盾壳霉几丁质酶和葡聚糖酶酶动力学性质初步研究

盾壳霉几丁质酶和葡聚糖酶酶动力学性质研究表明:几丁质酶和葡聚糖酶的酶促反应,在本试验计量范围内,其反应速度与酶量和底物浓度均呈正相关,但两种酶促反应适宜的温度和pH值有较大差。几丁质酶降解几丁质的最适反应温度为50℃,最适pH值为8.0,葡聚糖酶降解葡聚糖的最适反应温度为60℃,最适pH值为3.0。     

β-1,3-1,4葡聚糖酶基因双拷贝工程菌株的构建与发酵研究

本试验旨在研究β-1,3-1,4葡聚糖酶双拷贝基因毕赤酵母工程菌株的构建及发酵。首先,通过对质粒pGAP-glu-opt进行PCR扩增获得密码子优化的β-1,3-1,4葡聚糖酶基因glu-opt,用EcoR I和Not I双酶切后与毕赤酵母表达载体pPIC9K连接,获得重组质粒p9K-glu-opt。该重组质粒经Sac I线性化后转化毕赤酵母GS115菌株,利用不含组氨酸的MDS平板和摇瓶培养,筛选重组菌株,命名为GS115/9K-glu-opt。制备GS115/9K-glu-opt感受态细胞,再用线性化的重组质粒pPIC-glu-opt二次转化,在含有抗生素Zeocin的YPDS平板上筛选glu-opt基因双拷贝重组菌株GS115/2xglu-opt。双拷贝重组菌在10 L发酵罐中进行高密度发酵培养及甲醇诱导表达,β-1,3-1,4葡聚糖酶最高酶活达到21 600 U/mL,约为单拷贝工程菌株X33/pPIC-glu-opt发酵活力的1.44倍;蛋白表达量达8.4 g/L,约为X33/pPIC-glu-opt的1.68倍。     

Callose and β‐1,3‐glucanase inhibit Phytophthora cinnamomi in a resistant avocado rootstock

Phytophthora root rot (PRR) of avocado, caused by Phytophthora cinnamomi, is a significant threat to sustainable production wherever the crop is grown. Resistant rootstocks in combination with phosphite applications are the most effective options for managing this disease. Recently, the mechanisms underpinning PRR resistance have been investigated by the avocado community. Here, biochemical assays and confocal and scanning electron microscopy were used to investigate early defence responses in PRR resistant and ‐susceptible avocado rootstocks. Zoospore germination and subsequent hyphal growth for the pathogen were significantly inhibited on the surface of resistant avocado roots. When penetration occurred in the resistant R0.06 rootstock, callose was deposited in the epidermal cells, parenchyma and cortex of roots. In addition, β‐1,3‐glucanase was released early (6 h post‐inoculation, hpi) in response to the pathogen, followed by a significant increase in catalase by 24 hpi. In contrast, susceptible R0.12 roots responded only with the deposition of lignin and phenolic compounds incapable of impeding pathogen colonization. In this study, PRR resistance was attributed to a timely multilayered response to infection by P. cinnamomi.     

Influence of environmental pH modulation on efficiency of apoplastic PR proteins during Fusarium culmorum– wheat seedling interaction

Variation of apoplastic pH by Fusarium culmorum and its influence on the production, activity and isoenzymes patterns of the pathogenesis‐related (PR) proteins β1,3‐glucanase, chitinase and peroxidase enzymes were detected in apoplastic fluids (AFs) from infected wheat seedlings. The time course in the 24–48 h interval post infection was characterized by an increase in activity and isoenzymatic differential induction of the selected PR proteins and by a concomitant rise of apoplastic pH. Chitinase attained maximum activity at pH 8·0 in the case of inoculated seedlings. Optimal β1,3‐glucanase activity in the pH range 6·0–8·0, was observed at pH 7·0. Peroxidase was strongly affected by pH, with enzyme activity having a maximum rate at pH 6·0 and thereafter rapidly declining at higher pH. Maximum peroxidase activity paralleled the appearance of the complete isoenzymatic pattern. In order to investigate the biological role of PR proteins in AFs, the in vitro antifungal activity was evaluated. In the interval 0–6 h, pH of macroconidia suspensions rose up to 7·2. AFs revealed inhibitory activity against germinating macroconidia of F. culmorum by decreasing the germination efficiency of macroconidia apical compartments, while this effect was compensated by an increased germination capacity of middle compartments. Present results suggest that during infection of wheat seedlings by F. culmorum the pH modulation favours host colonization by enhancing the activity of pectin lyase, and simultaneously inhibits the capacity of the host to oppose the pathogen by interfering with peroxidases which represent an important component of the defence arsenal.     

Effect of micronutrients on B‐N‐Oxalyl l‐α, β‐diamino propionic acid level and its biosynthesis in Lathyrus Sativus

The detoxification of L. sativus grains by spraying of 0.5 ppm cobalt (nitrate) and 20 ppm molybdehum (ammonium molybdate) salts at the maximun flowering stage ‐ a suggestion based on preliminary findings ‐ has been confirmed in this investigation. Regulatory mechanisms of these micronutrients at the enzymatic level were also studied. On the basis of these observations, the involvement of a hitherto unknown biosynthetic pathway of BOAA cannot be ruled out.     

展示在巴斯德毕赤酵母细胞表面的内切-1,4-β-葡聚糖酶的酶学性质

本研究目的是利用毕赤酵母细胞表面展示技术来改善内切-1,4-β-葡聚糖酶的酶学性质。结果显示,SDS-PAGE显示展示酶(DEG)的分子量为34.1kDa,对CMC-Na有底物专一性。与游离酶(FEG)相比,DEG的最适作用温度和最适作用pH值未发生变化,分别为70℃和4.0;对底物的亲和力降低;pH 2.5~8.5时酶活残留率为60%,比游离酶高0.5倍;温度70℃时酶活无损失,残留率为102%,同等条件下比游离酶高1.5倍;DEG重复使用10次以后保留活性仍为65%;而且展示酶对Co2+、Mn2+、Hg2+等重金属离子的耐受力也大大增强。本研究结果表明,展示内切-1,4-β-葡聚糖酶的酶学性质得到大幅度改善,其酸热稳定性、重金属离子耐受性和操作稳定性均有所提高,DEG作为一种酵母全细胞生物催化剂在各种纤维素降解领域具有很广阔的应用前景。     

The effects of pelleting and glucanase supplementation in hulled barley based diets on feed manufacture,broiler performance,and digesta viscosity

Feeding broilers barley-based diets requires special consideration primarily due to effects on increased digesta viscosity and decreased nutrient digestion. Pelleting and glucanase supplementation are commonly performed prior to feeding broilers barley-based diets; however, the interaction of these practices is complex. The objective of this study was to establish a comprehensive evaluation of glucanase efficacy including: degree of processing, activity postpelleting, broiler performance, and digesta viscosity. Treatments were arranged in a 5 (diet formulation) × 2 (processing) factorial in a randomized complete block design with 8 replications/treatment. The 5 diet formulations consisted of positive control (PC), negative control (NC), glucanase A (GA) 125 or 1,000 β-Glu-U/kg feed, and glucanase B (GB) 1,000 β-Glu-U/kg feed. The PC and NC diets differed in metabolizable energy by 150 kcal/kg and enzymes were added to NC formulations. Diets were either fed as unprocessed mash or ground pellets. Diet formulation × processing did not interact for feed intake (FI), FCR, or total tract viscosity (P > 0.05); however, a trend was observed for ending bird weight, demonstrating that for ground pellets, GA 1,000 β-Glu-U/kg feed was improved relative to NC and similar to PC (P = 0.0903). Benefits associated with GB were not of similar magnitude, perhaps in part due to a 50% decrease in activity postpelleting. In addition, GA benefits were not suggested for unprocessed mash. The main effect processing was significant (P < 0.0001) demonstrating that broilers fed ground pellets resulted in greater pen ending bird weight, FI, and bird live weight gain (LWG) compared to birds fed unprocessed mash diets. Evaluations of glucanase should go beyond in vitro activity and include live bird performance using feed that has undergone pelleting.