Induction of β-N-acetylhexosaminidase in Aspergillus oryzae

Summary Extracellular -N-acetylhexosaminidase in basic specific activity 1.5 U/mg protein was induced 15 – 35 times (up to 50 U/mg protein) by mixture of chitooligomers (crude chitin hydrolysate), 10 – 20 times (20 – 30 U/mg protein) by N-acetylglucosamine, and 10 times (14 U/mg protein) by chitosan in Aspergillus oryzae. Addition of NaCl (15 – 23 g/l) to the cultivation medium enhanced the induction in 10 – 20 %.     

Induction of β-oxidation enzymes and microbody proliferation in Aspergillus nidulans

Aspergillus nidulans is able to grow on oleic acid as sole carbon source. Characterization of the oleate-induced β-oxidation pathway showed the presence of the two enzyme activities involved in the first step of this catabolic system: acyl-CoA oxidase and acyl-CoA dehydrogenase. After isopicnic centrifugation in a linear sucrose gradient, microbodies (peroxisomes) housing the β-oxidation enzymes, isocitrate lyase and catalase were clearly resolved from the mitochondrial fraction, which contained fumarase. Growth on oleic acid was associated with the development of many microbodies that were scattered throughout the cytoplasm of the cells. These microbodies (peroxisomes) were round to elongated, made up 6% of the cytoplasmic volume, and were characterized by the presence of catalase. The β-oxidation pathway was also induced in acetate-grown cells, although at lower levels; these cells lacked acyl-CoA oxidase activity. Nevertheless, growth on acetate did not cause a massive proliferation of microbodies in A. nidulans. Received: 8 March 1996 / Accepted: 5 August 1996     

Induction of xylanase and β-xylosidase in Cellulomonas flavigena growing on different carbon sources

When Cellulomonas flavigena CDBB-531 was grown on glucose, xylose, glycerol, solka floc, sugarcane bagasse or xylan, xylanase activity was found only in the fermentation broth, while -xylosidase activity was always associated with the cells. Both enzymes were inducible, sugar-cane bagasse was the best inducer, solka floc and avicel were moderately good, while xylan was poor. A synergistic effect on xylanase and -xylosidase synthesis was observed when cellulose and hemicellulose were used together as carbon sources. When this strain was grown on glucose, cellobiose, arabinose or xylose, only low levels of both enzymes were detected. These results indicate that xylanase and -xylosidase were carbon-source-repressed by readily metabolizable substrates. The effect of glycerol on enzymes that were already induced was studied. The addition of glycerol caused a significant decrease in the levels of xylanases, while -xylosidase activity remained unchanged.     

The production of β-glucosidase in shake-flasks by Aspergillus wentii

Studies in shake-flasks showed that Aspergillus wentii produces the maximum activity of β-glucosidase among the cultures tested. The activity against cellobiose was about 2–3 fold that against 4NPG. Aspergillus wentii produced a maximum activity of 16.5 U/ml in 14 days on malt extract. It also produced a comparable amount on other simple soluble sugars, which indicates that it is constitutive and does not require an inducer. Peptone was found to the best nitrogen source for β-glucosidase production. Optimum C/N ratio was found to be 7.3. Phosphate, magnesium and trace metals did not play significant roles in the production of β-glucosidase when they were used with malt extract as a carbon source. An inoculum of 6% (v/v) of 20-h-old culture grown on malt extract produced the maxium β-glucosidase activity.     

Production of β-glucosidase by Aspergillus terreus

The production of -glucosidase by Aspergillus terreus was investigated in liquid shake cultures. Enzyme production was maximum on the 7th day of growth (2.18 U/ml) with the initial pH of the medium in the range of 4.0–5.5. Cellulose (Sigmacell Type 100) at 1.0% (wt/vol) gave maximum -glucosidase activity among the various soluble and insoluble carbon sources tested. Potassium nitrate was a suitable nitrogen source for enzyme production. Triton X-100 at 0.15% (vol/vol) increased the enzyme levels of A. terreus. The test fungal strain showed an ability to ferment glucose to ethanol.     

Isolation of mutants of Aspergillus awamori with enhanced production of extracellular xylanase and β-xylosidase

Plate screening tests were designed for the selection and isolation of mutant strains of the fungus Aspergillus awamori CMI 142717 showing over-production and constitutive synthesis of xylanase and -xylosidase. Following mutation by N-methyl-N-nitro-N-nitrosoguanidine, nitrous acid and UV (254 nm), two generations of mutants were isolated and cultured in shake fiasks containing glucose, ball-milled oat straw or oat speit xylan as carbon source. Growth of a number of selected mutants in shake flask culture on medium containing oat spelt xylan produced the highest titres of xylanase and -xylosidase. Thus, xylanase producton by mutant AANTG43 was 132 U/ml when the Somogyl-Nelson (alkaline copper) method of measuring reducing sugar released was used, or 1160 U/ml using the dinitrosalicylic acid method of reducing sugar analysis. These values were 8-fold higher than those produced by the wild type. A 20-fold improvement in -xylosidase production was produced by mutant AANO19 (3.51 U/ml). The titres for these two enzyme activities are the highest recorded so far in the literature. Mutant AANTG43 also produced high levels of xylanase (49.8 U/ml) in submerged culture in a fermenter and showed a substantial improvement in the overall productivity of enzyme compared to the wild type strain.The authors are with the Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, UK.     

Fed-batch biotransformation of β-ionone by Aspergillus niger

Aspergillus niger IFO 8541 was found to be an efficient biocatalyst for the biotransformation of -ionone into hydroxy and oxo derivatives. The reaction had to be carried out with an inoculum made of about 4 × 10⁷ fresh spores/l and with a preliminary growth period giving at least 3 g/l biomass. The fungus developed in the form of pellets when cultivated as free mycelium; entrapment of the microorganism in calcium alginate beads was an efficient way to mimic this feature in an aerated, stirred bioreactor. The biotransformation was carried out using a fed-batch mode of operation involving sequential precursor addition. -Ionone stopped the fungal growth and was converted into metabolites only when the carbon source remained present in the medium; it was fully oxidized after sucrose exhaustion. These conditions allowed recovery of about 2.5 g/l aroma compounds after 230 h cultivation with a molar yield close to 100%.     

Production of β-fructofuranosidase by Aspergillus japonicus in batch and fed-batch cultures

Summary A comparison of -fructofuranosidase (FFase, EC 3.2.l.26) production by Aspergillus japonicus TIT-90076 in batch and fed-batch cultures was investigated in shaken flasks. Results showed that fed-batch cultivation of A. japonicus using intermittent sucrose supply produced more FFase than batch culture, and the maximal enzyme production was 910 units ml^–1, which was about 20% higher than that in the batch cultures.     

Coexpression of β-mannanase and xylanase genes in Escherichia coli

[目的]β-甘露聚糖酶和木聚糖酶都属于半纤维素酶,它们已经同时运用于工农业生产的许多领域.构建β-甘露聚糖酶和木聚糖酶共表达菌株并进行相关评价.[方法]通过设计一个共同的酶切位点,将菌株Bacillus subtilis BE-91中的β-甘露聚糖酶和木聚糖酶基因串联到表达载体pET28a(+)上,转化大肠杆菌构建了一株能够共表达β-甘露聚糖酶和木聚糖酶的菌株B.pET28a-man-xyl.[结果]菌株诱导21h后,发酵液中β-甘露聚糖酶和木聚糖酶的酶活分别为713.34 U/mL和1455.83 U/mL,是胞内酶活的11.8倍和2.53倍.[结论]SDS-PAGE分析、水解圈活性检测和胞外酶与胞内酶酶活检测表明:两个酶均以功能蛋白独立分泌到胞外.此外,与β-甘露聚糖酶和木聚糖酶单独酶解半纤维素相比,复合酶的酶解效果更好.菌株的成功构建为复合酶制剂(半纤维素酶制剂)的研究和生产奠定基础.     

Induction of resistance against pathogens by β-aminobutyric acid

Among the many types of plant stressors, pathogen attack, mainly fungi and bacteria can cause particularly severe damage both to individual plants and, on a wider scale, to agricultural productivity. The magnitude of these pathogen-induced problems has stimulated rapid progress in green biotechnology research into plant defense mechanisms. Plants can develop local and systemic wide-spectrum resistance induced by their exposure to virulent (systemic acquired resistance—SAR) or non-pathogenic microbes and various chemical elicitors (induced systemic resistance—ISR). β-Aminobutyric acid (BABA), non-protein amino acid, is though to be important component of the signaling pathway regulating ISR response in plants. After treatment with BABA or various chemicals, after infection by a necrotizing pathogen, colonization of the roots by beneficial microbes many plants establish a unique physiological state that is called the “primed” state of the plant. This review will focus on the recent knowledge about the role of BABA in the induction of ISR against pathogens mainly against fungi.     

Induction of β-family chemokines mRNA in human embryonic astrocytes by inflammatory cytokines

The cellular infiltration found during CNS inflammation consists of monocytes and activated T cells, suggesting the presence of cell-specific chemotactic signals during inflammatory responses. Astrocyte chemokine expression might contribute to site-specific leukocyte infiltration within the CNS. To investigate the factors that regulate astrocyte chemokine expression, we examined the ability of human fetal astrocytes to induce β-family chemokine mRNA. Astrocyte-derived monocyte chemoattractant protein-1 (MCP-1), RANTES, macrophage inflammatory protein-1α (MIP-1α), and MIP-1β mRNA were easily induced by lipopolysaccharide and/or the proinflammatory cytokines (IFNγ and/or TNF-α), respectively. Addition of both IFNγ and TNF-α together did not lead to an additive effect but resulted in the inhibition of MCP-1 and MIP-1β mRNA expression, indicating that interaction between chemokines and cytokines may play a key role in regulating the local immune response of resident and infiltrating cells at the site of lesion. Interestingly, ultraviolet light-inactivated measles virus, but not cytomegalovirus, strongly induced expression of MCP-1, RANTES, MIP-1α, and MIP-1β mRNA in human embryonic astrocytes, especially MCP-1 and MIP-1β. An association occurs between the β-family chemokine expression in astrocytes and inflammatory factors/virus, suggesting a possible role for β-family chemokines in the pathogenesis of CNS inflammatory disease.     

Induction of regulatory T cells by a murine β-defensin

β-Defensins are antimicrobial peptides of the innate immune system produced in the skin by various stimuli, including proinflammatory cytokines, bacterial infection, and exposure to UV radiation (UVR). In this study we demonstrate that the UVR-inducible antimicrobial peptide murine β-defensin-14 (mBD-14) switches CD4(+)CD25(-) T cells into a regulatory phenotype by inducing the expression of specific markers like Foxp3 and CTLA-4. This is functionally relevant because mBD-14-treated T cells inhibit sensitization upon adoptive transfer into naive C57BL/6 mice. Accordingly, injection of mBD-14, comparable to UVR, suppresses the induction of contact hypersensitivity and induces Ag-specific regulatory T cells (Tregs). Further evidence for the ability of mBD-14 to induce Foxp3(+) T cells is provided using DEREG (depletion of Tregs) mice in which Foxp3-expressing cells can be depleted by injecting diphtheria toxin. mBD-14 does not suppress sensitization in IL-10 knockout mice, suggesting involvement of IL-10 in mBD-14-mediated immunosuppression. However, unlike UVR, mBD-14 does not appear to mediate its immunosuppressive effects by affecting dendritic cells. Accordingly, UVR-induced immunosuppression is not abrogated in mBD-14 knockout mice. Together, these data suggest that mBD-14, like UVR, has the capacity to induce Tregs but does not appear to play a major role in UVR-induced immunosuppression. Through this capacity, mBD-14 may protect the host from microbial attacks on the one hand, but tame T cell-driven reactions on the other hand, thereby enabling an antimicrobial defense without collateral damage by the adaptive immune system.     

Hyper production of β-glucosidase by an Aspergillus sp.

Summary An Aspergillus sp. was isolated which secreted high levels of -glucosidase in growth medium. The maximum activity(10 IU/ml of -glucosidase and 22.6 IU/ml of cellobiase) was obtained in cellulose medium supplemented with wheat bran. The pH and temperature optima for this enzyme were 4.5 and 65°C respectively.NCL Communication No. 3616     

Detection of endogenous β-glucuronidase activity in Aspergillus niger

 An endogenous β-glucuronidase that hydrolyses the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc) in Aspergillus niger is reported. The activity was induced when the fungus was grown in media containing xylan, but was either very low, or absent, when grown on glucose. Endogenous β-glucuronidase was primarily located in newly formed hyphae, and was apparent at pH values between 3 and 6. Hydrolysis of X-gluc was sensitive to the inhibitor D-saccharic acid 1,4-lactone and was irreversibly inactivated by heating. The bacterial uidAβ-glucuronidase reporter gene was strongly expressed in the hyphae of transformed A. niger but, in contrast to the endogenous activity, the enzyme was also active at pH 7–8.5. Histochemical localization of uidA expression in A. niger, without interference from the endogenous β-glucuronidase activity, was achieved by staining at this pH. Received : 22 March 1995/Received last revision : 17 August 1995/Accepted : 22 August 1995     

Over-production of β-glucosidase by Aspergillus niger mutant from lignocellulosic biomass

The maximum yield of -glucosidase by A. niger KK2 mutant, grown on the basal medium for 7 days, was 514 I U g^–1 ground rice straw, and was about twice those obtained from wheat straw or bran by previous researchers. Optimal activity of -glucosidase was at 60–70 °C and pH 4.8.     

Synthesis of derivatives of methyl β-sophoroside

Selective tritylation of methyl β-sophoroside (1) and subsequent acetylation gave the 3,4,2′,3′,4′-penta-O-acetyl-6,6′-di-O-trityl derivative, which was O-detritylated, and the product p-toluenesulfonylated, to give methyl 3,4,2′,3′,4′-penta-O-acetyl-6,6′-di-O-p-tolylsulfonyl-β-sophoroside (4) in 63% net yield. Compound 4 was also obtained in 69% yield by p-toluenesulfonylation of 1, followed by acetylation. Several, 6,6′-disubstituted derivatives of 1 were synthesized by displacement reactions of 4 with various nucleophiles. Treatment of 4 with sodium methoxide afforded methyl 3,6:3′,6′-dianhydro-β-sophoroside. Several 6- and 6′-monosubstituted derivatives of 1 were prepared, starting from the 4,6-O-benzylidene derivative of 1.