Asymmetric reduction of prochiral ketones by cell-free systems from alcaligenes eutrophus

A strain of Alcaligenes eutrophus has been isolated from the soil by enrichment culture technique with nerolidol ( 1 ), a sesquiterpene alcohol, as the sole source of carbon and energy. Fermentation of nerolidol ( 1 ) by this bacterium in a mineral salts medium resulted in the formation of two major metabolites, viz. geranylacetone ( 2 ) and an optically active alcohol, (S)-(+)-geranylacetol ( 3 ). Nerolidol ( 1 )-induced cells readily transformed 1,2-epoxynerolidol ( 4 ) and 1,2-dihydroxynerolidol ( 5 ) into geranylacetone ( 2 ). These cells also exhibited their ability to carry out stereospecific reduction of 2 into (S)-(+)-geranylacetol ( 3 ). Oxygen uptake studies clearly indicated that nerolidol-induced cells oxidized compounds 2, 3, 4, 5 and ethyleneglycol ( 7 ). Based on the nature of the metabolites isolated, the ability of nerolidol-induced cells to convert compounds 4 and 5 into geranylacetone ( 2 ), and oxygen uptake studies, a pathway for the microbial degradation of nerolidol ( 1 ) has been proposed. The proposed pathway envisages the epoxidation of the terminal double bond, opening of the epoxide and cleavage between C-2 and C-3 in a manner similar to the periodate oxidation of cis-diol. The cell-free extract prepared from nerolidol-induced cells readily carried out the asymmetric reduction of compound 2 to an optically active alcohol ( 3 ) in the presence of NAD(P)H. The cell-free extract carried out both oxidation and reduction reactions at two different pH values and exhibited wide substrate specificity towards various steroids besides terpenes.     

Microbial synthesis and properties of poly(3-hydroxybutyrate-co-4-hydroxybutyrate)

Microbial synthesis of copolymers of [R]-3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB), P(3HB-co-4HB), by Alcaligenes eutrophus, Alcaligenes latus, and Comamonas acidovorans from various carbon sources has been studied. The copolyester compositions varied from 0 to 100 mol% 4HB, depending on the microorganism and the combination of carbon substrates supplied. The thermal and physical properties of compositions with 0–100 mol% 4HB were investigated. The copolyesters represented a wide variety of polymeric materials, from hard crystalline plastic to very elastic rubbers, depending on composition. The copolyester films with high 4HB fractions (64–100 mol% 4HB) exhibited the characteristics of a thermoplastic elastomer, and the tensile strength increased from 17 to 104 MPa as the 4HB fraction increased. The enzymatic degradation of P(3HB-co-4HB) films was studied in an aqueous solution of extracellular polyhydroxybutyrate (PHB) depolymerase from Alcaligenes faecalis or lipase from Rhizopus delemer. The erosion rate of P(3HB-co-4HB) films was strongly dependent on the copolymer composition. In addition, environmental degradation of P(3HB-co-4HB) films in sea water was investigated.     

Nitrogen removal characteristics of heterotrophic nitrification-aerobic denitrification by Alcaligenes faecalis C16

Alcaligenes faecalis C16 was found to have the ability to heterotrophically nitrify and aerobical y denitrify. In order to further understand its nitrogen removal ability and mechanism, the growth and ammonium removal response were investigated at different C/N ratios and ammonium concentrations in the medium with citrate and acetate as carbon source separately. Furthermore, experiments of nitrogen sources, production of nitrogen gas and enzyme assay were conducted. Results show that the bacterium converts NH4+-N and produces NH2OH during the growing phase and nitrite accumulation is its distinct metabolic feature. A. faecalis C16 is able to tolerate not only high ammonium concentration but also high C/N ratio, and the ammonium tolerance is associated with carbon source and C/N ratio. The nitrogen balance under different conditions shows that approximately 28%–45%of the initial ammonium is assimilated into the cells, 44%–60%is denitrified and several percent is converted to nitrification products. A. faecalis C16 cannot utilize hydroxylamine, nitrite or nitrate as the sole nitrogen source for growth. However, nitrate can be used when ammonium is simultaneously present in the medium. A possible pathway for nitrogen removal by C16 is suggested. The preliminary enzyme assay provides more evidence for this nitrogen removal pathway.     

Identification of interaction site of pseudoazurin with its redox partner, copper-containing nitrite reductase from Alcaligenes faecalis S-6

Pseudoazurin, a low molecular weight protein containing a singletype I copper, functions as an electron donor to a copper-containingnitrite reductase (NIR) in a denitrifying bacterium Alcaligenesfaecalis S-6. To elucidate the proteinprotein interaction betweenthese two copper-containing proteins, each of nine out of 13lysine residues on the surface of pseudoazurin were independentlyreplaced by alanine or aspartate, and the effects of the mutationson the interaction with NIR, as well as the physicochemicalproperties of pseudoazurin, were analyzed. All of the mutatedpseudoazurins showed optical spectra and oxidation-reductionpotentials almost identical to those of wildtype pseudoazurin,suggesting that none of the replacements of these lysine residuesaffected the environment around the type I copper site. Kineticanalysis of electron transfer between mutated pseudoazurinsand NIR reveals that the lysine mutations have very little effecton the rate of electron transfer to NIR, but substitution atresidues 10, 38, 57 and 77, all close to the copper site, substantiallydecreases the affinity of pseudoazurin for NIR. This suggeststhat pseudoazurin interacts with NIR through the region closeto the type I copper site. The refined X-ray structures of Lys38Aspand Lys10Asp/Lys38Asp show that the molecular structure hasindeed changed little. A new space group is observed for theLys109Ala mutant crystal. Crystal packing interactions changefor the Lys10Asp/Lys38Asp mutant but remain the same for Lys38Aspand Lys59Ala mutants.     

游离DHA酯化富集的脂酶催化工艺研究

研究了脂酶催化游离DHA和甘油酯化的工艺。结果表明:以Rhizomucor miehei脂酶催化的一步法工艺的最适条件为FFA∶甘油=1∶3、脂酶量1%和水分5%,其酯化率、三酰甘油(TAG)含量和TAG中DHA富集量分别为71.6%、22.98%和47.53%;以Rhizomucor miehei脂酶催化的二步法工艺和以Rhizomucor miehei脂酶、Alcaligenes sp.脂酶共同催化的混合酶法工艺不能改变TAG中DHA的百分含量,但可显著提高反应的酯化率和TAG含量。其中,二步法工艺可将酯化率和TAG含量分别提高到84.66%和91.86%,而混合酶法工艺则可分别将其进一步增加到97.68%和93.39%。     

一株粪产碱杆菌(Alcaligenes faecalis)产热凝胶的发酵条件

通过对一株粪产碱杆菌WX-C12(Alcaligenes     

Piggery wastewater treatment using Alcaligenes faecalis strain No. 4 with heterotrophic nitrification and aerobic denitrification

Alcaligenes faecalis strain No. 4, which has heterotrophic nitrification and aerobic denitrification abilities, was used to treat actual piggery wastewater containing high-strength ammonium under aerobic conditions. In a continuous experiment using a solids-free wastewater (SFW) mixed with feces, almost all of the 2000 NH4+ -N mg/L and 12,000 COD mg/L in the wastewater was removed and the ammonium removal rate was approximately 30 mg-N/L/h, which was 5-10 times higher than the rates achieved by other bacteria with the same abilities. The denitrification ratio was more than 65% of removed NH4+ -N, indicating that strain No. 4 exhibited its heterotrophic nitrification and aerobic denitrification abilities in the piggery wastewater.     

粪产碱菌对阿魏酸厌氧降解的影响

粪产碱菌在以阿魏酸为唯一碳源的厌氧发酵中培养7 d,阿魏酸的降解率约为70%;研究秸秆厌氧发酵产气（以产气量反应菌群的活性）发现,体系接种5%粪产碱菌后,菌群的活性最强,生物气增量最大,达130 mL,比接种3%和7%粪产碱菌的体系提高85.71%和116.67%;同时体系的产酸效率和阿魏酸的降解率均显著提升,分别比接种3%和7%粪产碱菌的体系提高136%和110.71%以及25%和33.33%。傅里叶红外光谱检测表明：厌氧发酵体系接种5%粪产碱菌后秸秆中木质素、阿魏酸的特征官能团结构被有效破坏。秸秆厌氧发酵体系接入粪产碱菌可以有效降解阿魏酸等木质素降解衍生物、解除木质素及其降解产物对厌氧菌群的毒性同时提高产气效率,具有应用价值。     

羟基脂肪酸聚酯产生菌选育

以特定方法筛得羟基脂肪酸聚酯（ＰＨＡＳ）产生株ＡＭＢ－００１，初步鉴定为产碱菌（Ａｌｃａｌｉｇｅｎｅｓ）属。经紫外线与亚硝基胍诱变，变异株ＡＵＮ－３９产聚羟丁酸（ＰＨＢ）和聚羟丁戊酸共聚物（ＰＨＢＶ）的产率分别为细胞干重的３５％和４７．５％．透射电镜观察表明ＡＵＮ－３９胞内积累的ＰＨＢ粒子可充满胞内空间。