卷枝毛霉Δ6-脂肪酸脱氢酶基因的克隆及在酿酒酵母中的高效表达

为获得产高γ 亚麻酸的酿酒酵母工程菌株,应用RT PCR技术,从卷枝毛霉中扩增出△6 脂肪酸脱氢酶 基因,亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES2.0,在大肠杆菌中筛选到含有目的基因的重组质粒 pYES412,用醋酸锂方法转化到酿酒酵母缺陷型菌株INVScI中,在SC ura合成培养基中筛选到转化酵母,在合适 的培养基及培养条件下,加入外源底物亚油酸,经半乳糖诱导后,收集菌体,通过气相色谱对转化酵母进行脂肪酸 色谱分析,结果表明:γ 亚麻酸占总脂肪的50.07%。迄今为止,这是国内外△6 脂肪酸脱氢酶基因在酿酒酵母表 达量最高的报道。     

总状毛霉凝乳酶的研制及初步应用

从１７株产凝乳酶的霉菌中初筛得到产酶量较高的菌株,再进行^６０Ｃｏ－γ射线诱变,得到产酶量较初筛菌株提高８０％的诱变菌株─—总状毛霉Ｒ１３２。确定了诱变株Ｒ１３２的液体发酵产酶工艺及提取纯化路线,获得适于应用的部分纯化酶液样品。用该酶制作的Ａｄａｍ干酪与车间生产样品对照进行成熟期理化性质分析的结果基本相同。     

Sterol composition of chitosomes from yeast cells of Mucor rouxii: Comparison with whole cells

Abstract The free sterol composition of chitosomes and whole yeast cells of Mucor rouxii was determined by gas-liquid chromatography (GLC). The results obtained showed that, in contrast to whole cells, chitosomes are particularly rich in ergosterol: 87% vs. 29% of total free sterols. Three other sterols were present in varying amounts in both chitosomes and whole cells. These were identified by GLC as cholesterol, stigmasterol and sitosterol. These results indicate that the yeast cells preferentially accumulate ergosterol in chitosomes providing these microvesicles with a unique composition that may be important for the structure of the organelle.     

Stress-induced morphological and physiological changes in γ-linolenic acid production by Mucor fragilis in batch and continuous cultures

Batch and continuous cultures conditions were studied in order to increase γ-linolenic acid production by Mucor fragilis CCMI 142, in response to the presence of ethanol. Continuous cultures were used to add ethanol pulses to steady state pellet cultures. It was demonstrated that pellet size, which allowed homogeneous fungal cultures, can be obtained by means of pH adjustment, thus enabling steady state continuous growth at 2.90±0.05.

The 5 and 2% (v/v) ethanol pulses induced hyphal morphological changes with arthrospore formation. A 1% (v/v) pulse of ethanol did not immediately affect growth, but induced morphological changes, which led to autolysis at the pellet core. A 0.5% (v/v) pulse of ethanol did not affect neither the morphology nor the physiology of the microorganism to any significant extent. The 0.5 and 1% (v/v) ethanol pulses resulted in an increase in the proportion of γ-linolenic acid production up to 11%. Data from batch cultures showed a higher enhancement of ethanol, attaining 30% of γ-linolenic acid.

The increase of γ-linolenic acid content observed in batch and continuous conditions appears to be a response associated with stress induced by the ethanol which seems to be of value as an industrial medium component.     

云南香格里拉叉襀科昆虫病原菌的鉴定

叉襀科Nemouroidea是襀翅目中数量最多的一个科,叉襀科昆虫也是水生生态系统环境监测的重要指示生物.为明确云南香格里拉叉襀科稚虫自然病死虫体上的病原菌并研究该病原菌对叉襀科稚虫的致病性,本研究利用常规组织分离法分离云南香格里拉叉襀科稚虫病原菌,结合形态学与分子生物学方法对两株病原菌进行鉴定,利用柯赫法则及二项式回...     

Integrative transformation by homologous recombination in the zygomycete Mucor circinelloides

Summary Transformation of a Mucor circinelloides Leu^– strain with the plasmid pAD45, harbouring the wild-type allele (leuA⁺) and a chymosin gene, led to the identification of mitotically stable transformants after one to three vegetative growth cycles on non-selective medium. Southern analysis of the stable transformed strains demonstrated that the vector is integrated, as an intact molecule, into the resident Mucor leuA locus. Retransformation of Escherichia coli with genomic DNA restricted with enzymes having no or only a single recognition site within the inserted sequence did not permit isolation of plasmids or fragments carrying the leuA or chymosin gene.     

In vitro efficacy of some fungicides on mycelial growth of Alternaria alternata and Mucor pyriformis

Various chemical fungicides, systemic and non-systemic, were tested against fruit rot pathogens viz. Alternaria alternata and Mucor pyriformis for the evaluation of inhibition of mycelial growth. In A. alternata, among the systemic fungicides used, hexaconozole showed highest inhibition of mycelial growth followed by carbendazim and least effective was myclobutanil. While in M. pyriformis, hexaconozole showed highest inhibition and least effective was bitertanol. Among the non-systemic fungicides tested in both A. alternata and M. pyriformis, mancozeb showed highest inhibition of mycelial growth followed by capton and the least inhibition was shown by zineb.     

On the activity loss of hydrolases in organic solvents: I. Rapid loss of activity of a variety of enzymes and formulations in a range of organic solvents

Dehydrated enzyme powders have been used extensively as suspensions in organic solvents to catalyze synthetic reactions. Prolonged enzyme activity is necessary to make such applications commercially successful. However, it has recently become evident that the stability and thus activity of many enzymes is compromised in organic solvents. Herein we explore the stability of various hydrolases (i.e., lipases from Mucor meihei and Candida rugosa, -chymotrypsin, subtilisin Carlsberg, and pig-liver esterase) and various formulations (lyophilized powder, cross-linked enzyme crystals, poly(ethylene glycol)-enzyme conjugates) in different organic solvents. The results show a roughly exponential activity decrease for all enzymes and formulations studied after exposure to organic solvents. Inactivation was observed independent of the enzyme, formulation details, and the solvent. In addition, no relationship was found between the magnitude of inactivation and the value of initial activity. Thus, quite active formulations lost their activity as quickly as less active formulations. The estimated half-times (t_1/2) for all enzymes and preparations ranged from 1.8 h for subtilisin C. co-lyophilized with methyl-β-cyclodextrin to 61.6 h for the most stable poly(ethylene glycol)--chymotrypsin preparation. The data here presented indicates that the inactivation is likely not related to changes in enzyme structure and dynamics.     

Microbial metabolism. Part 13. Metabolites of hesperetin

The fungal culture, Mucor ramannianus (ATCC 2628) transformed hesperitin (1) to four metabolites: 4'-methoxy-5,7,8,3'-tetrahydroxyflavanone (8-hydroxyhesperetin) (2), 5,7,3',4'-tetrahydroxyflavanone (eriodictyol) (3), 4'-methoxy-5,3'-dihydroxyflavanone 7-sulfate (hesperetin 7-sulfate) (4) and 5,7,3'-trihydroxyflavanone 4'-O-α-quinovopyranoside (eriodictyol 4'-O-α-quinovopyranoside) (5). The structures were established by spectroscopic methods.     

卷枝毛霉孢子原生质体的制备与再生

为了构建高产γ-亚麻酸的卷枝毛霉稳定遗传转化体系,利用酶解法对卷枝毛霉(Mucor circinelloides sp.)EIM-10的孢子进行原生质体制备。研究酶液组成、渗透压稳定剂、酶解温度、酶解时间等对卷枝毛霉孢子原生质体形成和再生的影响,建立了制备卷枝毛霉孢子原生质体的最适条件:1%纤维素酶和2%溶壁酶为酶解体系,0.5mol/L NaCl作为渗透压稳定剂,酶解温度32℃,酶解时间2.5 h,再生培养基为0.5 mol/L NaCl高渗培养基。用双层平板培养法进行原生质体再生,在此条件下原生质体的形成量为1.2×106个/mL,再生率为70.5%。