Effect of hydrolytic and oxidative enzymes produced by solid-state fermentation on greige linen fabric

Solid state fermentation (SSF) was applied for production of fungal enzyme preparations from Phanerochaete chrysosporium, Aspergillus oryzae, Aspergillus giganteus and Trichoderma virens using cotton seed-coat fragment waste as a carbon source and enzyme inducer. Lignin-holocellulose matrix of cotton seed coat fragment proved to be effective in inducing production of ligninolytic, cellulolytic and xylanolytic enzymes in solid-state fermentation. The effect of the enzymes produced by SSF on greige linen fabric is discussed and evaluated. In the first experiment the hydrolytic and accompanying oxidative enzymes in the buffer extract of the whole SSF cultures were used for fabric treatment. In the second trial, the enzymes produced in situ (whole SSF material—mixture of fungal biomass, residual substrate and enzymes) were used for the treatment. Weight loss, reducing sugar liberation and removal of colouring materials were measured. The results showed that at equal enzyme charges the intact SSF materials were more efficient than the enzyme extracts. Of the six strains evaluated, Ph. chrysosporium VKM F-1767 was the most effective in removing colouring matters from greige linen fabric.     

Kinetic Analysis of Aspergillus oryzae Cultivations on Starch

Cultivation of Aspergillus oryzae on starch is described as a combination of two rate processes: Starch hydrolysis and the cellular activities of the fungi including growth, enzyme production and maintenance. Kinetic models are presented to describe growth, enzyme production, starch hydrolysis and uptake of the hydrolysis products. Numerical values of the model parameters indicated that the rate controlling step of A. oryzae growth on starch was not starch hydrolysis, but the substrate uptake process. Glucose was one of the starch hydrolysis products. About 35% of the substrate consumed for biomass synthesis was glucose. Its accumulation in the medium did not cause repression of the starch hydrolysing enzymes. Steady state starch hydrolysis rates increased with initial starch concentration in the medium. Starch hydrolysing enzymes of A. oryzae have extensive industrial uses. This study may help in a more detailed understanding of the kinetic aspects of the production of these enzymes.     

A two-step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus

A two-step bioconversion process of ferulic acid to vanillin was elaborated combining two filamentous fungi, Aspergillus niger and Pycnoporus cinnabarinus. In the first step, A. niger transformed ferulic acid to vanillic acid and in the second step vanillic acid was reduced to vanillin by P. cinnabarinus. Ferulic acid metabolism by A. niger occurred essentially via the propenoic chain degradation to lead to vanillic acid, which was subsequently decarboxylated to methoxyhydroquinone. In 3-day-old cultures of P. cinnabarinus supplied with vanillic-acid-enriched culture medium from A. niger as precursor source, vanillin was successfully produced. In order to improve the yields of the process, sequential additions of precursors were performed. Vanillic acid production by A. niger from ferulic acid reached 920 mg l⁻¹ with a molar yield of 88% and vanillin production by P. cinnabarinus from vanillic acid attained 237 mg l ⁻¹ with a molar yield of 22%. However, the vanillic acid oxidative system producing methoxyhydroquinone was predominant in P. cinnabarinus cultures, which explained the relatively low level in vanillin.     

Structural features of the glycogen branching enzyme encoding genes from aspergilli

A maltose binding protein, p78, was purified to homogeneity from Aspergillus nidulans by a single column chromatography step on cross-linked amylose. The partial amino acid sequence was highly homologous to the glycogen branching enzymes (GBEs) of human and yeast, and p78 did show branching enzyme activity. The genomic gene and its cDNA encoding GBE (p78) were isolated from the A. nidulans genomic and cDNA libraries. Furthermore, a cDNA encoding A. oryzae GBE was entirely sequenced. A. nidulans GBE shared overall and significant amino acid sequence identity with GBEs from A. oryzae (83.9%), Saccharomyces cerevisiae (61.1%) and human (63.0%), and with starch branching enzymes from green plants (55–56%).     

Cellobiohydrolase I (Cel7A) from Trichoderma reesei has chitosanase activity

Cellobiohydrolase CBH I (Cel7A) from the filamentous fungus Trichoderma reesei (TrCBHI), which is a member of glycoside hydrolase family (GHF) 7, was expressed in Aspergillus oryzae. We found that the recombinant enzyme showed significant chitosanase activity, as well as cellulase activity, and acted in an endo-type manner on soluble polymeric substrate. Furthermore, another GHF7 CBH I from Aspergillus aculeatus (AaCBHI) expressed in A. oryzae also had chitosanase activity, while endoglucanase EG I (Cel7B) from T. reesei had no activity towards chitosan. To our knowledge, this is the first report of GHF7 enzymes possessing chitosanase activity.     

Enzymes and chelating agent in cotton pretreatment

Desized cotton fabric and cotton seed-coat fragments (impurities) have been treated with commercial cellulase (Celluclast 1.5 L), hemicellulase–pectinase (Viscozyme 120 L) and xylanase (Pulpzyme HC) enzymes. Seed-coat fragments hydrolyzed much faster than the cotton fabric itself. This relative difference in hydrolysis rates makes possible a direct enzymatic removal of seed-coat fragments from desized cotton fabric. Addition of chelating agents such as ethylenediamine-tetra-acetic acid (EDTA) markedly enhanced the directed enzyme action. Pretreatments carried out in acidic solution at pH 5 increased the lightness of seed-coat fragments, contrary to the samples treated in neutral medium at pH 7. Alkaline scouring resulted in darker seed-coat fragments except for the samples pretreated with Pulpzyme HC plus EDTA. This effect is similar to that observed in the biobleaching process in pulp and paper industry.     

我国黄曲霉遗传多样性与产毒特性

黄曲霉(Aspergillus flavus)是一种广泛分布的腐生真菌, 是黄曲霉毒素B (aflatoxin B, AFB)和圆弧偶氮酸(cyclopiazonic acid, CPA)的主要产生菌, 也是动植物的条件致病菌。全球的玉米、花生和棉籽均不同程度地遭到黄曲霉及其毒素的污染。黄曲霉菌株间在形态学、遗传学和产毒特性上变异较大, 且其居群遗传结构也尚不明确。为了揭示黄曲霉居群遗传结构及其产毒素特性的规律, 本研究选取了从我国26省区(包括大小兴安岭)不同环境中分离的黄曲霉88株, 结合模式菌株和国际权威菌株9株, 基于钙调蛋白基因(CaM)和β-微管蛋白基因(benA)进行多基因序列分型(multi-locus sequence typing, MLST), 使用MEGA 6.0和Structure 2.3.4软件进行系统发育学分析和居群结构推导, 并结合菌株的产毒特性(AFB和CPA)进行比较分析。结果显示本研究的97株黄曲霉可分为3个居群, 即黄曲霉居群I、黄曲霉居群II和米曲霉居群, 该97株黄曲霉共有17个序列型(sequence type, ST), 其中我国的88株菌分布于15个序列型。米曲霉居群均不产AFB, 黄曲霉居群I和II的菌株绝大多数都产AFB和CPA, 其产毒特性只具有菌株特异性, 与居群和序列型无关。黄曲霉菌株产毒特性与地理分布或农作物类型间存在一定关系。我国东北玉米产区、西北干旱棉花产区和南方花生产区的黄曲霉居群I和II菌株均产AFB和CPA, 我国青海可可西里和四川阿坝地区的黄曲霉仅产CPA而不产AFB, 不产AFB的米曲霉居群大部分来自我国气候和地理环境多样的华北地区, 该地区也是我国农村传统酿造黄豆酱的地区。     

Oligosaccharide and alkyl β-galactopyranoside synthesis from lactose with Caldocellum saccharolyticum β-glycosidase

The synthetic utility of the thermostable β-glycosidase from Caldocellum saccharolyticum was investigated. The ability of the enzyme to catalyze oligosaccharide and β-galactopyranoside synthesis from lactose was compared with that of the readily commercially available, moderately thermostable β-galactosidase (β- -galactoside galactohydrolase, EC 3.2.1.23) from Aspergillus oryzae. Generally, the C. saccharolyticum enzyme showed significantly greater resistance to inactivation by heat and organic solvent and better yields of product. Although the A. oryzae enzyme gave better oligosaccharide yields at lower lactose concentrations, at higher concentrations (above 50% w/w) the C. saccharolyticum enzyme was significantly better, yielding a sugar mixture containing 42% by weight of tri- plus tetra-saccharides, from a 70% w/w lactose solution, compared with 31% by weight of oligosaccharides with the A. oryzae enzyme. In ethyl galactoside synthesis from ethanol and lactose, neither enzyme appeared to hydrolyze the product to any great extent. Under all conditions tested, the product yield did not peak, even at long reaction times, when most of the lactose had been consumed. The C. saccharolyticum enzyme, however, gave slightly higher product yields and could be used at higher ethanol concentrations without serious loss of activity.     

Ligninolytic and Nonligninolytic Mineralization of Trinitrotoluene by Several White Rot Basidiomycetes

The explosive 2,4,6-trinitrotoluene (TNT) is considered a toxic environmental pollutant that contaminates the soil and ground water. The white rot fungus Phanerochaete chrysosporium is well known for the degradation of TNT under ligninolytic condition. Very few, if any, studies have been done using other white rot fungi. In this study four fungal species, namely, P. chrysosporium, Kuehneromyces mutabilis, Hypholoma fasciculare, and Phlebia radiata, were used to investigate TNT degradation. All fungi were grown under ligninolytic (low-nitrogen) and nonligninolytic (high-nitrogen) conditions containing 25 parts per million (ppm) (0.11 mM) of TNT. Analysis by high-performance liquid chromatography (HPLC) showed biotransformation of TNT under both conditions. Complete degradation occurred under ligninolytic conditions (peroxidase enzymes were present) by P. chrysosporium and P. radiata. A nitrite release assay at 6 days indicated the denitrifying abilities of all the tested varieties of white rot fungi. For both ligninolytic and non-ligninolytic conditions, mass-balance studies showed biotransformation of 0.5 μ Ci ¹⁴C-labeled TNT with pregrown mycelial pellets of all fungal species, in which 5% to 15% of the TNT was converted to CO₂. These studies show that TNT may be degraded by several other species of white rot fungi and provided additional information on the biodegradation of nitroaromatic compounds in the environment.     

Hydrolysis of diethyl diferulates by a tannase from Aspergillus oryzae

Diferulic acid forms cross-links in naturally occurring plant cell wall polymers such as arabinoxylans and pectins. We have used model ethyl esterified substrates to find enzymes able to break these cross-links. A tannase from Aspergillus oryzae exhibited esterase activity on several synthetic ethyl esterified diferulates. The efficiency of this esterase activity on most diferulates is low compared to that of a cinnamoyl esterase, FAEA, from Aspergillus niger. Of the diferulate substrates assayed, tannase was most efficient at hydrolysing the first ester bond of the 5–5- type of dimer. Importantly and unlike the cinnamoyl esterase, tannase from A. oryzae is able to hydrolyse both ester bonds from the 8–5-benzofuran dimer, thus forming the corresponding free acid product. These results suggest that tannases may contribute to plant cell wall degradation by cleaving some of the cross-links existing between cell wall polymers.     

Deracemization of aryl ethanols and reduction of acetophenones by whole fungal cells of Aspergillus terreus CCT 4083, A. terreus CCT 3320 and Rhizopus oryzae CCT 4964

The enantioselective deracemization of a number of p-substituted aryl ethanols and the reduction of p-substituted acetophenones were carried out with whole fungal cells of Aspergillus terreus CCT 4083, A. terreus CCT 3320 and Rhizopus oryzae CCT 4964 giving the corresponding alcohols in enantiomeric excesses up to >99%.     

Hydrophilization of immobilized model enzymes suggests a widely applicable method for enhancing protein stability in polar organic co-solvents

β-Galactosidases from Escherichia coli, Kluyveromyces lactis and Aspergillus oryzae were used to characterize the potential for enzyme stabilization of a two-step strategy: (i) immobilization on glutaraldehyde-agarose (Glut90), (ii) subsequent generation of a hydrophilic nano-environment by reaction with polyaldehyde-dextran polymer (Glut90-Pal), followed by polyamine-dextran polymer (Glut90-Pal-Pam). The derivatives were characterized by kinetics parameters, co-solvent (ethanol and acetone) and temperature stability. Hydrophilization achieved important co-solvent stabilization in all cases. One of the most remarkable results obtained was a 25-fold increase in the half-life of the A. oryzae Glut90-Pal-Pam derivative in 50% (v/v) acetone. Stabilization achieved in very drastic co-solvent concentrations is directly related to the hydrophilization of the nano-environment. The K_M values show that the hydrophilic shell appears to behave as an open structure and may create a “partition effect” that protects the enzymes from denaturation. These results show the potential of hydrophilization for building up additional stabilization of immobilized enzymes which would make possible the development of industrial applications.     

Non-Ligninolytic TNT Mineralization in Contaminated Soil by Phanerochaete chrysosporium

The explosive 2,4,6-trinitrotoluene (TNT) is widely used and results in widespread soil contamination. The white-rot fungus Phanerochaete chrysosporium has been shown to degrade TNT, using the peroxidase enzyme. In this study, we report peroxidase-independent degradation of TNT by non-ligninolytic P. chrysosporium. Significant disappearance of TNT from highly contaminated soil using P. chrysosporium has been observed. Soil highly contaminated with TNT (2270 ppm [10 mM]) was diluted to 100 ppm (0.44 mM) with malt extract medium. Pregrown (48 hours) mycelial pellets of P. chrysosporium were added in 100 mL malt extract medium and incubated in Gledhill flasks. Analysis by high-performance liquid chromatography (HPLC) was conducted on soil extracts at specific time points to estimate the disappearance of TNT from contaminated soil incubated with P. chrysosporium. When the pregrown mycelial pellets were added, TNT disappeared within 48 hours. The dissolved concentration of 2-amino-4,6-dinitrotoluene (2Am-DNT) increased up to the third day, then declined before its final disappearance by day 10. Results show that the pregrown mycelial pellets of P. chrysosporium mineralized up to 17.3±6.3% [¹⁴C]-TNT within 30 days.     

Asymmetric synthesis of arylselenoalcohols by means of the reduction of organoseleno acetophenones by whole fungal cells

A series of novel organoseleno acetophenones (3a–f) have been synthesized. The microbial reduction of the seleno ketones (3) has been evaluated using whole cells of Rhizopus oryzae CCT 4964, Aspergillus terreus CCT 3320, A. terreus CCT 4083 and Emericella nidulans CCT 3119. These microorganisms showed Prelog and anti-Prelog stereoselectivity, leading to the arylselenoalcohols in moderate to high enantiomeric excesses. The organoselenium compounds were compatible with the biocatalytic conditions employed.     

Functional characterization of a salt- and thermotolerant glutaminase from Lactobacillus rhamnosus

The deamination of glutamine is a crucial step in the production of enzymatically hydrolyzed plant proteins to reach high glutamic acid yields. The required glutaminase activity usually is provided by addition of technical enzymes or by in situ generation from fungi, yeast or bacteria (i.e. Aspergillus oryzae in soy sauce production). We screened food-grade Lactobacilli for potential glutaminase activity and selected the enzyme found in Lactobacillus rhamnosus for further characterization. Glutaminase from L. rhamnosus was induced by growing the microorganism on hydrolyzed wheat gluten, a glutamine-rich protein source. Glutamine deaminating activity (glutaminase, EC 3.5.1.2) was found to be membrane-bound and lost its activity gradually upon solubilization. Functional studies of the glutaminase showed an optimal working pH of 7.0 and maximum activity at 50 °C. High salt-tolerance of the enzyme was observed, i.e. the presence of 5% (w/v) salt increased glutaminase activity almost two-fold and 90% of the initial activity still remained at 15% (w/v) salt. The glutaminase activity showed typical Michaelis–Menten behavior with an affinity constant K_m of 4.8±0.4 mM for glutamine and a V_max of 101±2 U/l.     

Production of tannase from Aspergillus ruber under solid-state fermentation using jamun (Syzygium cumini) leaves

Tannase producing fungal strains were isolated from different locations including garbages, forests and orchards, etc. The strain giving maximum enzyme yield was identified to be Aspergillus ruber. Enzyme production was studied under solid state fermentation using different tannin rich substrates like ber leaves (Zyzyphus mauritiana), jamun leaves (Syzygium cumini), amla leaves (Phyllanthus emblica) and jawar leaves (Sorghum vulgaris). Jamun leaves were found to be the best substrate for enzyme production under solid-state fermentation (SSF). In SSF with jamun leaves, the maximum production of tannase was found to be at 30 °C after 96 h of incubation. Tap water was found to be the best moistening agent, with pH 5.5 in ratio of 1:2 (w/v) with substrate. Addition of carbon and nitrogen sources to the medium did not increase tannase production. Under optimum conditions as standardized here, the enzyme production was 69 U/g dry substrate. This is the first report on production of tannase by A. ruber, giving higher yield under SSF with agro-waste as the substrate.     

Induction of tannin acyl hydrolase (EC 3.1.1.20) activity in some members of fungi imperfecti

The potential of gallotannin, methyl gallate, gallic acid, and pyrogallol to induce tannin acyl hydrolase (EC 3.1.1.20) activity in Aspergillus niger, Aspergillus fischerii, Fusarium solani, and Trichoderma viride has been investigated. The maximum induction ratios recorded were A. fischerii (26.7), F. solani (26.1), and T. viride (40.7) when the fungi were induced with gallotannin, gallic acid, and methyl gallate, respectively. A. niger was devoid of basal enzyme activity. The gallotannin tolerance limits for A. niger, A. fischerii, F. solani, and T. viride were determined by progressively increasing the gallotannin concentration and were found to be 20, 4, 3, and 3%, respectively. F. solanii induced with 3% gallotannin has been recommended for the production of tannase enzyme.     

Release of ferulic acid from agroindustrial by-products by the cell wall-degrading enzymes produced by Aspergillus niger I-1472

Aspergillus niger I-1472 was grown on sugar beet pulp to produce cell wall polysaccharide-degrading enzymes, including feruloyl esterases. Compared to enzymatic activities measured in commercially available mixtures previously used for the release of ferulic acid, the A. niger enzymes were more various. These enzymes were tested to release ferulic acid from sugar beet pulp, maize bran, or autoclaved maize bran. They were as efficient as the commercial mixture to release ferulic acid from sugar beet pulp. On the other hand, they were much more efficient to release ferulic acid from maize bran after autoclaving pretreatment, as 95% of ferulic acid ester were solubilized. Thus, A. niger enzymes exhibited a high interest in the release of ferulic acid from various agro-industrial by-products.     

黄曲霉毒素生物合成的遗传调控研究进展

黄曲霉毒素是一类具有较强毒性和致癌力的次级代谢产物,在小麦、水稻、玉米和花生等多种粮食、油料、饲料和食品中检出率均比较高。因此,黄曲霉毒素不仅给人和动物的健康造成极其严重的威胁,而且也给食品和饲料等行业造成了巨大的经济损失。黄曲霉毒素主要由黄曲霉和寄生曲霉产生。自上个世纪60年代首次发现黄曲霉毒素以来,研究者在黄曲霉毒素合成途径、降解、合成机制和致病机理等方面做了大量研究。本文主要综述近年来国内外以黄曲霉为对象的黄曲霉毒素合成的遗传调控机制研究进展。从转录调控、蛋白翻译后修饰、信号转导途径、参与生长发育和形态建成的蛋白和其他酶等方面对黄曲霉毒素合成机制展开综述,为今后进一步深入系统研究黄曲霉毒素合成机制奠定基础,同时为制定防治黄曲霉及其毒素的策略提供理论基础。