Single amino acid substitutions can further increase the stability of a thermophilic L-lactate dehydrogenase

Lactate dehydrogenases are of considerable interest as stereospecificcatalysts in the chemical preparation of enantiomerically pure-hydroxyacid synthons. For such applications in synthetic organicchemistry it would be desirable to have enzymes which tolerateelevated temperatures for prolonged reaction times, to increaseproductivity and to extend then applicability to poor substrates.Here, two examples are reported of significant thermostabilizations,induced by sitedirected mutagenesis, of an already thermostableprotein, the L-lactate dehydrogenase (EC 1.1.1.27 [EC] , 35 kDa permonomer subunit) from Bacillus stearothermophilus. Thermal inactivationof this enzyme is accompanied by irreversible unfolding of thenative protein structure. The replacement of Argl71 by Tyr stabilizesthe enzyme against thermal inactivation and unfolding. Thisstabilizing effect appears to be based on improved interactionsbetween the subunits in the core of the active dimeric or tetramericforms of the enzyme. The thermal stability of L-lactate dehydrogenasevariants with an active site Arg residue, either in the 171(wild-type) or in the 102 position, is further increased bysulfate ions. The two stabilizing effects are additive, as foundfor the Argl71Tyr/ Gln1O2Arg double mutant, for which the stabilityof the protein in 100 mM sulfate solution reaches that of L-lactatedehydrogenases from extreme thermophiles. All mutant proteinsretain significant catalytic activity, both in the presenceand absence of stnhilfoing salts, and are viable catalysts inpreparative scale reactions.     

凝胶过滤色谱法研究酶解过程中木聚糖分子结构的变化

采用凝胶过滤色谱法研究了木聚糖在酶解过程中分子量分布的变化。结果表明,木聚糖酶对高分子量组分的木聚糖的降解能力较差,而主要降解分子量介于15000～3000之间组分的木聚糖,使得这部分木聚糖变为分子量更低的木聚糖组分。随着酶解时间的延长,酶解得率上升,酶解产物中低聚木糖含量增多,未被酶解的木聚糖的平均聚合度上升,当酶解时间超过4h时,这种变化趋势缓慢。随着酶解轮次的增加,酶解产物中可溶性木聚糖的含量越来越少,木聚糖被降解的难度越来越大。在实际生产中,用来作为低聚木糖生产的木聚糖其聚合度应在20～100之间,酶解时间以4h为宜,重复利用未水解木聚糖的轮次以2轮为宜。     

碱法麦草浆木聚糖酶处理后木质素及碳水化合物的变化

采用UV光谱、凝胶色谱、X—射线衍射及扫描电镜等技术对碱法麦草浆的酶解浆和酶解液进行分析,探讨了木聚糖酶对碱法麦草浆处理后浆中木质素和碳水化合物的变化情况。研究认为,木聚糖酶对碱法麦草浆漂白作用主要是因为木聚糖酶降解了浆中部分木聚糖和残余木质素中的LCC结构,从而使后序漂白中漂液更易渗透,木质素的溶出更多、更易。     

产胞外耐高温纤维素酶细菌的获得和酶的纯化及性质研究

对从高温堆肥中分离得到的一株产胞外耐高温纤维素酶的细菌进行了生理生化鉴定,结果显示其属于嗜热脂肪芽孢杆菌。对其所产纤维素酶进行了分离纯化,该酶反应的最适温度约为66℃,最适pH值为7.0,至少有8个亚基。     

木质素的提纯及其在脲醛树脂胶粘剂中的应用

采用连续浸提和半纤维素酶处理方法,从工业碱木质素（M1）中脱去糖类物质后得到纯度不等的木质素样品M2和M3,同时进行木质素脲醛树脂的制备。结果表明,木质素经过抽提和酶处理后得到木质素M3其纯度为97％,当其添加量为甲醛和尿素总量的35％时,合成的胶粘剂的粘结性能优良,具有资源环保意义。     

化学预处理对玉米秸秆酶解糖化效果的影响

研究了化学预处理对玉米秸秆酶解糖化效果的影响.结果表明效果最好的是:40目玉米秸秆粉用0.5%的稀盐酸在121℃预处理60 min后,加入纤维素酶2.5 g·(100 g)-1和木聚糖酶液2 mL·(100 g)-1,在pH 4.8的乙酸-乙酸钠缓冲液体系中,液固比为10、50℃下糖化48 h,玉米秸秆的总糖产率达48.5%、纤维素和半纤维素的转化率达80.8%.     

Computer-based engineering of thermostabilized antibody fragments

We used the molecular modeling program Rosetta to identify clusters of amino acid substitutions in antibody fragments (scFvs and scAbs) that improve global protein stability and resistance to thermal deactivation. Using this methodology, we increased the melting temperature (T_m) and resistance to heat treatment of an antibody fragment that binds to the Clostridium botulinum hemagglutinin protein (anti-HA33). Two designed antibody fragment variants with two amino acid replacement clusters, designed to stabilize local regions, were shown to have both higher T_m compared to the parental scFv and importantly to retain full antigen binding activity after 2 hr of incubation at 70°C. The crystal structure of one thermostabilized scFv variants was solved at 1.6 Å and shown to be in close agreement with the RosettaAntibody model prediction.     

碱抽提强化木聚糖酶处理对麦草浆二氧化氯漂白的助漂作用

研究了碱抽提强化的木聚糖酶处理对麦草浆二氧化氯漂白的助漂作用。结果显示：木聚糖酶预处理对ClO2脱木素的改善效果不明显,无论是脱木素前预处理（XD）还是脱木素后处理（DX）,ClO2脱木素效率提高不大;采用XDP和DXP漂序,木聚糖酶对麦草浆ClO2漂白的助漂效果不明显,白度提高仅1.5个百分点左右,但可以节省H2O2用量25%～33.3%;采用XDEpP和DXEpP漂序,经H2O2强化的碱抽提Ep后,木聚糖酶对麦草浆ClO2漂白的助漂效果显著增强,与对照样相比,可以提高白度4.5个百分点,同样漂序采用木聚糖酶处理可节省ClO2用量17%～20%。纸浆滤液的紫外-可见吸收光谱分析表明,酶处理后所释放出的发色物质中含有木素或木素衍生物。     

大孔树脂D380固定化橄榄绿链霉菌E-86来源木聚糖酶的研究

选择有代表性的 1 4种吸附和离子交换树脂进行了橄榄绿链霉菌E 86来源的木聚糖酶固定化试验 ,筛选出固定化效果较好的 72 4和D3 80两种树脂 ;对含伯氨基的离子交换树脂D3 80采用戊二醛进行交联固定化 ,研究了其固定化条件。结果表明 ,戊二醛浓度为1 % ,处理 3 0min ,加酶量为 0 8～ 1mL ,酶液pH 5 8,2 5℃ ,5～ 1 0h固定化处理效果最好 ,获得的固定化酶活力可达 64U/ g(载体 )。     

木霉菌株利用作物秸秆产油脂研究进展

木霉能分泌产生纤维素酶、木聚糖酶和木质素酶,并能产生和积累微生物油脂,因此利用木霉能够分解木质纤维素类的作物秸秆,并利用分解木质纤维素产生的单糖、合成和积累油脂.该文介绍了微生物分解秸秆产油的意义,综述了木霉菌作为发酵秸秆产生微生物油脂的研究对象优势和利用木霉生产微生物油脂的研究现状及进展.