Formation and activities of xylanhydrolysing enzymes of Humicola grisea var thermoidea

A thermophilic fungus, Humicola grisea var thermoidea, produced in liquid culture two endoxylanases (1,4--d-xylan-xylanohydrolase, EC 3.2.1.8) with M _r of 95 (Xyl I) and 13 (Xyl II) kDa. PAGE of the crude culture filtrate and of each fraction obtained by gel filtration produced three and one band, respectively. Cross-reaction of the culture filtrate and each fraction with polyclonal antibodies prepared against Xyl II produced two and one precipitin bands, respectively. Hydrolysis of wheat straw and rice husk xylan was maximal using a combination of Xyl I and Xyl II. The products formed after hydrolysis, xylo-oligosaccharides and traces of xylose, indicated an endotype enzyme action and the co-operative activities of the xylanases.     

Acid trehalase deficiency and extracellular trehalose utilization in Candida utilis

Biochemical characterization of a trehalase, detected in the mid-exponential growth phase of Candida utilis NCIM Y500, has indicated that it was a neutral trehalase and possibly the only trehalase present in this strain. Unlike Saccharomyces cerevisiae and other C. utilis strains, this strain without acid trehalase grew quite well in minimal or complete medium containing trehalose as the sole source of carbon. Both these observations were contradictory to the findings reported for acid trehalase mutants of S. cerevisiae and C. utilis. The trehalase system of the strain is suggested to be similar to that of fungi.     

Purification and partial characterization οf a new β-xylosidase from Humicola grisea var. thermoidea

Summary The thermophilic fungus Humicola grisea var. thermoidea produces a mycelium-associated β-xylosidase activity when grown in liquid-state cultures on media containing oat spelt xylan as the carbon source. The β-xylosidase was purified to apparent homogeneity by gel filtration and anion exchange chromatography. Its molecular weight was 37 and 50 kDa, as determined by MALDI/TOF mass spectrometry and SDS-PAGE, respectively. The purified enzyme exhibited maximum activity at 55 °C and pH 6.5. It was also active at pH 8.8, retaining 60% of its activity after 6 h of incubation at 50 °C. β-xylosidase was strongly inactivated by NBS and slightly activated by DTT and β-mercaptoethanol. The enzyme was highly specific for PNPX as the substrate. The purified β-xylosidase showed K _m and V _max values of 1.37 mM and 12.98 IU ml⁻¹, respectively.     

Expression and processing of a major xylanase (XYN2) from the thermophilic fungus Humicola grisea var. thermoidea in Trichoderma reesei

AIMS: To express a gene encoding a heterologous fungal xylanase in Trichoderma reesei. METHODS AND RESULTS: Humicola grisea xylanase 2 (xyn2) cDNA was expressed in Trichoderma reesei under the main cellobiohydrolase I (cbh1) promoter (i) as a fusion to the cellobiohydrolase I (CBHI) secretion signal and (ii) the mature CBHI core-linker. The recombinant xylanase (HXYN2) was secreted into the cultivation medium and processed in a similar fashion to the endogenous T. reesei xylanases, resulting in an active enzyme. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: HXYN2 was successfully processed in T. reesei. Composition of the culture medium affected the HXYN2 yields, favouring Avicel-lactose as a carbon source. Best yields (about 0.5 g l(-1)) in shake flask cultivations were obtained from a transformant where xyn2 was fused directly to the CBHI secretion signal.     

Production and Characteristics of Avicel-Disintegrating Endoglucanase from a Protease-Negative Humicola grisea var. thermoidea Mutant

Mutational experiments were performed to decrease the protease productivity of Humicola grisea var. thermoidea YH-78 using UV light and N-methyl-N′-nitro-N-nitrosoguanidine. A protease-negative mutant, no. 140, exhibited higher endoglucanase activity than the parent strain in mold bran culture at 50°C for 4 days. The culture extract rapidly disintegrated filter paper but produced a small amount of reducing sugar. About 30% of total endoglucanase activity in the extract was adsorbed onto Avicel. The electrophoretically homogeneous preparation of Avicel-adsorbable endoglucanase (molecular weight, 128,000) showed intensive filter-paper-disintegrating activity but did not release reducing sugar. The preparation also exhibited a highly synergistic effect with the cellulase preparation from Trichoderma reesei in the hydrolysis of microcrystalline cellulose. This endoglucanase was observed via scanning electron microscopy to disintegrate Avicel fibrils layer by layer from the surface, yielding thin sections with exposed chain ends. A mutant, no. 191, producing higher protease activity and an Avicel-unadsorbable, Avicel-nondisintegrating endoglucanase was isolated. The purified enzyme (molecular weight, 63,000) showed no disintegrating activity on filter paper and Avicel and a less synergistic effect with the T. reesei cellulase in hydrolyzing microcrystalline cellulose than did the former enzyme. Endoglucanase was therefore divided into two types, Avicel disintegrating and Avicel nondisintegrating.     

Purification and properties of trehalase from the thermophilic fungus Humicola lanuginosa.

Trehalase (alpha,alpha-Trehalose glucohydrolase, EC 3.2.1.28) was partially solubilized from the thermophilic fungus Humicola lanuginosa RM-B, and purified 184-fold. The purified enzyme was optimally active at 50 degrees C in acetate buffer at pH 5.5. It was highly specific for alpha,alpha-trehalose and had an apparent Km = 0.4 mM at 50 degrees C. None of the other disaccharides tested either inhibited or activated the enzyme. The molecular weight of the enzyme was around 170 000. Trehalase from mycelium grown at 40 and 50 degrees C had similar properties. The purified enzyme, in contrast to that in the crude-cell free extract, was less stable. At low concentration, purified trehalase was afforded protection against heat-inactivation by "protection against heat-inactivation by "protective factor(s)" present in mycelial extracts. The "protective factor(s)" was sensitive to proteolytic digestion. It was not diffusible and was stable to boiling for at least 30 min. Bovine serum albumin and casein also protected the enzyme from heat-inactivation.     

杓兰果胶杆菌海藻糖酶的克隆表达及应用

海藻糖酶在工业发酵和食品医药等领域应用广泛,我国缺乏性能优良工业品种的海藻糖酶,同时在应用研究领域还不够深入。本研究从自然界筛选获得一株产酸性海藻糖酶的杓兰果胶杆菌(Pectobacterium cypripedii),克隆获得其海藻糖酶基因PCTre,在大肠杆菌(Escherichia coli)BL21(DE3)体系中实现了重组表达,在5L发酵罐上28h产酶达到4130U/mL。酶学性质研究显示,PCTre能特异性水解海藻糖,最适反应温度和pH分别是35℃和5.5,在pH4.0、4.5、5.0处理8h后残余酶活仍超过80%,表现出良好的耐酸性。同时该酶还显示出优良的有机溶剂耐受性,在20%(V/V)乙醇溶液中处理24h仍能保留60%的酶活。进一步在含有20%(V/V)乙醇和7.5%海藻糖的模拟发酵体系中,当添加500U/L PCTre,可在16h内完全水解海藻糖,具有良好的应用于工业乙醇发酵的潜力。     

Purification and biochemical characterization of β-xylosidase from Humicola grisea var. thermoidea

Abstract β-d-Xylosidase production was maximal for Humicola grisea var. thermoidea grown on xylan as the sole carbon source. The main β-d-xylosidase activity was localised in the periplasm. β-Xylosidase was purified from crude extracts by heat treatment, ammonium sulfate precipitation and chromatography on DEAE-cellulose and Sephadex G-100. The purified enzyme was a monomer of molecular mass estimated to be 43 kDa by SDS-PAGE and gel filtration. Optima of pH and temperature were 6.0 and 50 °C, respectively. The enzyme activity was stimulated by Ca²⁺, Fe²⁺, and Mg²⁺. The purified β-xylosidase did not exhibit xylanase, carboxymethylcelullase, galactosidase, glucosidase, fucosidase or arabinosidase activities. The purified β-xylosidase hydrolysed xylobiose and xylo-oligosaccharides of up to five monosaccharide units. The enzyme had a K _m of 0.49 mM for p -nitrophenyl- β -d-xylopyranoside and was not inhibited by its product, xylose.     

Temperature response of trehalase from a mesophilic (Neurospora crassa) and a thermophilic (Thermomyces lanuginosus) fungus

The purified trehalases of the mesophilic fungus, Neurospora crassa, and the thermophilic fungus, Thermomyces lanuginosus, had similar temperature and pH optima for activity, but differed in molecular weight, electrophoretic mobility and Michaelis constant. At lower concentration, trehalases from both fungi were inactivated to similar extent at 60°C. While purified trehalase of T. lanuginosus was afforded protection against heat-inactivation by proteinaceous protective factor(s) present in mycelial extracts, by bovine serum albumin and by casein, these did not afford protection to N. crassa trehalase against heat inactivation. Both trehalases exhibited discontinuous Arrhenius plots with temperature of discontinuity at 40°C. The activation energy calculated from the slope of the Arrhenius plot was higher for the T. lanuginosus enzyme. The plots of apparent K _m versus 1/T for trehalases of N. crassa and T. lanuginosus were linear from 30° to 60°C.The results show that purified trehalases of the mesophilic and the thermophilic fungus are distinct. Although, these exhibit similar thermostability of their catalytic function at low concentration, distinctive thermal stability characteristics of thermophilic enzyme become apparent at high protein concentration. This could be brought about in the cell by the enzyme itself, or by other proteins.     

A highly thermostable trehalase from the thermophilic bacterium <Emphasis Type="Italic">Rhodothermus marinus</Emphasis>

Trehalases play a central role in the metabolism of trehalose and can be found in a wide variety of organisms. A periplasmic trehalase (α,α-trehalose glucohydrolase, EC 3.2.1.28) from the thermophilic bacterium Rhodothermus marinus was purified and the respective encoding gene was identified, cloned and overexpressed in Escherichia coli. The recombinant trehalase is a monomeric protein with a molecular mass of 59 kDa. Maximum activity was observed at 88°C and pH 6.5. The recombinant trehalase exhibited a K _m of 0.16 mM and a V _max of 81 μmol of trehalose (min)⁻¹ (mg of protein)⁻¹ at the optimal temperature for growth of R. marinus (65°C) and pH 6.5. The enzyme was highly specific for trehalose and was inhibited by glucose with a K _i of 7 mM. This is the most thermostable trehalase ever characterized. Moreover, this is the first report on the identification and characterization of a trehalase from a thermophilic bacterium.     

Lipase production of a new thermophilic fungus,Humicola lanuginosa var. catenulata

We isolated a new thermophilic fungus from soil and identified it as Humicola lagunisoa var. catenulata, a new variety of Humicola lanuginosa. We cultured the fungus and found that these were two kinds of lipases in the culture filtrate.     

Evaluation of hydrolysis products of xylans by xylan-degrading enzymes from Humicola grisea var. thermoidea and Aspergillus fumigatus Fresenius

Xylan-degrading enzyme activities were isolated from crude extracts of solid-state cultures of Aspergillus fumigatus Fresenius (Xyl I, Xyl III, Xyl IV and Xyl V) and Humicola grisea var. thermoidea (Xyl II) by chromatographic procedures. The pattern of hydrolysis of different xylans and pulps varied from traces of xylose to xylooligomers. The products formed suggest an endo-type enzyme mode of action. Some enzymes showed debranching and transglycosidase activities.     

Intracellular changes of trehalose content in toluene tolerant Pseudomonas sp. BCNU 171 after exposure to toluene

Pseudomonas sp. BCNU 171, when grown with 10% (v/v) toluene for 2 h, accumulated approximately 7.7 mM trehalose probably arising by the action of trehalose-6-phosphate synthase (E.C. 2.4.1.15). Trehalose may thus play a significant role in the tolerance of the Pseudomonas sp. to toluene.     

Mycelial amylase activities of thermophilic species ofRhizomucor,Humicola andPapulaspora

Amylase activities in mycelia ofRhizomucor pusillus, Humicola grisea var.thermoidea, Humicola lanuginosa andPapulaspora thermophilia do not correspond directly with previously-measured extracellular values, and appear to decline within the time period corresponding to reduction in mycelial dry weight. The results are compared with previously-reported data on extracellular amylase.     

Biochemical characterization of neutral trehalase activity in Saccharomyces boulardii

Lyophilized cells of the non-pathogenic yeast Saccharomyces boulardii are used in many countries for the treatment of several types of diarrhoea and other gastrointestinal diseases. Although the cells must be viable, their mechanism of action is unknown. The disaccharide trehalose is a protectant against several forms of environmental stress in yeast and is involved in maintaining cell viability. There is no information on the enzymes involved in degradation of trehalose in S. boulardii. The aim of the present study was to characterize trehalase activity in this yeast. Cells of S. boulardii grown in glucose exhibited neutral trehalase activity only in the exponential phase. Acidic trehalase was not detected in glucose medium. Cells grown in trehalose exhibited acid and neutral trehalase activities at all growth stages, particularly in the exponential phase. The optimum pH and temperature values for neutral trehalase activity were determined as 6.5 and 30 °C respectively, the half-life being approximately 3 min at 45 °C. The relative molecular mass of neutral trehalase is 80 kDa and the K _m 6.4 mM (±0.6). Neutral trehalase activity at pH 6.5 was weakly inhibited by 5 mM EDTA and strongly inhibited by ATP, as well as the divalent ions Cu⁺⁺, Fe⁺⁺ and Zn⁺⁺. Enzyme activity was stimulated by Mg⁺⁺ and Ca⁺⁺ only in the absence of cAMP. The presence of cAMP with no ion additions increased activity by 40%. This revised version was published online in July 2006 with corrections to the Cover Date.     

Induction of non-diapause eggs in Bombyx mori by a trehalase inhibitor

The effect of validoxylamine A (VAA), a potent and specific trehalase inhibitor, on the induction of non-diapause in Bombyx mori was examined. The VAA induced non-diapause eggs and prevented the glucogen accumulation in the eggs. Trehalase activity of the pupal ovary was effectively inhibited by the VAA injection.     

感染布氏白僵菌后油松毛虫血淋巴中海藻糖酶活性、海藻糖和葡萄糖含量的变化

为了揭示病原真菌白僵菌Beauveria侵染昆虫过程中如何利用虫体内糖类物质作为自身营养, 本研究测定了布氏白僵菌Beauveria brongniartii (Sacc.) Petch （2382菌株）感染油松毛虫Dendrolimus tabulaeformis Tsai et Liu幼虫后, 虫体血淋巴中酸性海藻糖降解酶活性及海藻糖和葡萄糖含量的变化。油松毛虫4龄幼虫感染菌株孢子悬浮液后, 血淋巴中酸性海藻糖降解酶的活性明显高于对照组, 感染后第3天酶活性达到最大值（0.2786 U/mg）, 此后第4-6 天酶活性逐渐降低; 染菌后的6 d中, 血淋巴中海藻糖含量显著低于对照组, 同样在感染后第4天其含量逐渐降低, 第6天时降到最低值。相比之下, 处理组血淋巴中的葡萄糖含量显著高于对照组; 处理组其含量在第1-3天内呈现快速升高趋势, 在第3天达到最大值（7.7615 mmol/L）, 然后逐渐降低。结果说明, 白僵菌侵入昆虫血淋巴后, 菌株代谢产生酸性海藻糖降解酶, 将血淋巴中的海藻糖水解成为葡萄糖, 然后为真菌利用, 破坏了虫体内的血糖平衡, 这是一个相互连接的生理代谢和生化反应过程。