吸附金(Au3+)的真菌筛选

通过筛选获得3株吸附Au³⁺能力较强的真菌。对其中一株芽枝状枝孢(Cladosporimmcladosporioides AS 3.3995)进行了吸附Au³⁺的条件研究。结果表明,溶液的pH值、温度、时间和Au³⁺的浓度对菌的吸附作用有影响。最适pH为5以下,温度在30—50℃之间。该菌的最大吸附量为140 mgAu³⁺/g(干细胞)。电镜观察表明,Au³⁺在细胞壁的表面上慢慢地还     

Δ12-脂肪酸脱氢酶基因在大肠杆菌中的表达*

把高山被孢霉 (Mortierellaalpina)和深黄被孢霉 (Mortierellaisabellina)的Δ¹²-脂肪酸脱氢酶基因亚克隆到大肠杆菌表达载体pET21a中 ,获得重组表达载体pMACL12和pMI CL12 ,并用氯化钙方法将重组表达载体转化到大肠杆菌BL21 (DE3)中。筛选阳性克隆进行培养 ,然后分离其细胞膜蛋白 ,并构建体外表达体系 ,同时加入外源性底物油酸进行表达。经气相色谱 (GC)分析表明 ,分别有 17.87%和 17     

Ca2+参与水霉菌丝细胞壁修饰的证据

以细胞壁崩溃酶-Driselflse短时间处理水霉(Saprozegma ferax)菌丝,pH5．0时可使原生质从菌丝亚顶端喷出,pH6．0～8．0时则不导致该现象发生;适当浓度EGTA的存在,可提高pH5．0时酶解引起的原生质喷出频率、使pH6．0～8．0时生长菌丝的顶端原生质也喷出、并且喷出多发生在菌丝最顶端;外加CaCl₂．不抑制菌丝顶端原生质的喷出,排除了Ca²⁺抑制酶活性的可能。随后的跟踪观察显示,长时间以缺Ca²⁺培养介质培养菌丝,同样能够导致菌丝顶端原生质喷出。上述研究结果表明,培养介质中Ca²⁺和H⁺对菌丝完整性的维持起调节作用,细胞壁上的Ca²⁺可能参与了水霉菌丝细胞壁物理特性的修饰。     

微生物发酵合成L－缬氨酸－15N

采用含有稳定同位素^１５Ｎ－硫铵为主要氮源的专用发酵培养液配方和相应的工艺条件,在国内首次用微生物直接发酵研制成Ｌ－缬氨酸－^１５Ｎ高丰度精制产品。产品^１５Ｎ丰度９７．６８％,反比原料^１５Ｎ－硫铵丰度下降０．５３％,Ｌ－缬氨酸－^１５Ｎ产酸率最高达４％以上（未校正）。每克^1５Ｎ－硫铵可得到１克以上的Ｌ－缬氨酸－^１５Ｎ（分析值）。提取精制收率平均为８０－９０％（     

木霉GXC产β-葡聚糖酶条件和酶学性质

研究了木霉GXC产 β 葡聚糖酶的条件。结果表明 ,最适产酶碳源为麸皮 ,氮源为硫酸铵 ;产酶的最适条件为 :初始pH为 4 0～ 5 0 ,30℃培养 44h。粗酶液经硫酸铵沉淀、SephadexG 2 5、SephadexG 1 0 0和DEAE SephadexA 50柱层析得到纯β 葡聚糖酶 ,SDS PAGE凝胶电泳显示一条带 ,测得分子量为 35kD。该酶最适反应pH5 0 ,最适反应温度为 60℃ ,在 40℃以下、pH4 0～ 5 0酶活力相对稳定。 5 0mmol L以下的Ca²⁺、Zn²⁺和Fe²⁺,以及 1 0 0mmol L以下的Co²⁺对酶活力有激活作用 ;而Cu²⁺和Fe³⁺具有抑制作用。     

Cu2+对喜树愈伤细胞中喜树碱合成的影响

喜树碱是一种从木本植物喜树(Camptotheca acuminata)中分离得到的抗癌活性物质,通过细胞培养合成喜树碱是喜树碱生产的一条重要途径。研究Cu²⁺对喜树愈伤细胞培养中喜树碱积累的影响,结果表明,在B5培养基中添加0.008mg/mL Cu-Cl²时,对喜树愈伤细胞的生长没有明显影响,但是对喜树愈伤细胞合成喜树碱的促进作用最强,喜树碱含量比未诱导前增加了约30倍。Cu²⁺阻止培养细胞后期过氧化物酶活力的下降,并抑制花青素的生成。与黑暗培养相比,光照条件下添加Cu²⁺更利于喜树愈伤细胞诱导合成喜树碱。     

L－缬氨酸发酵供氧与补料过程控制的研究

高产缬氨酸的北京棒杆菌(corynebacterium pekinense)突变株125菌株,在2．6L自控发酵罐上分批培养结果表明,当发酵中后期DO为零时,产酸量较多。也可用kL。值为指标来调节过程的供氧强度,Kla=90．75h^-1时,产酸量最高。同时比较了恒速连续补加葡萄糖液,当F=3．75g／b时,产酸较高。由此获得了该菌株L一缅氨酸发酵的低供氧与恒速补糖的控制模式。总糖量为16．85％的发酵,可使产酸量达38．2g／L,转化率为22．67％。本文对有关试验结果,进行了发酵动力学的分析和讨论。     

维生素C生产用菌系2980产酸菌基因转移的筛选模型及转座子Tn5诱变

维生素C的生产目前国内广泛采用由产酸菌(Gluconobacter oxydans)与伴生菌(Bacillus megaterium)组成的2980菌系,在2980中G.oxydans单独生长传代困难,其生长和产酸需要B.megaterium参与。以Bacillus subtilis Ki-2-132(pUB110)作为伴生菌与原2980的G.oxydans组合,获得稳定产酸的新菌系。在此基础上建立了适合我国混菌发酵产酸菌外源基因(Kanr)转移的筛选模型。同时报道了以携带有自杀性载体P1：：Tn5的大肠杆菌E.coli W3110为供体菌对G.oxydans进行Tn5诱变的条件和结果。     

用构建的新型BmNPV载体在家蚕高效表达人β-干扰素

构建了家蚕核多角体病毒(Bombyx mori nuclear polyhedrosis virus,BmNPV)新型载体pBm92,该载体将多角体蛋白基因的起始密码ATG改变为ATT,然后在多角体蛋白基因的+12位后连接有5个外源基因的克隆位点。将HulFN-β基因克隆在多角体蛋白基因的+12位后,构建了pBmIFN+12;同时构建了HuIFN－β克隆在－3位后的转移载体pB．mIFN－3。将两种转移载体DNA分别与BmNPV基因组DNA共转染Bm—N细胞。利用重组病毒不产生多角体蛋白的特征,筛选重组病毒。用HuIFN－β基因探针与重组病毒DNA进行杂交鉴定。重组病毒BmIFN+12感染Bm-N细胞,其上清IFN活性为2．0×10⁶Iu／ml,将BmIFN+12注射5龄家蚕虫体,表达水平为5．O×10⁷Iu／ml,是HulFN-β基因克隆在多角体蛋白基因的-3位后获得的重组病毒表达量的2—4倍。构建的新型BmNPv载体能够在家蚕高效地表达HuIFN-β。家蚕虫体生产的rHulFN-β蛋白具有天然HuIFN-β的抗原性。     

胞外Ca2+促进粟酒裂殖酵母细胞增殖作用的研究

研究了胞外Ca²⁺对粟酒裂殖酵母Schizosaccharomyces pombe)细胞增殖的影响。实验结果首次证明胞外Ca²⁺能明显促进粟酒裂殖酵母的增殖,其作用方式主要是缩短了粟酒裂殖酵母的生长延滞期。当起始的接种细胞密度升高至使粟酒裂殖酵母的生长延滞期消失时,外加Ca²⁺的作用也消失。EGTA可抑制粟酒裂殖酵母的细胞增殖,而外加Ca²⁺能够有效消除EGTA的抑制作用,进一步说明胞外Ca²⁺是粟酒裂殖酵母增殖所必需的。此外,外加EGTA除了可延长细胞增殖的延滞期外,还能显著降低指数期细胞分裂的速率以及达到稳定期时培养液中的细胞总数,提示缺Ca²⁺还影响粟酒裂殖酵母细胞的分裂。     

Kinetics of kojic acid fermentation byAspergillus flavus link S44-1 using sucrose as a carbon source under different pH conditions

Kojic acid production byAspergillus flavus strain S44-1 using sucrose as a carbon source was carried out in a 250-mL shake flask and a 2-L stirred tank fermenter. For comparison, production of kojic acid using glucose, fructose and its mixture was also carried out. Kojic acid production in shake flask fermentation was 25.8 g/L using glucose as the sole carbon source, 23.6 g/L with sucrose, and 6.4 g/L from fructose. Reduced kojic acid production (13.5 g/L) was observed when a combination of glucose and fructose was used as a carbon source. The highest production of kojic acid (40.2 g/L) was obtained from 150 g/L sucrose in a 2 L fermenter, while the lowest kojic acid production (10.3 g/L) was seen in fermentation using fructose as the sole carbon source. The experimental data from batch fermentation and resuspended cell system was analysed in order to form the basis for a kinetic model of the process. An unstructured model based on logistic and Luedeking-Piret equations was found suitable to describe the growth, substrate consumption, and efficiency of kojic acid production byA. flavus in batch fermentation using sucrose. From this model, it was found that kojic acid production byA. flavus was not a growth-associated process. Fermentation without pH control (from an initial culture pH of 3.0) showed higher kojic acid production than single-phase pH-controlled fermentation (pH 2.5, 2.75, and 3.0).     

Kinetics and modelling of kojic acid production by Aspergillus flavus Link in batch fermentation and resuspended mycelial system

An unstructured model based on logistic and Luedeking-Piret equations was proposed to describe growth, substrate consumption and kojic acid production by Aspergillus flavus Link strain 44-1 in batch fermentation and also in a resuspended cell system. The model showed that kojic acid production was non-growth associated. The maximum kojic acid and cell concentrations obtained in batch fermentations using the fermenter with optimized dissolved oxygen control (32.5 g/l and 11.8 g/l, respectively) and using a shake-flask (36.5 and 12.3 g/l, respectively) were not significantly different. However, the maximum specific growth rate and a non-growth-associated rate constant for kojic acid formation (n) for batch fermentation using the fermenter (0.085/h and 0.0125 g kojic acid/g cell.h, respectively) were approximately three and two times higher than the values obtained for fermentation using a shake-flask, respectively. Efficient conversion of glucose to kojic acid was achieved in a resuspended pellet or mycelial system, in a solution containing only glucose with citrate buffer at pH 3.5 and at a temperature of 30 °C. The resuspended cell material in the glucose solution was still active in synthesizing kojic acid after prolonged incubation (up to about 600 h). The rate constant of kojic acid production (n) in a resuspended cell system using 100 g glucose/l was almost constant at an average value of 0.011 g kojic acid/g cell.h up to a cell concentration of 19.2 g/l, above which it decreased. A drastic reduction of n was observed at a cell concentration of 26.1 g/l. However, the yield based on glucose consumed (0.45 g/g) was similar for all cell concentrations investigated.     

Studies on Kojic Acid and its Related γ-Pyrone Compounds

The Mannich reaction of kojic acid in both acidic and basic media was studied. Mono-Mannich derivatives (substitution at position 6) were obtained; the reactivity in basic medium was found to be somewhat greater than in acidic medium. Reduction of the Mannich derivatives with zinc dust and acetic acid gave a 6-methyl kojic acid (6-methyl-5-hydroxy-2-hydroxymethyl-γ-pyrone).

Mono-Mannich base of pyromeconic acid was also prepared in a manner similar to that of kojic acid. From this Mannich base, maltol (2-methyl-3-hydroxy-γ-pyrone) was obtained by the reduction with zinc dust and acetic acid.     

Isolation of a kojic acid-producing fungus capable of using starch as a carbon source

A fungal strain (S33-2), able to grow on cooked starch and produce a substantially high level of kojic acid, was isolated from morning glory flower ( Bixa orellana ). The fungus was characterized and identified as Aspergillus flavus. The effect of different types of starch (sago, potato and corn starch) on growth of strain S33-2 and kojic acid production was examined using shake flasks. It was found that strain S33-2 grew well on all types of starch investigated. However, kojic acid production was highest when corn starch was used, with the maximum kojic acid obtained being comparable to fermentation using glucose. The highest kojic acid production (19·2 g l⁻¹) was obtained when 75 g l⁻¹ corn starch was used. This gave a yield, based on starch consumed, and an overall productivity of 0·256 g g⁻¹ and 0·04 g l⁻¹ h⁻¹, respectively.     

Kojic acid-tripeptide amide as a new tyrosinase inhibitor

Twenty two kojic acid-tripeptide amides were prepared using a solid-phase Fmoc/tBu strategy with Rink Amide SURE(R) resin. To effectively obtain kojic acid-tripeptide amide conjugates, the coupling conditions of kojic acid to the tripeptide on the resin were optimized. The tyrosinase inhibitory activity of kojic acid-tripeptide amides and the effect of the amino acid sequence on the activity were compared with those of kojic acid-tripeptide acids. The stability of kojic acid-tripeptide amides were then compared with those of kojic acid and kojic acid-tripeptides acids. As a consequence, kojic acid-FWY-NH(2) proved to be the best compound, with the highest inhibitory activity, which was maintained over different storage times under various temperatures and pHs.     

Combining induced mutation and protoplasting for strain improvement of Aspergillus oryzae for kojic acid production

By combining induced mutation, using NTG and UV irradiation, and protoplasting of a wild type strain of Aspergillus oryzae ATCC 22788, a hyper-producing strain was obtained that accumulated 41 g kojic acid l(-1) in shake-flasks, which was 100-fold higher than that in the wild type strains. Similar production of kojic acid was obtained in 5 l stirred-tank fermentations.     

曲酸高产菌株的诱变选育

以紫外线和氮离子注入作为诱变手段对曲酸产生菌黄曲霉CICC2242进行诱变处理,筛选出一株高产菌株N83。曲酸产量由初始的12．4g／L提高到19．4g／L,提高了56．5％;遗传稳定性实验结果表明,N83遗传性状稳定良好。该结果表明氮离子注入诱变是获得高产曲酸的有效途径。     

Solid-phase synthesis of kojic acid-tripeptides and their tyrosinase inhibitory activity, storage stability, and toxicity

A small library of kojic acid-tripeptides (Ko-X1X2X3) was prepared by solid-phase parallel synthesis and assayed to evaluate their tyrosinase inhibitory activity. Most of the kojic acid-tripeptides showed better activities than kojic acid. Kojic acid-FWY was the best compound, and it exhibited 100-fold tyrosinase inhibitory activity compared with kojic acid. In addition, their storage stabilities were approximately 15 times higher and their toxicity was lower than that of kojic acid.     

Improvement of kojic acid production in Aspergillus oryzae B008 mutant strain and its uses in fermentation of concentrated corn stalk hydrolysate

A strain designated M866, producing kojic acid with a high yield, was obtained by combining induced mutation using ion beam implantation and ethyl methane sulfonate treatment of a wild type strain of Aspergillus oryzae B008. The amount of kojic acid produced by the strain M866 in a shaking flask was 40.2 g/L from 100 g/L of glucose, which was 1.7 times higher than that produced by wild strain (23.58 g/L). When the mixture of glucose and xylose was used as carbon source, the resulting kojic acid production was raised with the increasing of glucose ratios in the mixture. With concentrations of glucose at 75 g/L and xylose at 25 g/L mixed in the medium, the production of kojic acid reached 90.8 %, which was slightly lower than with glucose as the sole source of carbon. In addition, the kojic acid fermentation of the concentrated hydrolysate from corn stalk was also investigated in this study, the maximum concentration of kojic acid accumulated at the end of the fermentation was 33.1 g/L and this represents the yield based on reducing sugar consumed and the overall productivity of 0.36 g/g and 0.17 g/L/h, respectively.     

Kojyl thioether derivatives having both tyrosinase inhibitory and anti-inflammatory properties

This study was conducted to examine the tyrosinase inhibitory and anti-inflammatory activities of kojic acid derivatives. A series of kojic acid derivatives containing thioether, sulfoxide, and sulfone linkages were synthesized. In the tyrosinase assay, kojyl thioether derivatives containing appropriate lipophilic alkyl chains (pentane, hexane, and cyclohexane) showed potent inhibitory activity. However, sulfoxides and sulfones exhibited decreased activity. Similar experimental results were obtained with inhibitory activities of NO production being induced by LPS. The presence of thioether linkage and appropriated lipophilic acid moiety was critical for the tyrosinase inhibitory and anti-inflammatory activities.