【作者】 王春林；

【导师】 李德发； 陆文清；

【作者基本信息】 中国农业大学 ， 动物营养与饲料科学， 2005， 博士

【摘要】 通过物理和化学复合诱变处理扬奇青霉（Penicillium janczewskii）,筛选高产α-半乳糖苷酶（GAL）变异株,从培养的环境因素和培养基组分两方面优化了固态发酵（SSF）产酶条件,并进行中试生产,探讨了α-半乳糖苷酶粗制剂的性质及其在肉鸡日粮中的应用。首先建立了测定饲用青霉GAL活力的对硝基酚比色法,建议反应液pH为5.5,温度为40℃,时间10 min,比色波长405 nm。对出发菌株进行紫外线、甲基磺酸乙酯、亚硝基胍多次重复连续的诱变处理,获得产GAL活力较高的5个菌株。对菌株连续传代试验以及循环交替培养,筛选获得稳定产GAL的青霉菌株（Penicillium sp.）MAFIC-6,其基础产酶活力为147 U/g。青霉MAFIC-6合成α-半乳糖苷酶在一定程度上仍受葡萄糖阻遏,也受蔗糖、蜜二糖和棉籽糖部分诱导,说明该菌属于半组成型突变株。三角瓶和托盘SSF试验研究了影响产酶的各种环境因素。结果表明,培养基的初始适宜pH在5.5～6.5,发酵温度为26～32℃,水分宜控制在55%左右,在250 mL三角瓶中装曲量为50.7 g,培养75 h时,产α-半乳糖苷酶活力最高。托盘发酵研究了料曲温度和含水量变化规律,发现随着发酵进程,料曲内部温度可持续升高,水分则减少。对SSF培养基组分优化结果表明,苹果渣、甜菜渣、甘蔗渣部分替代麸皮时能显著提高酶产量,酵母粉和大豆粕是较好的氮源。在培养基中添加0.1%的Tween-80、Triton-100和EDTA均能显著地提高α-半乳糖苷酶产量。按四因素三水平正交设计安排试验。结果显示,豆粕、甜菜渣、硫酸铵和磷酸氢二钾各因素/水平依次为16%、4%、2%和2%时,酶活力达到172 U/g以上。保持硫酸铵含量不变,选择豆粕、甜菜渣和磷酸氢二钾的三因素/水平进行响应面分析试验。结果表明,当培养基中含豆粕19.20%、甜菜渣3.89%和磷酸氢二钾2.92%时,得到最高理论酶活力为174.98 U/g,与实验结果相符。分别在自然状态和调控状态下开展托盘SSF中间规模试验。结果表明,在自然SSF条件下,酵曲温度和含水量变化幅度较大,酵曲温度最高达到43℃,最后料曲水分损失22%,收获GAL活力为128.5 U/g;在调控状态下,通过调节曲房空气的温度和相对湿度,可以维持相对稳定的温度和含水量,酵曲高峰期温度仅维持在33.5～36.5℃,酵曲水分含量下降幅度较小。最后收获GAL活力达到166.3 U/g,比自然发酵酶活提高了29.4%。通风换气和喷无菌水是调节发酵过程中温度和水分的有效措施。GAL粗酶制剂稳定pH范围在3.5～6.5,最适温度50～60℃,在70℃以下时稳定,经过猪胃肠液仍能保持活力70%以上,金属离子影响酶活力。GAL对底物（对硝基苯酚-α-D-吡喃半乳糖苷）Km为1.38 mmol L^-1,V_m为2.58 mmol L^-1s^-1。粗酶制剂中含有其它多种非淀粉多糖酶,在常温能稳定储存。在玉米-豆粕型日粮中添加GAL对肉鸡营养物质利用、生产性能、血清学参数利器官重量的影响进行了研究。结果表明,GAL能改善日粮代谢能水平和营养物质消化率,显著提高肉仔鸡早期生长阶段的生产性能,日粮能量与GAL添加水平间存在一定的互作效应。更多还原

【Abstract】 A hyperproducing α-galactosidase (GAL) strain was screened from Penicillium janczewskii through physical and chemical compound mutagenesis. The conditions of solid-state fermentation (SSF), including cultivating environmental factors and culture compositions, were optimized, and the pilot-scale production of GAL in SSF was performed. Moreover, the properties and applications in broiler on the enzyme preparation were studied. Firstly, the assay of feed Penicillium GAL activity was established as the para-nitrophenol spectrophotometeric method. It was suitable to specify GAL determination under the conditions of pH 5.5, 40℃, 10 min reaction at 405 nm. The original strain was treated repeatedly with ultraviolet light, ethyl methane sulfonate, and nitrosoguanidine in turn, and five strains with hyperproducing GAL were selected. With sequential generation, then circulatory and alternative culture, Penicillium sp. MAFIC-6 was obtained since it is stable and high production. Its basic GAL activity is about 147 U/g. GAL synthesis from MAFIC-6 is repressed by glucose to a certain extent, whereas induced by sucrose, melibiose and raffinose, which suggested the strain is a partially constitutive. Various factors effecting enzyme production were studied in Erlenmeyer’s flasks and trays. The results showed that the best GAL activity could be available with 50.7 g substrate on the initial pH 5.5-6.5 and 55% moisture content, incubating at 26~32℃ for 75 h. Tests on SSF in trays disclosed the changes of temperature and water in the course of fermenting process. When the inner temperature of the culture rises, the water content of culture drops. Apple pulp, beet pulp, and bagasse can partially substitute wheat bran as carbon sources for GAL production. Yeast meal and soybean meal are better nitrogen sources than others. Adding 0.1% Tween-80, Triton-100, or EDTA to the culture markedly improved GAL activity. An orthogonal design was employed to optimize the composition of the culture. GAL activity reached the highest (172 U/g) when the levels of soybean meal, beet pulp, (NH4)2SO4, and K2HPO4 were 16%, 4%, 2% and 2% respectively in the medium. The following response surface methodology indicated that the recipe of soybean meal at 19.20%, beet pulp at 3.89%, and K2HPO4 at 2.92% amounted to the theoretical value 174.98 U/g, coinciding with the practical result. In natural SSF the temperature and water of culture varied greatly and the final activity was 128.5 U/g. However, under controlled condition the temperature and water changed little and the final activity was 166.3 U/g that increased 29.4% compared with the natural. Ventilation and spraying sterile water may be effective measures to mediate temperature and water balance. The crude GAL is optimal at pH 3.5~6.5 and 50~60℃. Metal ions affected GAL activity. The Km and Vm of GAL to para-nitrophenyl-a-D-galactopyranoside is 1.38 mmol L-1 and 2.58 mmol L-1 s-1. Finally, the effects of GAL supplementation of corn-soybean meal diet on nutrient utilization, performance, serum indices and organ weight in broilers were studied. The results indicated that adding GAL enhanced metabolizable energy and nutrient digestibility to the diets, and obviously improved performance of broilers in the early growth stage. The interaction of GAL and energy can be also detected by some parameters determined.更多还原