Thermostability of β-xylosidase from Aspergillus sydowii MG49

Heating of Aspergillus β-xylosidase at 85°C ± 1°C and pH 5.5–6.0 (optimum for activity), causes irreversible, covalent thermoinactivation of the enzyme, involving oxidation of the thiol groups that are required for catalysis. Exogenous addition of cysteine, DTT, GSH and mercaptoethanol stabilizes the enzyme by extending its half-life. A similar effect is also exhibited by bivalent cations like Mg²⁺, Mn²⁺, Co²⁺, Ca²⁺and Zn²⁺ while, on the other hand Cu²⁺ accelerates thermoinactivation. Chemical modification of crude β-xylosidase with cross-linking agents like glutaraldehyde or covalent immobilization to a nonspecific protein like gelatin and BSA also enhances enzyme thermostability. These results suggest that addition of thiols and bivalent metal ions to a crude β-xylosidase preparation or immobilization/chemical modification enhances its thermal stability, thus preventing loss of catalytic activity at elevated temperatures.     

Catalytic properties of the immobilized Talaromyces thermophilus β-xylosidase and its use for xylose and xylooligosaccharides production

The present study explores the efficiency of Talaromyces thermophilus β-xylosidase, in the production of xylose and xylooligosaccharides. The β-xylosidase was immobilized by different methods namely ionic binding, entrapment and covalent coupling and using various carriers. Chitosan, pre-treated with glutaraldehyde, was selected as the best support material for β-xylosidase immobilization; it gave the highest immobilization and activity yields (94%, 87%, respectively) of initial activity, and also provided the highest stability, retaining 94% of its initial activity even after being recycled 25 times. Shifts in the optimal temperature and pH were observed for the immobilized β-xylosidase when compared to the free enzyme. The maximal activity obtained for the immobilized enzyme was achieved at pH 8.0 and 53 °C, whereas that for the free enzyme was obtained at pH 7.0 and 50 °C. The immobilized enzyme was more thermostable than the free β-xylosidase. We observed an increase of the K_m values of the free enzyme from 2.37 to 3.42 mM at the immobilized state. Native and immobilized β-xylosidase were found to be stimulated by Ca²⁺, Mn²⁺ and Co²⁺ and to be inhibited by Zn²⁺, Cu²⁺, Hg²⁺, Fe²⁺, EDTA and SDS. Immobilized enzyme was found to catalyze the reverse hydrolysis reaction, forming xylooligosaccharides in the presence of a high concentration of xylose. In order to examine the synergistic action of xylanase and β-xylosidase of T. thermophilus, these two enzymes were co-immobilized on chitosan. A continuous hydrolysis of 3% Oat spelt xylan at 50 °C was performed and better hydrolysis yields and higher amount of xylose was obtained.     

Enzymatic synthesis of mannobioses and mannotrioses by reverse hydrolysis using α-mannosidase from Aspergillus niger

Various manno-oligosaccharides including and

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were formed when a highly concentrated mannose solution was incubated in the presence of α-mannosidase from Aspergillus niger. and

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were isolated by activated carbon chromatography followed by high performance liquid chromatography using an amino-silica column. In addition to the above oligosaccharides,

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, and

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were also isolated.     

Effect of carbon source on the biochemical properties of β-xylosidases produced by Aspergillus versicolor

The filamentous fungus Aspergillus versicolor produced large amounts of mycelial β-xylosidase activity when grown on xylan or xylose as the only carbon source. The presence of glucose drastically decreased the level of β-xylosidase activity, while cycloheximide prevented the induction of the enzymes by xylan or xylose. The β-xylosidases induced by xylose or xylan were purified by a simple protocol involving DEAE-cellulose chromatography and ammonium sulphate precipitation. The purified enzymes were acidic proteins, with carbohydrate contents of 21% for that induced by xylose, and 47% for that induced by xylan. Their apparent molecular masses, estimated by gel filtration, and optimal temperatures for β-xylosidase activities, were about 60 and 100 kDa, and 40 and 45 °C, respectively, for the enzymes induced by xylose and xylan. Xylose-induced β-xylosidase exhibited an optimum pH of 6.0, while that of the xylan-induced enzyme was 5.5. Both purified β-xylosidases exhibited also β-galactosidase, β-glucosidase and -arabinosidase activities. In addition to synthetic substrates, the enzymes hydrolysed xylobiose and xylotriose, suggesting a physiological role. K_M values for p-nitrophenyl β- -xylopyranoside were 0.32 mM, for the xylose-induced β-xylosidase, and 0.19 mM for the xylan-induced one. Xylose competitively inhibited both β-xylosidases, with K_I values of 5.3 and 2.0 mM, for the enzymes induced by xylose or xylan, respectively.     

Improvement of isoflavone aglycones production using β-glucosidase secretory produced in recombinant Aspergillus oryzae

β-Glucosidase (BGL1) from Aspergillus oryzae was efficiently produced in recombinant A. oryzae using sodM promoter-mediated expression system. The yield of BGL1 was 960 mg/l in liquid culture, which is 20-fold higher than the yield of BGL1 produced using the yeast Saccharomyces cerevisiae. Recombinant BGL1 converted isoflavone glycosides into isoflavone aglycones more efficiently than β-glucosidase from almond. In addition, BGL1 produced isoflavone aglycones even in the presence of the insoluble form of isoflavone glycosides.     

Purification and characterization of a thermostable intracellular β-xylosidase from the thermophilic fungus Sporotrichum thermophile

An intracellular β-xylosidase from the thermophilic fungus Sporotricum thermophile strain ATCC 34628 was purified to homogeneity by Q-Sepharose and Mono-Q column chromatographies. The protein properties correspond to molecular mass and pI values of 45 kDa and 4.2, respectively. The enzyme is optimally active at pH 7.0 and 50 °C. The purified β-xylosidase is fully stable at pH 6.0–8.0 and temperatures up to 50 °C and retained over 58% of its activity after 1 h at 60 °C. The enzyme hydrolyzes β-1,4-linked xylo-oligosaccharides with chain lengths from 2 to 6, releasing xylose from the non-reducing end, but is inactive against xylan substrates. The apparent K_m and V_max values from p-nitrophenyl β-d-xylopyranoside are 1.1 mM and 114 μmol p-nitrophenol min⁻¹ mg⁻¹, respectively. Alcohols inactivate the enzyme, ethanol at 10% (v/v) yields a 30% decrease of its activity. The enzyme is irreversibly inhibited by 2,3-epoxypropyl β-d-xylobioside while alkyl epoxides derived from d-xylose were not inhibitors of the enzyme. The enzyme catalyses the condensation reaction using high donor concentration, up to 60% (w/v) xylose.     

Some factors affecting production of pectic enzymes by Aspergillus niger

The effects of varying cultural conditions were assessed for the production of pectic enzymes in a strain of Aspergillus niger, isolated from decaying orange fruit. Polygalacturonase and pectinmethylesterase were found to be inducible by polygalacturonic acid and pectin in the medium, respectively. Ammonium sulphate was the best nitrogen source for the production of both enzymes. There were variations in enzyme levels produced in culture filtrates with age of the culture, the highest levels being in 4-day-old cultures. The temperature and pH also had marked effects on the production of pectic enzymes with the best conditions being 40°C and pH 5, respectively. Surface culture technique gave appreciable enzyme yield, while agitation had an inhibitory effect on enzyme production.     

Aerial mycelia of Aspergillus oryzae accelerate α-amylase production in a model solid-state fermentation system

Aspergillus oryzae is commonly used in solid-state fermentation (SSF) and forms abundant aerial mycelia. Previously, we have shown that aerial mycelia are extremely important for the respiration of this fungus during growth on a wheat-flour model substrate. In this paper, we show that aerial mycelia of this fungus give a strong increase in fungal biomass and α-amylase production. Cultures of A. oryzae on wheat-flour model substrate produced twice the amounts of fungal biomass and α-amylase, when aerial mycelia were formed. Utilization of these findings in commercial solid-state fermenters requires further research; results from packed beds of grain indicate that aerial mycelia are of limited importance there. Probably, substrate pre-treatment and an increase in bed voidage are required.     

柚子皮黑曲霉的分离鉴定及产酶特性研究

对柚子皮上自然生长的黑曲霉进行分离鉴定,并探讨其产酶特性。以平板稀释法从柚子皮上分离出一株霉菌菌株,通过观察其形态特征和培养特征,对照《真菌鉴定手册》判定该菌株的种属;采用鉴定培养基法对其产酶特性进行分析。根据柚子皮的成分特性,以干柚子皮为主要原料,该菌为生产菌株,采用固态发酵法探究培养基的成分、柚子皮含量、培养基初始含水量及发酵时间4个因素对纤维素酶活力的影响。结果表明,该菌株为黑曲霉(Aspergillus nige),可产淀粉酶、蛋白酶、纤维素酶、果胶酶;固态发酵培养基中添加柚子皮12g,麸皮0.5 g和(NH_4)_2SO_40.5 g,培养基初始含水量保持在68.5 mL/100 g,培养时间控制在60 h左右时纤维素酶产量较高。     

黑曲霉菌渣综合利用研究进展

黑曲霉菌渣是黑曲霉经过工业发酵所需产物之后的副产物,资源储量丰富。目前利用黑曲霉生产的制剂种类繁多,对菌渣综合利用的报道却相对较少。已有的研究表明黑曲霉菌渣不仅可作为饲料添加剂、有机肥料或土壤改良剂得到直接应用,也可用于精制,作为食品、药品和化妆品等行业的主要原材料,实现高附加值转化应用。还可作为生物吸附剂用于去除水体中的污染物等。本文结合目前国内外对黑曲霉菌渣在各领域研究及利用现状,综述了黑曲霉菌渣高附加值转化与利用的研究进展,可以为工业发酵废渣资源化利用提供理论参考。     

Immobilization of the glycosidases: α- -arabinofuranosidase and β- -glucopyranosidase from Aspergillus niger on a chitosan derivative to increase the aroma of wine. Part II

The purpose of this paper was to study the immobilization of two glycosidases, α- -arabinofuranosidase (EC 3.2.1.55) and β- -glucopyranosidase (EC 3.2.1.21), contained in a commercial preparation and purified as reported in Part I. The procedure which proved to be the best is simple and inexpensive to perform, employing the chitosan derivative, glyceryl-chitosan, especially synthesized and characterized, as a support. The glycosidases were adsorbed on this support and cross-linked with glutaraldehyde to prevent them from being released into the wine. Subsequent reduction of the biocatalyst with sodium borohydride allowed for improved stability. Finally, the immobilized glycosidases were compared with free ones in terms of optimum pH and temperature, stability over time, and kinetics parameters (K_m and V_max) after which they were employed for aromatizing a model wine solution containing aromatic precursors.     

新一代糖化酶高产菌株的选育及其工业应用

【目的】建立对糖化酶生产菌种黑曲霉随机突变文库进行筛选的方法,以获得糖化酶酶活提高的突变菌株。【方法】以一株可产糖化酶的黑曲霉菌株Aspergillus niger X1为出发菌株,经硫酸二乙酯诱变获得突变文库,采用葡萄糖的结构类似物——2-脱氧葡萄糖进行筛选,并在筛选过程中逐渐提高2-脱氧葡萄糖浓度,定向选育具有2-脱氧葡萄糖抗性、高产糖化酶的突变株。【结果】获得的高产突变菌株DG36摇瓶发酵糖化酶产量比出发菌株A.niger X1提高22.2%–33.8%,经工业水平50 m~3罐发酵测试,突变株DG36发酵128 h糖化酶活可达49094 U/m L,在相同发酵时间内,其酶活较出发菌株A.niger X1提高32.8%,发酵时间缩短16.9%。【结论】本研究开发了一种以2-脱氧葡萄糖为抗性标记选育高产糖化酶突变株的方法,所得突变株DG36遗传性状稳定,与出发菌相比具有菌丝粗壮、产酶期提前、糖化酶活高、发酵时间短、有利于发酵后处理的优点。     

过表达Na,K-ATPase基因增强了黑曲霉对硅灰石的风化能力

【目的】探究和证实黑曲霉钠钾ATP酶(NKA)在硅灰石风化过程中的作用。【方法】以野生型黑曲霉(WT)为原始菌株构建黑曲霉Na,K-ATPaseα1基因(NKAα1)高表达菌株oeNKA。通过测定不同时间点(0d、2d、4d、6d)oeNKA和WT生物量、培养液pH值和矿物风化释放的Ca~(2+)浓度,并用X-ray diffraction (XRD)对风化后的矿物残渣进行检测,比较oeNKA和WT菌株对硅灰石这种硅酸盐矿物的风化效果。【结果】oeNKA菌株的NKAα1基因相对表达量和酶活分别为WT菌株的103倍和1.76倍。在持续6d的培养过程中,oeNKA与WT的菌丝体生物量变化趋势相同,在培养第2天时WT显著高于oeNKA,随时间差异逐渐缩小并在第6天达到最低;培养液pH值变化趋势相同,分别下降至3.64和3.87;oeNKA风化硅灰石时所释放Ca~(2+)浓度(1011.36±47.78μg/g)约为WT (248.30±25.21μg/g)的4倍;XRD检测图谱显示菌株oeNKA对硅灰石风化作用更明显。【结论】NKAα1过表达菌株oeNKA对硅灰石的风化能力显著高于WT菌株,且黑曲霉的NKA与硅灰石的风化有密切关联。     

Temperature and carbon source affect the production and secretion of a thermostable β-xylosidase by Aspergillus fumigatus

The effect of the temperature of growth and carbon source on the production and secretion of β-xylosidase (EC 3.2.1.37) by the thermotolerant fungi Aspergillus fumigatus was studied in submerged cultures. In cultures developed at optimal temperature (30 °C), the enzyme was predominantly cell-bound, while in cultures developed at higher temperature (42 °C), the β-xylosidase activity was predominantly found in the cell-free filtrates. The use of corn cob powder instead of xylan as substrate increased considerably the secretion of enzyme. The highest level of extracellular β-xylosidase (45 U/ml or 360 U/mg protein) was obtained in 3% corn cob cultures grown at 42 °C for 72 h. The partially purified enzyme was active and stable at high temperatures. The presence of high titres of β-xylosidase activity in association with xylanase in the culture filtrates enhanced the efficiency of the pulp hydrolysis process.     

黑曲霉产木聚糖酶的分离纯化与鉴定研究

木聚糖酶的分离纯化是对其进行酶学研究和分子改良研究的基础。利用实验室选育的黑曲霉菌株Aspergillus niger SM24/a进行木聚糖酶发酵,粗酶液经过(NH_4)_2SO_4分级沉淀Bio-Gel P6除盐、UNO sphere Q阴离子交换和Enrich SEC70凝胶色谱层析四个步骤的分离纯化,成功获得了3种木聚糖酶蛋白定义为X-Ⅰ、X-Ⅱ和X-Ⅲ。随着纯化步骤的增加,各组分酶比活力得到显著提高,其数值分别为37.41、34.56和53.96 U/mg,纯化倍数分别为3.96、3.66和5.72。经质谱分析和蛋白氨基酸序列比对,初步认定X-Ⅰ属于糖基水解酶第十家族内切-β-1,4-木聚糖酶,X-Ⅱ和X-Ⅲ均属于糖基水解酶第十一家族木聚糖酶。     

Optimization of media for β-fructofuranosidase production by Aspergillus niger in submerged and solid state fermentation

The kinetics of β-fructofuranosidase (Ffase) production by Aspergillus niger in submerged (SmF) and solid-state fermentation (SSF) systems was investigated. The maximum productivity of Ffase (81.8 U/l per h) was obtained in SSF for 72 h while it was 18.3 U/l per h in SmF for 120 h. The productivity of extra cellular Ffase produced in SSF was 5-fold higher than in SmF. Optimization of fermentation medium for Ffase production was carried out using De Meo's fractional factorial design with seven components such as (NH₄)₂SO₄, KH₂PO₄, FeSO₄, MgSO₄ · 7H₂O, sucrose, urea and yeast extract. The media designed for SmF after two steps of optimization supported the growth of A. niger and higher productivity of Ffase (58.3 U/l per h) than with the medium before optimization. The optimized medium of SmF when used in SSF, did not improve the Ffase productivity and therefore medium for SSF was optimized independent of SmF. After two optimization steps, the media was defined for SSF which supported the growth and high level of Ffase productivity (149.1 U/l per h) in SSF compared to the medium before optimization (81.8 U/l per h) and optimized medium for SmF (58.3 U/l per h). Our results suggested that the optimized media for SmF and SSF for the production of Ffase have to be different.     

Biodegradation of organochlorine pesticide, endosulfan, by a fungal soil isolate, Aspergillus niger

Endosulfan is a chlorinated pesticide widely used in India for the protection of cotton, tea, sugarcane and vegetables. The persistence of endosulfan in environment and toxic effects on biota necessitate its removal. The role of soil fungi in recycling organic matter prompted us to attempt biodegradation of endosulfan using fungi. This study aims at enrichment, isolation and screening of fungi capable of metabolizing endosulfan. In all, 16 fungal isolates were obtained by enrichment of soil samples that had seems exposed to endosulfan before. Isolates were screened by a gradient plate assay, and results were confirmed by broth assay. On the basis of tolerance to endosulfan, an isolate, identified as Aspergillus niger was selected for further studies. The culture could tolerate 400 mg ml⁻¹ of technical grade endosulfan. Complete disappearance of endosulfan was seen on 12 days of incubation. Evolution of carbon dioxide during endosulfan metabolism has indicated the complete mineralization of endosulfan. Change in pH of culture broth to acidic range supported the biological transformation. Thin layer chromography (TLC) analyses revealed the formation of various intermediates of endosulfan metabolism including endosulfan diol, endosulfan sulfate, and an unidentified metabolite. The toxic intermediate, endosulfan sulfate, was also metabolized, further resulting in complete mineralization of endosulfan. Direct desulfurization of endosulfan sulfate or a novel pathway could be the mechanism of endosulfan and endosulfan sulfate degradation in Aspergillus niger. The fungal strain isolated by us could prove valuable for bioremediation of endosulfan contaminated soils and waters.     

高温高压条件下黑曲霉孢子的失活参数计算

高温高压灭菌是黑曲霉孢子灭活的主要方式。通过研究黑曲霉孢子在高温高压过程中的死亡率,计算黑曲霉孢子的比热死速率常数和活化能。结果表明,黑曲霉孢子在97、115、121 ℃时的比热死速率常数分别为0.028 39、0.041 59、0.065 92/min,热死的活化能为38 324.479 4 J/mol。为利用高温高压灭活大量曲霉孢子提供了理论基础。     

Enantioselective ester hydrolase from Sphingobacterium sp. 238C5 useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis

A novel enzyme, β-phenylalanine ester hydrolase, useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis was characterized. The enzyme purified from the cell free-extract of Sphingobacterium sp. 238C5 well hydrolyzed β-phenylalanine esters (S)-stereospecifically. Besides β-phenylalanine esters, the enzyme catalyzed the hydrolysis of several α-amino acid esters with l-stereospecificity, while the deduced 369 amino acid sequence of the enzyme exhibited homology to alkaline d-stereospecific peptide hydrolases from Bacillus strains. Escherichia coli transformant expressing the β-phenylalanine ester hydrolase gene exhibited an about 8-fold increase in specific (S)-β-phenylalanine ethyl ester hydrolysis as compared with that of Sphingobacterium sp. 238C5. The E. coli transformant showed (S)-enantiomer specific esterase activity in the reaction with a low concentration (30 mM) of β-phenylalanine ethyl ester, while it showed both esterase and transpeptidase activity in the reaction with a high concentration (170 mM) of β-phenylalanine ethyl ester and produced β-phenylalanyl-β-phenylalanine ethyl ester. This transpeptidase activity was useful for β-phenylalanine β-peptide synthesis.