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.     

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.     

Optimization for xylanase and cellulase production from Aspergillus niger ATTC 6275 in palm oil mill wastes and its application

Optimization of enzyme production from Aspergillus niger ATCC 6275 under both submerged and solid-substrate cultivation was investigated. Results from submerged cultivation using palm oil mill effluent revealed that pretreatment of ground palm cake did not improve enzyme production. Addition of 0.60g NH4NO3/l generated maximum activity of xylanase and cellulase (CMCase). The optimum aeration rate was 1.2 v/v min. Under solid-substrate cultivation, the results indicated that heating and alkali treatment of the ground palm cake gave no further improvement in enzyme production. The optimal N-source was 2% urea. Optimal initial moisture contents for xylanase and CMCase activities were 60% and 50% respectively, with temperature optima of 30°C and 35°C, respectively. The optimal inoculum size was 1× 108 spores/g palm cake with an initial pH of 4.5–5.0. The maximum activities of xylanase (282.9U/g) and CMCase (23.8U/g) were obtained under the optimum conditions. Solid-substrate cultivation was a better method for the production of enzyme, particularly xylanase, from A. niger ATCC 6275. The application of these enzymes to decanter effluent showed the separation of oil and grease and suspended solids from the effluent. This is comparable to the result achieved from using the commercial xylase preparation Meicelase and superior to the effect of Sumyzyme.     

Effect of growth temperature on lipid fatty acids of four fungi (Aspergillus niger,Neurospora crassa,Penicillium chrysogenum,andTrichoderma reesei)

The effect of growth temperature on the lipid fatty acid composition was studied over a temperature range from 35 to 10° C with 5° C intervals in four exponentially growing fungi: Aspergillus niger, Neurospora crassa, Penicillium chrysogenum, and Trichoderma reesei. Fatty acid unsaturation increased in A. niger, P. chrysogenum, and T. reesei when the temperature was lowered to 20–15, 20, and 26–20° C, respectively. In A. niger and T. reesei, this was due to the increase in linolenic acid content. In P. chrysogenum, the linolenic acid content increased concomitantly with a more pronounced decrease in the less-unsaturated fatty acid, oleic acid, and in palmitic and linoleic acids; consequently, the fatty acid content decreased as the temperature was lowered to 20° C. In T. reesei, when the growth temperature was reduced below 26–20° C, fatty acid unsaturation decreased since the mycelial linolenic acid content decreased. In A. niger and P. chrysogenum, the mycelial fatty acid content increased greatly at temperatures below 20–15° C. In contrast, in N. crassa, fatty acid unsaturation was nearly temperature-independent, although palmitic and linoleic acid contents clearly decreased when the temperature was lowered between 26 and 20° C; concomitantly, the growth rate decreased. Therefore, large differences in the effects of growth temperature on mycelial fatty acids were observed among various fungal species. However, the similarities found may indicate common regulatory mechanisms causing the responses. Received: 1 March 1995 / Accepted: 8 May 1995     

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。     

黑曲霉单宁酶高活性菌株的诱变选育*

以黑曲霉（Ａｓｐｅｒｇｉｌｌｕｓ ｎｈｉｇｅｒ）Ｎｏ．１３为出发菌株,经紫外线诱变处理,获得一株制备原生质体的起始菌,该菌株单宁酶活性比Ｎｏ．１３提高５５％,并对其制备原生质体的条件进行了研究,在优化方案基础上,紫外诱变原生质体,诱变株经筛选,最后得到一株具有稳定遗传性的单宁酶高活性菌株,在摇瓶培养基中进行生物转化实验,连续传代１０次,结果显示发酵液中没食子酸浓度始 维持在２２．８－２３．９ｍｇ／     

黑曲霉N14植酸酶基因在巴斯德毕赤酵母中的高效表达

从黑曲霉N14基因组DNA中扩增出植酸酶phyA基因表达片段 ,并将其克隆到pMD18-T载体中。以此片段构建了pPIC9K-phyA重组表达载体 ,目的片段以正确的阅读框架插入到pPIC9K的多克隆位点EcoRⅠ和NotⅠ之间。重组表达载体经XbaⅠ线性化处理 ,电击转化毕赤酵母 ,经G418抗性筛选、酶活性测定、PCR鉴定和SDS-PAGE分析 ,获得了两株产酶活性分别为 14 39583u mL发酵液 (PP N1422 )和14 89083u/mL发酵液 (PP-N1444)的高产工程菌 ,其酶活性分别是出发菌株酶活性 (422u/mL)的 34113倍和 35286倍 ,重组酵母具有很好的遗传稳定性。重组植酸酶在pH值 2.5～3.0和 5.0～5.5时酶活性最高 ,且在pH4.5～6.5之间均有相当高的酶活性 ,最适作用温度为55℃。     

β-葡萄糖苷酶的分离纯化和性质研究

β－葡萄糖苷酶是纤维素酶的重要组分之一,它不仅可水解纤维二糖和寡糖,更可解除纤维二糖对β－１,４－内切葡聚糖酶和外切葡聚糖酶的抑制,提高水解速率和程度．利用ＳｅｐｈａｄｅｘＧ－１５０和ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０层析法从黑曲霉变异株Ｌ－２２中分离提纯了β－葡萄糖苷酶,该酶是由两个分子量相同的亚基组成的二聚体,每个亚基分子量为２０３ｋＤ．该酶最适ｐＨ为４．８,ｐＨ稳定范围在３．６～６．４;最适温度是６０℃,温度稳定范围为４～６０℃;酶分子含糖量为８．３５％．它是一个酸性β－葡萄糖苷水解酶,专一性地水解β－糖苷键．而不水解α－糖苷键,对短链底物表现了相对高的活力．用动力学分析和共价化学修饰方法探讨了与该酶活力有关的必需基团．由ｐＨ对ｌｇＶｍ和ｌｇＶｍ／Ｋｍ的影响,推测出酶活性部位至少有两个可解离基团为酶活性所必需,它们在酶－底物复合物中的ｐＫｅｓ１和ｐＫｅｓ２的值分别为４．０和５．６,在游离酶中的ｐＫ值分别为４．２和５．９．由此可初步判断这两个可解离基团可能为组氨酸和含羧基的氨基酸,它们与酶的催化和底物结合可能有关．     

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。