嗜热子囊菌利用短链有机酸生产角质酶

以嗜热子囊菌(Thermobifida fusca WSH03-11)发酵生产角质酶为模型,研究微生物利用市政污泥厌氧酸化所产短链有机酸为碳源发酵生产高附加值产品的可能。发现：(1)以丁酸、丙酸和乙酸为碳源时,有机酸和氮元素浓度分别为8.0 g/L和1.5 g/L有利于角质酶的生产;而以乳酸为碳源时,最适有机酸和氮源浓度分别为3.0 g/L和1.0 g/L;(2)改变诱导物角质的浓度,以丁酸、丙酸、乙酸和乳酸为碳源,分别比优化前提高了31.0%、13.3%、43.8%和73.2%;(3)在四种有机酸中,T. fusca WSH03-11利用乙酸的速率最快,平均比消耗速率是丙酸的1.3倍,丁酸的2.0倍及乳酸的2.2倍;以丁酸为碳源时的酶活(52.4 U/mL)是乳酸的1.7倍、乙酸的2.5倍和丙酸的3.2倍;角质酶对乳酸的得率(12.70 u/mg)分别是丁酸的1.4倍、丙酸的3.0倍和乙酸的3.8倍;(4)以混合酸为碳源生产角质酶,T. fusca WSH03-11优先利用乙酸,而对丁酸的利用受到抑制。进一步研究发现,混合酸中0.5 g/L的乙酸将导致丁酸的消耗量降低66.7%。这是首次利用混合酸作碳源发酵生产角质酶的研究报道。这一研究结果进一步确证了利用市政污泥厌氧酸化所产有机酸为碳源发酵生产高附加值产品的可行性,为以廉价碳源生产角质酶奠定了良好的基础。     

一株嗜热子囊菌产生的碱性耐热过氧化氢酶及其应用潜力

研究了一株嗜热子囊菌产过氧化氢酶的摇瓶发酵条件,并对其在纺织工业中的应用潜力进行了评价。以20 g/L糊精和1%(V/V)乙醇为混合碳源时,过氧化氢酶酶活达到1594 u/Ml,比以糊精和乙醇单独为碳源时过氧化氢酶的活力之和还高23%。改变培养基的初始Ph、提高发酵液中的溶氧水平及添加外源过氧化氢,过氧化氢酶的产量进一步提高到2762 u/Ml,比优化前提高了5.8倍。将嗜热子囊菌的过氧化氢酶同来源于牛肝、黑曲霉的过氧化氢酶进行了热(70℃, 80℃, 90℃)、碱(Ph 9.0, Ph 10.0, Ph 11.0)稳定性的比较。结果显示,产自嗜热子囊菌的过氧化氢酶对高温和强碱性的耐受性能明显优于其它来源的酶,在纺织染整工艺中具有良好的应用潜力。     

混合添加乙醇和H2O2对嗜热子囊菌产过氧化氢酶的影响

在以嗜热子囊菌( T. aurantiacus WSH03-01BC)生产过氧化氢酶的7L罐发酵研 究中,发现混合添加适量的乙醇(75%)和H2O2可以促进菌体产酶.在发酵36h和 48h分别添加0.8%(v/v)的乙醇时,酶活比对照提高了34.3%;当添加乙醇的总量超过2.4 %时 ,对菌体的生长及产酶有明显的抑制作用;在发酵36～60h恒速流加1.6%的乙醇,CAT的酶活 达到2519U/mL,单位细胞产酶能力提高了47.3%;在发酵36h～60h恒速流加1.6%的乙醇并在4 8h混合添加0.4%的H2O2时,CAT的酶活达到2786U/mL,比对照提高了50.1% .     

嗜热子囊菌JCM12803来源的双功能木聚糖/纤维素酶

纤维素和木聚糖的充分利用对于生物燃料的生产是非常重要的。文中利用PCR的方法从嗜热子囊菌Thermoascus crustaceus JCM12803中克隆到一个新颖的双功能木聚糖/纤维素酶基因Tcxyn10a,并将其在毕赤酵母Pichia pastoris GS115中实现高效异源表达。经过蛋白纯化和酶学性质研究分析,TcXyn10A的最适pH值和最适温度分别为5.0和65-70℃,能够在酸性至碱性(pH 3.0-11.0)条件下和60℃下保持稳定;对榉木木聚糖、小麦阿拉伯木聚糖、羧甲基纤维素钠和地衣多糖均有降解活性,比活分别为(1 480±26)U/mg、(2 055±28)U/mg、(7.4±0.2)U/mg和(10.9±0.4)U/mg;同源建模结构以及分子对接试验表明,双功能酶TcXyn10A只含有单一催化结构域,且木聚糖底物与纤维素底物共用一条催化通道。文中为探索双功能酶结构与其功能的关系提供了很好的素材。     

嗜热链霉菌过氧化氢酶的纯化及性质研究

嗜热链霉菌(Thermostreptostreptomyces sp.)T485的除去菌体的培养液,经硫酸铵盐析,Sepha- dex G—100、DEAE—Sephadex A-50及羟基磷灰石等柱层析,得到了凝胶电泳均-的过氧化氧酶,纯化了954倍,得率为7％。用浓度梯度PAGE测定分子量为152000,SDS—PAGE测定亚基分子量为57000,凝胶薄层等电点聚焦测定等电点为4．25。过氧化氢酶的反应最适温度为60℃,最适pH为7．0;对H2O2的K为50 mmol·L-1,Vmas值为6．0 mmol·min-1·mg-1。Nall3和Hg2+对酶活力有强烈抑制作用．Ca2+对酶活力有激活作用。测定了波长200—500nm的吸收光谱,在405nm处有明显的吸收峰。根据受NaN3,抑制和吸收光谱性质,推测它为含血红素酶。此外还测定了过氧化氢酶的氨基酸组成。     

嗜极菌的极端酶

近年来,嗜极菌极端酶的分离鉴定取得了很大进展。本文简述了极端酶的分离纯化及其某些生化特性、极端酶的稳定因素和应用。极端酶的发现与研究,拓宽了传统的生物催化及应用的范围。但是,关于极端酶的稳定机制及其工业应用,仍有许多难题需要解决。     

嗜热子囊菌两个中国新记录种

对采自云南的标本进行研究,描述了两种嗜热子囊菌中国新记录种,分别是太瑞斯梭孢壳霉[Thielavia terrestris (Apinis) Malloch & Cain]和丝衣霉状篮状菌[Talaromyces byssochlamydioides Stolk & Samson],附插图。研究标本保存在山东农业大学植物病理学标本室(HSAUP)。     

嗜热栖热菌α-葡萄糖苷酶基因的表达及酶学性质研究

用PCR方法从嗜热栖热菌(Thermus thermophilus)HB27中扩增出编码α-葡萄糖苷酶基因hbg,将其克隆到大肠杆菌(Escherichia coli)表达载体pET28a( )上,电击转化E.coliBL21(DE3),获得高效表达hbg基因的大肠杆菌重组菌。重组菌经IPTG诱导表达,SDS-PAGE检测表达蛋白相对分子质量约为59kD,与预期分子量相符。经镍柱和阴离子交换柱纯化的重组表达的α-葡萄糖苷酶HBG最适温度为95℃,最适pH值为5.0。     

产几丁质酶褐色喜热裂孢菌的分离鉴定及产酶特性初步研究

采用选择性培养基从土壤中分离到1株产几丁质酶的微生物菌株YX,经形态和分子鉴定为褐色喜热裂孢菌(Thermobifida fusca)。进一步在摇瓶中比较了T.fusca YX在纤维二糖、几丁质、或羧甲基纤维素钠为碳源的培养基中的产酶特性,YX菌株在5 L发酵罐中以几丁质为碳源的培养基发酵到22 h左右时发酵液几丁质酶活即可达到1.7 U/m L。本文首次报道褐色喜热裂孢菌能够产生几丁质酶,具有潜在的应用价值。     

Codon optimized Thermobifida fusca hydrolase secreted by Bacillus megaterium

Production and secretion of a 28,172 Da hydrolase from Thermobifida fusca (TFH) in Bacillus megaterium MS941 and WH323 was investigated in shake flask and pH controlled bioreactors. Successful production of heterologous TFH was achieved by adapting the original tfh gene to the optimal codon usage of B. megaterium. A codon adaption index close to one was reached. The codon optimized tfh was cloned into an open reading frame with DNA sequence for the N-terminal signal peptide of B. megaterium lipase A and a C-terminal His(6)-tag, all under the control of a xylose inducible promoter. Successful TFH production and secretion were observed using batch reactor cultivations with complex medium. Expression of the tfh gene from the P(xylA) promoter and secretion of produced TFH were compared in detail to batch reactor cultivations with semi-defined growth medium. For the first time, significant TFH secretion was achieved using a semi-defined medium in glucose limited fed batch cultivations yielding 10-fold higher cell densities compared to LB medium cultivation. Comparable volumetric TFH activities were obtained for both cultivation strategies. Surprisingly, measured specific TFH activities exhibited drastic discrepancies between preparations from LB and semi-defined medium grown B. megaterium. TFH recovery by Ni-chelate affinity chromatography resulted in higher purification factors when LB medium was used. These results indicated that secreted TFH is favorably produced by batch cultures of B. megaterium WH323 in LB medium.     

Enhanced cutinase production of Thermobifida fusca by a two-stage batch and fed-batch cultivation strategy

In this study, cutinase production by Thermobifida fusca WSH03-11 was investigated with mixed short-chain organic acids as co-carbon sources to demonstrate the possibility of producing high value-added products from organic wastes. T. fusca WSH03-11 was cultured with different combinations of butyrate, acetate, and lactate with a purpose of increasing cutinase activity. The optimum proportion of butyrate, acetate, and lactate was 4:1:3. In batch cultivation, acetate and lactate were consumed quickly, while the consumption of butyrate was depressed in the presence of acetate with a concentration higher than 0.5 g/L. Based on these results, a two-stage batch and fed-batch cultivation strategy was proposed: a batch culture with acetate and lactate as the co-carbon sources in the first 10 h, and then a fed-batch culture with a constant butyrate feeding rate of 12 mL/h during 11∼20 h. By this two-stage cultivation strategy, cutinase activity, dry cell weight, and consumption rate of butyrate were increased by 70%, 103.4%, and 4.3-fold, respectively, compared to those of the batch cultivation. These results provided a novel and efficient way to produce high value-added products from organic wastes.     

Enhanced cutinase production with Thermobifida fusca by two-stage pH control strategy

A mutant of Thermobifida fusca ATCC 27730 was used for cutinase production. Acetate was the most suitable carbon source for cell growth and cutinase production compared with others. The pH was one of the most important factors affecting cutinase yield and productivity. Batch cutinase fermentations by mutant Thermobifida fusca WSH04 at various pH values ranging from 7.0 to 7.9 were studied. Based on the effects of different pH values on the specific cell growth rate and specific cutinase formation rate, a two-stage pH control strategy was developed, in which the pH was set at 7.3 for the first 20 h, and switched to 7.6 afterwards. By applying this two-stage pH control strategy for cutinase fermentation, the maximal cutinase activity reached 19.8 U/mL.     

Chitin binding by Thermobifida fusca cellulase catalytic domains

Cellulose is a linear homopolymer of beta 1-4 linked glucose residues. Chitin is similar to cellulose in structure, and can be described as cellulose with the hydroxyl group on the C2 carbon replaced by an acetylamine group. Both cellulose and chitin form tightly packed, extensively hydrogen-bonded micro-fibrils. Up to now, binding of cellulase catalytic domains (CDs) to chitin has not been reported. In this article, binding of the CDs of Thermobifida fusca Cel6A, Cel6B, Cel48A, Cel5A, and Cel9A to alpha-chitin was investigated. The CDs of endocellulases, Cel6A and Cel5A did not bind to alpha-chitin; one exocellulase, Cel48A CD bound alpha-chitin moderately well; and the exocellulase Cel6B CD and the processive endocellulase Cel9A CD bound extremely tightly to alpha-chitin. Only mutations of Cel6B W329C, W332A and G234S and Cel9A Y206F, Y206S and D261A/R378K caused weaker binding to alpha-chitin than wild-type, and all these mutations were of residues near the catalytic center. One mutant enzyme, Cel9A D261A/R378K had weak chitinase activity, but no soluble products were detected. Chitotriose and chitotetraose were docked successfully to the catalytic cleft of Cel9A. In general, the positioning of the sugar residues in the model structures matched the cellooligosaccharides in the X-ray structure. Our results show that the binding of chitin by a cellulase can provide additional information about its binding to cellulose.     

Cloning, expression and identification of a new trehalose synthase gene from Thermobifida fusca genome

Trehalose is a disaccharide with two glucose mole-cules linked in an α,α-1,1-glycosidic linkage. The onlyreducing group in each of its glucose molecules has beenused up for the formation of α,α-1,1-glycosidic linkage,therefore trehalose is a nonreducing disaccharide withhigh stability against the disruption caused by such factorsas temperature and extreme pH of environment [1]. Ithas been well established that many organisms will copewith external stress conditions by increasing the levelo…     

Characterization of a new cutinase from Thermobifida alba for PET-surface hydrolysis

A new cutinase from Thermobifida alba (Tha_Cut1) was cloned and characterized for polyethylene terephthalate (PET) hydrolysis. Tha_Cut1 showed a high degree of identity to a T. cellulolysitica cutinase with only four amino acid differences outside the active site area, according to modeling data. Yet, Tha_Cut1 was more active in terms of PET surface hydrolysis leading to considerable improvement in hydrophilicity quantified based on a decrease of the water contact angle from 87.7° to 45.0°. The introduction of new carboxyl groups was confirmed and measured after esterification with the fluorescent reagent alkyl bromide, 2-(bromomethyl) naphthalene (BrNP), resulting in a fluorescence emission intensity increase from 980 to 1420 a.u. On the soluble model substrates p-nitrophenyl acetate (PNPA) and p-nitrophenyl butyrate (PNPB), the cutinase showed Km values of 213 and 1933 μM and k_cat values of 2.72 and 6.03 s^?1 respectively. The substrate specificity was investigated with bis(benzoyloxyethyl)terephthalate (3PET) and Tha_Cut1 was shown to release primarily 2-hydroxyethyl benzoate. This contrasts with the well-studied Humicula insolens cutinase which preferentially liberates terminal benzoic acid from 3PET.