequenced, and expressed in Escherichia coli. The gene product was purified in two steps with a high yield. The N-terminal amino acid sequence of the enzyme purified from E. coli agreed with that of the purified enzyme from strain SA1. The BDH of strain SA1 had high amino acid sequence homology to that of Ralstonia eutropha H16. The Km values for D(-)-3-hydroxybutyrate and NAD+ in the oxidation reaction were 4.5 x 10(-4) M and 8.9 x 10(-5) M, respectively. The Km values for acetoacetate and NADH in the reduction reaction were 2.4 x 10(-4) M and 2.9 x 10(-5) M, respectively.     

Enzymatic Hydrolysis of Starch from Jacatupé (Pachyrhizus erosus L. Urban) by Thermostable Amylolytic Enzymes

An alternative source of starch obtained from jacatupé (Pachyrhizus erosus L. Urban) a tropical tuber has been characterized enzymatically using amylolytic enzymes. The best hydrolysis conditions were obtained using α-amylase of 13.56U/ml at pH 7.0 and 80°C; amyloglucosidase of 177.5U/ml at a pH range from 3.5 to 4.0 and 70°C, respectively. Michaelis constants were determined in terms of substrate concentration; Km and Vmax for α-amylase and amyloglucosidase were found to be 16.8g/1, 1.49g/1, 11.2μmol/h and 3.10μmol/h, respectively. Scanning electron microscopy studies suggest that the enzymes tunnelled into the granular interior of the starch granules and then hydrolysed from within, along concentric holes - a well established phenomenon in the starch granules from classical crops.     

Cloning and expression of the gene encoding the thermophilic NAD(P)H-FMN oxidoreductase coupling with the desulfurization enzymes from Paenibacillus sp. A11-2

The gene encoding the NAD(P)H-flavin oxidoreductase (flavin reductase) which couples with the thermophilic dibenzothiophene (DBT)-desulfurizing monooxygenases of Paenibacillus sp. A11-2 was cloned in Escherichia coli and designated tdsD. Nucleotide sequence analysis suggested that the gene product consisted of 200 amino acids and showed about 30%, 27% and 26% amino acid sequence similarity to the major flavin reductase of Vibrio fischeri, the NADH dehydrogenase of Thermus thermophilus and several oxygen-insensitive NAD(P)H nitroreductases in the Enterobacteriaceae family, respectively. Both the growing and resting recombinant E. coli, in which tdsD was coexpressed with a set of desulfurizing genes, showed a rate of DBT removal about 5 times higher than the recombinants lacking tdsD. Maximal desulfurization was observed close to 45 degrees C and 55 degrees C in the resting cells and in the cell-free extraction reaction with the tdsD-coexpressing recombinants, respectively. In an organic/aqueous biphasic system, the coexpression of tdsD also markedly enhanced the rate of DBT removal.     

Cloning and expression of the gene encoding the thermophilic NAD(P)H-FMN oxidoreductase coupling with the desulfurization enzymes from Paenibacillus sp. A11-2

The gene encoding the NAD(P)H-flavin oxidoreductase (flavin reductase) which couples with the thermophilic dibenzothiophene (DBT)-desulfurizing monooxygenases of Paenibacillus sp. A11-2 was cloned in Escherichia coli and designated tdsD. Nucleotide sequence analysis suggested that the gene product consisted of 200 amino acids and showed about 30%, 27% and 26% amino acid sequence similarity to the major flavin reductase of Vibrio fischeri, the NADH dehydrogenase of Thermus thermophilus and several oxygen-insensitive NAD(P)H nitroreductases in the Enterobacteriaceae family, respectively. Both the growing and resting recombinant E. coli, in which tdsD was coexpressed with a set of desulfurizing genes, showed a rate of DBT removal about 5 times higher than the recombinants lacking tdsD. Maximal desulfurization was observed close to 45°C and 55°C in the resting cells and in the cell-free extraction reaction with the tdsD-coexpressing recombinants, respectively. In an organic/aqueous biphasic system, the coexpression of tdsD also markedly enhanced the rate of DBT removal.     

Sequence analysis of a beta-agarase gene (pjaA) from Pseudomonas sp. isolated from marine environment

The pjaA gene of Pseudomonas sp. W7 consists of an open reading frame of 1926 bp encoding beta-agarase, a protein of 642 amino acids and a molecular weight of 69,540 Da. The expressed protein of plasmid pEAG3-3, in which 259 amino acid residues from C-terminus of the overexpression plasmid (pEAG3) were eliminated, led to the complete loss of agarolytic activity.     

醋酸杆菌醇脱氢酶粗酶液的特性研究

乙醇脱氢酶(ADH)是醋酸杆菌发酵生产醋酸的关键酶.以硫酸铵为沉淀剂,采用盐析法对醋酸杆菌中乙醇脱氢酶进行初步的分离纯化,并研究其酶学特性.结果表明:ADH比活力从粗酶液的0.201 U/mg提高到0.460 U/mg,纯化倍数为2.289;其最适作用pH值为7.5～8.0,pH值为7.8时酶活力达到最大,pH值为7.0时酶较为稳定;最适作用温度为35℃,温度为30℃～40℃时酶活力较为稳定,温度超过45℃后酶活力急剧下降.通过对乙醇底物浓度对ADH活力影响的研究,ADH对乙醇的米氏常数Km为2.59×10-2mol/L.     

Characterization of NAD-dependent alcohol dehydrogenase enzymes of strawberry's achenes (Fragaria x ananassa cv. Elsanta) and comparison with respective enzymes from Methylobacterium extorquens

Achenes were isolated from strawberries fruits, while the methylotroph Methylobacterium extorquens (strain with CABI registration number IMI 369321), which has been isolated from strawberry (Fragaria x ananassa cv. Elsanta) callus cultures, was grown on a mixture of methanol (0.25% v/v) and 1,2-propanediol (0.75% v/v). The Alcohol Dehydrogenase (ADH) enzymatic activities of the achenes were assessed and the optimum pH for ADH activity was found to be pH 10.0. Enzyme assays were carried out in order to define the best substrate specificity at pH = 10.0. The best substrates were found to be ethanol (Km = 5.950 mM) and methanol (Km = 12.610 mM). Only enzymes from the bacterium showed capability of using aldehydes - especially formaldehyde - as substrates. A wide variety of metals as well as EDTA and NaN₃ were shown to decrease the enzymatic activity. Furthermore, SDS-PAGE Electrophoresis experiments showed Molecular Weight of 47.0 kDa for the Alcohol Dehydrogenase from achenes of strawberries (Fragaria x ananassa). Additional experiments were conducted in order to define certain thermodynamic properties of the enzymes, by using the dehydrogenation activities of these three enzyme sources which were calculated by measuring the absorption of NADH at 340 nm, in the Arrhenius equation.     

Cloning and nucleotide sequence of carbaryl hydrolase gene (cahA) from Arthrobacter sp. RC100

A carbaryl hydrolase gene (cahA) encoded on the plasmid pRC1 in Arthrobacter sp. RC100 was cloned and sequenced. The entire region of the deduced amino acid sequence was found to be homologous to that of an amidase family. Parts of the consensus sequences of the amidase gene have been identified in CahA from strain RC100. CahA was overexpressed in Escherichia coli JM109, and the enzyme was purified to homogeneity by protamine sulfate treatment, ammonium sulfate precipitation, and hydrophobic and anion-exchange chromatographies. The purified enzyme showed hydrolase activity toward 1-naphthylacetamide and isobutyramide but showed no activity toward 1-naphthylacetate. This is the first report of an amidase that is able to hydrolyze N-methylcarbamate pesticides.     

栽培管花肉苁蓉中苯乙醇苷的乙醇提取工艺

探索了用乙醇回流法从栽培管花肉苁蓉中提取苯乙醇苷类物质的工艺流程.通过单因素和响应面实验,对影响苯乙醇苷提取的因素进行了探讨和研究,运用响应面分析法确定了提取工艺的最优化条件：提取温度为60 ℃,料液比（g：mL）为1：12.8,提取时间为3.75 h,乙醇体积分数为68.2 %,提取次数2次.在此条件下苯乙醇苷的得率为5.76 %.     

紫竹梅红色素的提取与纯化技术

紫竹梅红色素属于花色苷类物质,色泽鲜艳,使用安全,是一种新型的天然色素.本试验以紫竹梅为原料,采用单因素试验设计和正交试验设计,对不同提取剂、提取剂的用量、酸性乙醇浓度、提取时间、提取温度对提取率的影响进行测试,以确定最佳提取条件,用大孔树脂吸附法进行纯化.从试验结果可以看出,提取剂的用量、酸性乙醇浓度、提取时间、提取温度四个因子中,以提取剂的用量、提取温度对色素提取率起主要影响.试验结果表明,用酸性乙醇作提取溶剂较好,最佳的提取工艺条件为:溶剂量为1∶2,酸性乙醇的浓度为醋酸0.3％、乙醇20％,在50℃的温度下,浸提3h; AB-8树脂对紫竹梅红色素具有较高的吸附量,用50％乙醇洗脱为最佳.     

Isolation and characterization of Bacillus sp. INT005 accumulating polyhydroxyalkanoate (PHA) from gas field soil

A gram-positive bacterium (designated strain INT005) that accumulated polyhydroxyalkanoate (PHA) was isolated from gas field soil. From its morphological and physiological properties and the partial nucleotide sequence (about 500 bp) of its 16S rDNA, it was suggested that strain INT005 was similar to several species of the genus Bacillus. We confirmed that strain INT005 is a Bacillus sp. The PHA productivities of strain INT005 were higher than those of Bacillus megaterium and Ralstonia eutropha at 37-45 degrees C reported to date, and it was suggested that the PHA synthase of INT005 may exhibit moderate thermostability. The bacterium had the ability to produce poly(3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyhexanoate), and poly(3-hydroxybutyrate-co-6-hydroxyhexanoate-co-3-hydroxyhexanoate) from the appropriate carbon sources. The PHA synthase from INT005 showed similar substrate specificity to those of class I and III PHA synthases and strain INT005 produced PHAs with various monomer compositions. From the analysis of monomer composition and PHA accumulation in the presence of acrylic acid, it was suggested that de novo fatty acid synthesis and beta-oxidation are involved in the PHA synthesis of Bacillus sp. INT005. Since Bacillus sp. INT005 could synthesize PHA even at 45 degrees C and PHAs with various monomer compositions, and only one report on the cloning of the synthesis-related genes from a Bacillus species (B. megaterium) has been published;Bacillus sp. INT005 is thought to be very valuable source of PHA synthesis-related genes.