~(60)Co―γ射线诱变褐色高温单孢菌的研究

以褐色高温单孢菌(Thermomonospora fusca)为出发菌株,通过60Co―γ射线诱变孢子悬液,采用透明圈法初筛和摇瓶培养复筛的方法,获得了一株纤维素酶高产菌株AV5,与出发菌株相比,其产酶能力提高1.8倍。     

Constitutive Expression of Thermobifida fusca Thermostable Acetylxylan Esterase Gene in Pichia pastoris

A gene encoding the thermostable acetylxylan esterase (AXE) in Thermobifida fusca NTU22 was amplified by PCR, sequenced and cloned into the Pichia pastoris X-33 host strain using the vector pGAPZαA, allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular AXE production, as high as 526 U/mL in the Hinton flask culture broth. The purified enzyme showed a single band at about 28 kDa by SDS-polyacrylamide gel electrophoresis after being treated with endo-β-N-acetylglycosaminidase H; this agrees with the predicted size based on the nucleotide sequence. About 70% of the original activity remained after heat treatment at 60 °C for three hours. The optimal pH and temperature of the purified enzyme were 8.0 and 60 °C, respectively. The properties of the purified AXE from the P. pastoris transformant are similar to those of the AXE from an E. coli transformant.     

Letter to the Editor

Abstract

The water sorption isotherms of the Erythrina fusca Lour bark at 30 and 40°C were determined over relative humidity ranging from 55 to 85%. The equilibrium moisture content was determined gravimetrically. The moisture sorption isotherms showed that the equilibrium moisture decreases with increase of temperature. Six models were used for to fit the experimental curves of equilibrium humidity. Parameters of each equation were determined by nonlinear regression analysis. The isosteric heat of moisture sorption was calculated using the Claussius-Clapeyron equation.     

中南半岛珍贵树种刀状檀

刀状檀是中国国内最早流行的黑酸枝木类树种。工作人员通过对树种植物学特征的调查研究并对比木材构造特点,认为刀状檀是东南亚黑酸枝木的代表树种,并可以以树种名称作为木种名在商业活动和鉴定领域使用。     

Cloning and functional analysis of cis-prenyltransferase from Thermobifida fusca

cis-Prenyltransferase catalyzes the synthesis of Z,E-mixed prenyl diphosphates by a condensation of isopentenyl diphosphate to an allylic diphosphate. A novel gene encoding a cis-prenyltransferase is cloned from Thermobifida fusca. It showed a unique substrate specificity accepting dimethylallyl diphosphate as a shortest allylic substrate, and synthesizes polyprenyl products up to C₇₀.     

Experimental Determination and Modeling of Sorption Isotherms of Erythrina Fusca Lour Bark

The water sorption isotherms of the Erythrina fusca Lour bark at 30 and 40°C were determined over relative humidity ranging from 55 to 85%. The equilibrium moisture content was determined gravimetrically. The moisture sorption isotherms showed that the equilibrium moisture decreases with increase of temperature. Six models were used for to fit the experimental curves of equilibrium humidity. Parameters of each equation were determined by nonlinear regression analysis. The isosteric heat of moisture sorption was calculated using the Claussius-Clapeyron equation.     

刀状黑黄檀与黑黄檀系同种异名之考证

刀状黑黄檀和黑黄檀无论是在木材解剖特征和植物学特征上均难以分辨,经木材解剖和树木形貌比对,证实二者系同一树种,所以应宜将二者合并,只保留刀状黑黄檀这一树种名称,黑黄檀作为异名。     

重组Thermomonospora fusca纤维素酶Cel6A在毕赤酵母中的表达及纯化

目的在毕赤酵母中表达Thermomonospora fusca纤维素酶Cel6A。方法提取Thermomonospora fusca基因组DNA作模板,PCR扩增纤维素酶Cel6A基因,克隆到毕赤酵母表达载体pGAPZαA上,用电击法将线性Cel6A-pGAPZαA核酸片段转化毕赤酵母X-33株,克隆PCR鉴定法筛选阳性转化株,培养传代。选择稳定株,培养3d后取培养液上清,用His-BindQuick Cartridge300提取表达的重组Thermomonospora fusca纤维素酶Cel6A。结果一步His-Bind Quick Cartridge300层析可提取电泳纯的重组Thermomonospora fusca纤维素酶Cel6A,SDS-PAGE分析显示相对分子质量约为60000,收率为200mg/L,用羧甲基纤维素法测活性为500U/mg,用滤纸法测活性为1U/mg。结论已在毕赤酵母中成功表达了Thermomonospora fusca纤维素酶Cel6A。     

共表达谷氨酰-tRNA还原酶增强染料脱色过氧化物酶在大肠杆菌中的表达活性

染料脱色过氧化物酶(dye-decoloorizing peroxidase,DyP)属于以血红素为辅基的新型过氧化物酶类,常因缺乏辅因子而导致催化活性低。将来源于褐色嗜热裂孢菌(Thermobifida fusca)的染料脱色过氧化物酶基因(TfuDyP)与大肠杆菌谷氨酰-tRNA还原酶基因(hemA),构建重组质粒phemA-DyP,转化至E.coli BL21中进行共表达。分别以2,2-联氮-二(3-乙基苯并噻唑-6-磺酸)二铵盐(ABTS)和顽固性染料活性蓝19(RB19)、溴酚蓝、溴甲酚绿为底物检测TfuDyP的催化活力以及染料脱色效率。结果表明,诱导后的共表达菌株pAD胞内血红素含量为9. 8μmol/L,而单独过表达基因TfuDyP的菌株pD仅为3. 4μmol/L。TfuDyP纯酶的全波长扫描分析表明,在菌株pAD中DyP酶与血红素的结合度相比pD有较大幅度的提升。pAD菌株表达的DyP酶活力较pD菌株提高了110%,酶活力的提高使其在染料脱色应用方面也得到增强。在pAD菌株培养基中分别添加谷氨酸(Glu)、FeCl2使得胞内血红素含量、DyP酶活力和染料脱色效率比未添加时进一步提高。以上结果为TfuDyP的功能开发奠定了基础,同时也为其他血红素依赖性过氧化物酶的研发提供借鉴。