重组毕赤酵母高密度发酵生产碱性果胶酶的策略

重组Pichia pastoris GS115表达碱性果胶酶的诱导阶段, 最佳初始菌体浓度和甲醇诱导浓度分别为122 g/L和20 g/L, 两者之间最佳比值范围是0.16~0.20 g/g (甲醇/菌体浓度). 在此基础上通过生长阶段甘油的指数流加, 以及诱导阶段基于甲醇比消耗速率和溶氧等参数进行甲醇流加的方式, 将甲醇与菌体浓度比例控制在0.171~0.195 g/g之间. 此时, 酶活达到430 u/mL, 生产强度为4.34 u/mL/h, 实现了碱性果胶酶高效生产。     

芽孢杆菌（Bacillus sp.No.74）大麻脱胶酶系的研究

研究了Bacillussp.No．74菌株,在以玉米粉为碳源,硫酸铁为氮源,35℃,摇瓶培养24h。经测定产生的脱胶酶系有：果胶酶、木聚糖酶、CMC酶、蛋白酶和α－淀粉酶,不产生β－葡萄糖苷酶和C1酶。经底物亲和性试验和紫外分光光度计测定,证明产生的果胶酶为聚半乳糖醛酸裂解酶,其最适作用pH9．6,温度50℃。对温度的稳定性与它所处介质有极密切的关系。     

恒细胞密度发酵策略提高重组毕赤酵母生产碱性果胶酶的表达效率

为了提高重组毕赤酵母生产碱性果胶酶(Alkaline polygalacturonate lyase,PGL)的比速率,开发了一种新的恒细胞密度发酵策略。通过不同的甲醇流加方式,实现发酵过程细胞密度的合理控制。实验结果表明:控制细胞密度为75 g/L的策略为最优,最终单位发酵液体积生产强度和单位菌体生产强度为6.11 U/(mL.h)和81.5 U/(g.h),分别比传统高密度发酵提高了42.1%和191.2%,最终PGL酶活为441.9 U/mL。此外,该策略还具有提高细胞活性和降低蛋白酶降解作用等优势。     

Aluminium reactions with polygalacturonate and related organic ligands

Aluminium (Al), in inorganic monomeric forms, has been recognised as a limiting factor for root growth in many acid soils. Plant tolerance to Al may be achieved by the detoxification (complexation) of Al by organic ligands present in the rhizosphere. The Al-complexing ability of seven organic ligands, citric, oxalic, gluconic, glucuronic, mucic, galacturonic and polygalacturonic (pectin) acids, was investigated. The proportion of organically-complexed Al was determined using colorimetric methods based on differences in reaction rate with pyrocatechol violet or aluminon. The colorimetric methods confirmed that citric acid forms a strong complex with Al at pH 4.2. In contrast, pectin and related organic ligands weakly complexed Al in acidic conditions. In an additional study, the Al-binding ability of pectin and Ca-pectate was compared at a biologically significant concentration of 32 µM Al. Only 29% of free Al remained in solution in the presence of Ca-pectate, while 54% remained when pectin was present. This suggests that Ca-pectate, rather than pectin, is responsible for binding Al in root cell walls and consequently plays an important role in Al toxicity to plants. Root growth of mungbean (Vigna radiata (L.) Wilczek) confirmed differences in the ability of citrate, oxalate and galacturonate to complex Al.     

Metabolism of polymethylgalacturonate in apple fruit cortical tissue during ripening

Changes in the composition and metabolism of polymethylgalacturonate were followed in ripening apples. After the onset of ethylene production and fruit softening total polygalacturonate decreased and the water soluble fraction increased. No change was detected in the overall degree of esterification but the esterification of the water soluble fraction increased. Incorporation of radioactivity from methionine-[¹⁴C] into Me groups on polygalacturonate continued during ripening but incorporation from inositol-[³H] decreased sharply. Cell separation probably depends upon the removal of low ester polygalacturonate from the middle lamella by exopolygalacturonase; the continued incorporation from methionine-[¹⁴C] is probably due to synthesis of new polymethylgalacturonate.     

Properties of polygalacturonate and cell cohesion in apple fruit cortical tissue

Examination of the hydrodynamic properties of polygalacturonate fractions from unripe and ripe apple tissue suggested that the wall bound fraction was degraded during ripening but that the soluble fraction was not. Esterification of cell wall preparations with CH₂N₂ caused solubilisation of polygalacturonate. Acid MeOH caused more extensive solubilization, but this reagent hydrolysed arabinofuranosyl linkages. Both reagents reduced the cohesion of EtOH extracted apple tissue. This effect could also be achieved by treatment with sodium polyphosphate at pH 4 but not by EDTA or chaotropic agents. Free carboxyl groups on polygalacturonate probably maintain cell cohesion through co-operative binding of Ca²⁺ ions. The integrity of primary wall structure is thought to depend upon non-covalent bonding between cellulose, protein and polygalacturonate.