Applications of Hydrolytic and Decarboxylating Enzymes in Biotransformations

Peptides, and oligosaccharides and glycosides, can be synthesised by making use of the ‘reverse hydrolytic activity’ of proteases and glycosidases respectively. In applying these enzymes to the practical synthesis of these classes of compound, several factors need to be considered, namely the need to shift the rate-determining step through the use of activated substrates, the need to minimise competing hydrolysis of these and the need to minimise hydrolysis of the products. In spite of these problems, the enzymatic methods have many attractive features, not least amongst which is the absolute control of stereochemistry in acyl transfer and glycosyl transfer respectively.     

Honeydew amino acids in relation to sugars and their role in the establishment of ant-attendance hierarchy in eight species of aphids feeding on tansy (Tanacetum vulgare)

Abstract. The ratio of the concentration of honeydew total amino acids to total sugars in the honeydew of eight species of aphids, all feeding on tansy, Tanacetum vulgare (L.), was determined and correlated with honeydew production and ant‐attendance. The honeydew of the five ant‐attended aphid species [Metopeurum fuscoviride (Stroyan), Trama troglodytes (v. Hayd), Aphis vandergooti (Börner), Brachycardus cardui (L.), Aphis fabae (Scopoli)] was rich in total amino acids, ranging from 12.9 to 20.8 nmol µL^?1 compared with the unattended aphid Macrosiphoniella tanacetaria (Kalt.) with only 3 nmol µL^?1. Asparagine, glutamine, glutamic acid and serine (all nonessential amino acids) were the predominant amino acids in the honeydew of all species. The total concentration of amino acids in the phloem sap of tansy was much higher (78.7 nmol µL^?1) then in the honeydew samples, and the predominant amino acids were glutamate (34.3%) and threonine (17.7%). A somewhat unexpected result was the finding that those aphid species with the highest total amino acid concentration in the honeydew always had the highest concentration of sugars. The lowest amino acid–sugar combined value was 104–28.8 nmol µL^?1 in the non ant‐attended species M. tanacetaria, and the highest value was an average of 270–89.9 nmol µL^?1 for the three most intensely attended aphid species M. fuscoviride, A. vandergooti and T. troglodytes. There is no evidence that any single amino acid or group of amino acids in the honeydew acted as an attractant for ant‐attendance in these eight aphid species. The richness of the honeydew (rate of secretion × total concentration of sugars), along with the presence of the attractant sugar melezitose, comprised the critical factors determining the extent of ant‐attendance of the aphids feeding on T. vulgare. The high total amino acid concentration in sugar‐rich honeydews can be explained by the high flow‐through of nutrients in aphids that are particularly well attended by ants.     

Characterization of Aspergillus niger catalase

Aspergillus niger catalase has been characterized by a variety of physical techniques including gel filtration, sedimentation rate and equilibrium methods and photon correlation spectroscopy. The catalase has a sedimentation coefficient (S₂₀⁰) of 14.2 ± 0.08 S and diffusion coefficient (D₂₀⁰) of 4.14 ± 0.35 × 10⁻⁷ cm² s⁻¹. The average molecular weight of the catalase from all available data including current sedimentation equilibrium measurements and two previous literature values is 345 000. The frictional ratio of the molecule assuming a hydration parameter similar to that of bovine liver catalase (.3 g H₂O g⁻¹) is 1.103, suggesting that Aspergillus niger catalase has an asymmetric structure with an axial ratio of approximately 3 (the Stokes radius is 5.83 ± 0.49 nm). The titration curve and amino acid analysis indicate that in the native conformation only 23% of the ionizable amino acid residues are titratable between pH 3 and 10.5. Denaturation with sodium n-dodecylsulphate increases the number of titratable groups to 46%. The ratio of anionic to cationic amino acid residues in Aspergillus niger catalase is 2.46 and the isoelectric point is 6.5. The optimum pH for catalytic activity is approximately 7.     

Ultraviolet mutagenesis of the cellulase producer Aspergillus niger using protoplasts

Abstract Ultraviolet mutagenesis of Aspergillus niger strain O97 was achieved using protoplasts. Protoplasts of A. niger O97 showed the same ultraviolet killing kinetics as intact cells. After mutagenesis, several mutants were found in regenerated collonies. These mutants differ from the original strain in spore colour and cellulase-producing ability. The most active strain, designated 97V3-3 has an altered spore colour, and its carboxymethylcellose-hydrolysing, filter-paper-degradation, cotton-de-gradation and β-glucosidase activities were increased by 45.4%, 19.1%, 28.2% and 18.2%, respectively.     

Biotransformation of germacrane-type sesquiterpenoids by Aspergillus niger

Three germacrane-type sesquiterpenoids, (+)-germacrone-4,5-epoxide, germacrone and (+)-curdione were biotransformed by Aspergillus niger to give hydroxylated guaiane-type sesquiterpenoids together with allylic alcohols and spirolactone.     

Histological, fine-structural and histochemical differences in the testicular glands of gobiid and blenniid fishes

The testicular gland (t.g.) is a glandular tissue situated adjacent to the testis of blenniid and several gobiid species. In the present study the t.g. of Blennius pavo Risso and Gobius niger L. were compared by histological and histochemical methods. In B. pavo the spermatozoa have to cross the t.g. to reach the vas deferens and thus they come into contact with the gland cells, whereas in G. niger the vas deferens is situated between the testis and the t.g. The fine structure and histo-chemistry of the t.g. cells reveal that in B.pavo the cells of the t.g. have exocrine as well as endocrine functions. The t.g. cells of B. pavo contain large amounts of lipids, form a secretion containing acid mucopolysaccharides, show positive reaction for acid phosphatase, and some cells stain for 3β-HSD and G6PD. The function of the t.g. of G. niger is exclusively endocrine. Characteristics of the gland cells of this species are well developed smooth ER and tubulovesicular or paracristal-line mitochondria. The stainings for 3β-HSD, G6PD and UDPGD give strong positive results in the whole t.g., indicating the presence of steroids and steroid glucuronides.     

黑曲霉糖化酶高产和低产菌株糖化酶基因调控区的克隆及其分析比较

以PCR合成的糖化酶高产菌株黑曲霉(Asp. Niger)T21糖化酶基因5’近端非编码区588bp(EcoRI-BamHI)的序列为探针,从T21染色体DNA中克隆到近2.0kb的糖化酶基因5’端非编码区序列,并以此序列为探针从糖化酶低产菌株黑曲霉3.795(T21的诱变出发株)的染色体DNA中克隆到1.5kb的糖化酶基因5’端非编码区序列。该二序列的分析测定结果表明,其结构特征与文献报道的黑曲霉糖化酶基因5’端非编码区的基本一致,被称为“核心启动子”(Core promoter)的TATAAAT框及GCAAT框,分别在翻译起始点的-109bp及-178bp处。此外,在曲霉amdS,amyB基因中已发现有调控功能的CCAAT序列存在于-449bp和-799bp处。高产和低产菌株糖化酶基因5’端非编码区序列的分析比较结果表明,有9个部位的碱基发生了变化。此实验结果为进一步研究黑曲霉糖化酶基因在转录水平上的调控规律打下了基础。     

植酸钠对黑曲霉柠檬酸发酵产酸的促进效应

研究了植酸钠对黑曲霉柠檬酸发酵产酸的促进效应。在葡萄糖全合成培养基中添加１％的植酸钠,可使产酸比对照提高２．４倍;在薯粉、玉米粉等天然培养基中添加１％植酸钠,柠檬酸产量分别提高１．７倍和１．３倍。酶活性测定分析表明,植酸钠对柠檬酸代谢途径中的几种关键酶的活性有影响。     

Modeling approach to control of carbohydrate metabolism during citric acid accumulation by Aspergillus niger: II. Sensitivity analysis

Steady state sensitivity analysis of a model of carbohydrate metabolism and anaplerotic synthesis of oxalacetate were, in Aspergillus niger under conditions of citric acid accumulation, carried out. The flux and metabolite concentration control structure of the system obtained shows that the hexokinase/substrate transport step is the main controlling step of the pathway. The quantitative contribution of the other enzyme catalyzed or transport steps are also discussed. These results allow the design of a proper strategy of biotechnological manipulation aimed at improvement of the process. (c) 1994 John Wiley & Sons, Inc.     

碳源对固定化细胞生产柠檬酸的影响

柠檬酸是利用微生物代谢生产的一种极为重要的有机酸．广泛应用于食品、饮料、化工、冶金、印染等各个领域。在国外,近10年来,利用固定化细胞生产柠檬酸已获得较广泛的研究^〔1－6〕,国内也有学者指出,柠檬酸发酵的趋向是利用固定化细胞进行连续化生产⑺。而国内这方面的研究报道很少〔8,9〕。我们利用海藻酸钙凝胶包埋固定化黑曲霉细胞生产柠檬酸．探讨了碳源种类及其浓度对固定化细胞生产柠檬酸的影响。现将结果报道如下。