植酸酶工程菌产酶条件优化研究

为研究影响植酸酶酵母工程菌的产酶因素，利用摇瓶发酵对毕赤酵母工程菌产植酸酶的最佳甲醇诱导量、诱导时间等因素进行实验分析。结果显示，残留甘油、甲醇浓度、诱导培养时间对植酸酶生产具有重要影响，其中甘油残留会使植酸酶分泌时间延迟，1.5% 甲醇具有最佳诱导效果，在第2天酶活可以达到约1 100 U/mL，低浓度甲醇诱导产酶缓慢积累，而高浓度甲醇对菌体产生毒害。该研究为进一步优化植酸酶工程菌高密度发酵工艺奠定了基础。     

转植酸酶基因海水小球藻生理生化的初步研究

从42个转植酸酶基因小球藻单克隆藻株中筛选出植酸酶产量较高的8个株藻,经过7次传代,各藻株的植酸酶活保持在稳定的状态,证实植酸酶基因在小球藻中获得了稳定表达.对转基因小球藻所产生植酸酶的性质进行了初步研究,结果表明此酶最适作用pH值为3.8和6.0,最适作用温度为65℃.在相同培养条件下,转基因藻与未转基因藻生理指标并无明显差异,说明外源植酸酶基因的转入对小球藻的生长及生理指标无明显影响.     

不同比生长速率对毕赤酵母发酵生产植酸酶的影响

不同比生长速率控制条件下毕赤酵母代谢流流向不同,从而导致产酶量的不同。为了得到毕赤酵母发酵生产植酸酶的最适比生长速率,对毕赤酵母工程菌50 L发酵生产植酸酶的工艺条件进行了研究。结果表明,通过调节甲醇的流加速度,控制菌体比生长速率为0.02 h^-1,发酵结束时,植酸酶酶活17 445 U/mL,比对照提高69.7 %,该比生长速率对发酵生产植酸酶最有利。     

无花果曲霉植酸酶发酵及酶动力学

目的：研究野生型无花果曲霉植酸酶的动力学性质。方法：通过限制培养基中的无机磷,无花果曲霉发酵获得植酸酶。纯化后,对酶的动力学性质进行了研究。结果：该酶作用底物植酸钠的Km值为64．7μmol/L,对于植酸钠,植酸酶的最适反应pH为5．5温度为50℃。结论：无花果曲霉植酸酶在pH3.5－8．040℃以下比较稳定;不同的金属离子螯合剂对酶活性影响不一样,Ca^2 对酶活力有轻微的激活作用,Al^3 几乎无影响,Mg^2 、Fe^2 、Cu^2 对酶活力有抑制作用,Co^2 、Hg^2 、EDTA有较强的抑制作用。     

多角体蛋白对植酸酶的包埋

植酸酶是水解植酸及其盐类生成肌醇和磷酸的一类酶的总称。作为一种新型饲料添加剂,植酸酶在动物营养及环境保护等领域具有很大的应用潜力。为了获得高活性的植酸酶app A,首先将app A基因克隆到载体p Fast Bac Dual中,构建转移载体p Fast Bac Dual-polyh-app A;然后转化大肠杆菌Escherichia coli DH10Bac感受态细胞;通过位点特异性转座,将app A基因整合到Bacmid穿梭载体中,构建表达质粒Bacmid:Ac-polyh/app A。最后,通过脂质体将表达质粒转染Sf9昆虫细胞。Western blotting和酶活测定法对app A的表达和活性进行分析,结果表明在昆虫细胞中app A酶表达成功,植酸酶能够被包埋进多角体内部,并具有较好的植酸酶活性,为植酸酶大规模生产和应用提供了可参考利用的技术。     

解淀粉芽孢杆菌中性植酸酶基因的克隆及其在毕赤酵母中的表达

采用PCR法从解淀粉芽孢杆菌BA11中克隆到一个中性植酸酶phyc基因,将该基因克隆到毕赤酵母表达载体pPIC9K上并电转化至宿主细胞GS115后进行诱导表达。SDS-PAGE试验表明:该重组中性植酸酶在毕赤酵母宿主细胞中实现了高效分泌性表达。植酸酶活性测定结果显示阳性克隆子在诱导72h酶活性达到最高值,活性为2330U/L。     

毕赤酵母表达重组植酸酶酶学性质的比较研究

通过酶学性质比较毕赤酵母GS115表达的E.coli K12植酸酶(appA)及其突变体植酸酶(appA NR)和黑曲霉植酸酶(phyA)的最适pH、温度、热稳定性及对胃蛋白酶和胰蛋白酶的耐受性.结果表明,在95℃,加热10 min,appA NR可残余90％左右的活力,而其他两种只残余20％～50％的活力;appAN...     

产植酸酶菌株的筛选、诱变及酶学性质初探

从富含有机质的环境中采集土样,通过植酸钙平板透明圈法初筛和维生素C-硫酸-钼酸铵-酒石酸锑钾法测定植酸酶酶活复筛,共筛选到5株产植酸酶的霉菌,对这5株菌株进行紫外线和亚硝基胍复合诱变处理,最终获得一株高产植酸酶突变株NTG-506,酶活达到112 U/mL,比原始出发菌株的酶活提高了3.2倍.突变株NTG-506在250mL三角瓶中发酵培养,在装液量60 mL,接种量2%时,培养4 d后酶活达到最高,为124.5 U/mL.植酸酶的最适反应温度35℃,经50℃处理10 min后剩余酶活为原来的57%.     

植酸酶固体发酵高产菌的筛选

经大量菌种初筛和复筛，得到一株在固体发酵产植酸酶较高力株Ｇ２，酶活达２．６千单位Ｇ２菌株特别适合于固体发酵，并对植酸酶固体发酵酶活测定方法进行了探讨。     

芽孢杆菌植酸酶基因在毕赤酵母中的胞内表达

根据已知的枯草芽孢杆菌WHNB02植酸酶phyC基因全序列设计一对引物,采用PCR法从含有该基因的pUC18-phyC质粒上获得了长约1.1kb不含有信号肽序列的植酸酶phyC基因表达片段.经T载体克隆及序列测定后,构建毕赤巴斯德表达载体pPIC3.5K-phyC,并电转化毕赤巴斯德酵母宿主菌GS115.经MD和MM平板筛选、酶活性测定,获得了阳性转化子,并进行了诱导表达.SDS-PAGE分析表明:表达产物分子量为42.01KD,表达量占细胞可溶性总蛋白的24%,并具有植酸酶的生物学活性.酶学性质分析结果显示:胞内表达的植酸酶酶促反应最适pH值为7.5;最适反应温度为70℃;经90℃处理10min,残留酶活性42% 均优于出发菌株天然植酸酶的相应性质.     

Expression, purification and characterization of a phyAm-phyCs fusion phytase

The phyA^m gene encoding acid phytase and optimized neutral phytase phyCs gene were inserted into expression vector pPIC9K in correct orientation and transformed into Pichiapastoris in order to expand the pH profile ofphytase and decrease the cost of production. The fusion phytase phyA^m-phyCs gene was successfully overexpressed in P. pastoris as an active and extracellular phytase. The yield of total extracellular fusion phytase activity is （25.4±0.53） U/ml at the flask scale and （159.1±2.92） U/ml for high cell-density fermentation, respectively. Purified fusion phytase exhibits an optimal temperature at 55 ℃ and an optimal pH at 5.5-6.0 and its relative activity remains at a relatively high level of above 70% in the range ofpH 2.0 to 7.0. About 51% to 63% of its original activity remains after incubation at 75 ℃ to 95 ℃ for 10 min. Due to heavy glycosylation, the expressed fusion phytase shows a broad and diffuse band in SDS-PAGE （sodium dodecyl sulfate-polyacrylamide gel electrophoresis）. After deglycosylation by endoglycosidase H （EndoHf）, the enzyme has an apparent molecular size of 95 kDa. The characterization of the fusion phytase was compared with those ofphyCs andphyA^m.     

植酸酶在水产养殖中的应用（综述）

从植酸酶对水产动物生长性能、磷及其它矿物质生物利用、水产动物饲料营养消化率、水产动物体成分、水产动物血液生化指标等的影响和降低磷酸二氢钙用量,减少环境污染方面综述了新型饲料添加剂植酸酶的作用。探讨了水产饵料中添加植酸酶的最适添加量问题和植酸酶的活性问题。从提高水产动物饲料利用率;减少环境污染,保护生态平衡等方面阐述了植酸酶在水产养殖中的应用前景。     

Ancestral lysozymes reconstructed, neutrality tested, and thermostability linked to hydrocarbon packing

The controversy surrounding the idea that neutral mutations dominate protein evolution is attributable in part to the inadequacy of the tools available to evolutionary investigators. With a few exceptions, most investigations into the force driving protein evolution have relied on indirect criteria for distinguishing neutral and non-neutral variants. To investigate a particular pathway of molecular evolution, we have reconstructed by site-directed mutagenesis likely ancestral variants of the lysozymes of modern game birds (order Galliformes), tested their activity and thermostability and determined their three-dimensional structure. We focused on amino acids at three positions that are occupied in all known game birds either by the triplet Thr 40, Ile 55, Ser 91, or by the triplet Ser 40, Val 55, Thr 91. We have synthesized proteins representing intermediates along the possible three-step evolutionary pathways between these triplets. Although all of these are active and stable, none of these intermediates is found in known lysozymes. A comparison of the structures and thermostabilities of the variants reveals a linear correlation between the side-chain volume of the triplet and the thermostability of the protein. Each pathway connecting the two extant triplet sequences includes a variant with a thermostability outside the range of the extant proteins. This observation is consistent with a non-neutral evolutionary pathway. The existence of variants that are more stable than the extant proteins suggests that selection for maximum thermostability may not have been an important factor in the evolution of this enzyme.     

A new way of enhancing the thermostability of proteases

Thermostability of enzyme can be enhanced by single amino acid substitutions. Recent advances in genetic engineering have made it possible to create novel proteins in a predictable manner where structural information for the protein is available. This 'protein engineering' has already been used to enhance enzyme thermostability, but it is usually not clear which amino acid substitutions should be made. We consider that the following approach should be helpful in engineering proteins with enhanced thermostability: highly conserved residues should be left unchanged; the sequences of known mesophilic and thermophilic proteins should be used to suggest the kinds of changes likely to increase thermostability; and substitutions should be made that increase internal hydrophobicity and that stabilize helices for strong internal packing. We describe here the use of this approach to alter the thermostability of the thermostable neutral protease of Bacillus stearothermophilus, the sequence of which is known. Surprisingly we find that a single mutation that decreases thermostability can require two mutations that increase stability to compensate for it. The effects on stability are not additive, suggesting cooperativity.     

微生物植酸酶对土壤有机磷组分含量及有效性的影响

为提高土壤有机磷素的有效性,并为微生物植酸酶应用于农业生产实践提供理论参考,采用室内培养的方法,研究了不同用量微生物植酸酶菌剂在不同培养时期对土壤有机磷组分含量及有效性的影响.结果表明,添加微生物植酸酶菌剂处理后的土壤活性、中等活性有机磷含量均高于对照,而且与植酸酶菌剂使用量显著相关;其含量随培养时间的延长呈增加趋势,培养至第32d后基本保持平稳;而土壤中稳性和高稳性有机磷含量均低于对照,与植酸酶菌剂使用量显著相关;其含量随培养时间的延长呈减少趋势,培养至第32d后基本保持平稳.添加微生物植酸酶各处理的有机磷总量均低于对照,并随微生物植酸酶添加量的增加而减小;而土壤有效磷含量均高于对照.因此微生物植酸酶可以促进土壤稳定性有机磷向活性有机磷转化,乃至向无机磷转化,从而提高了土壤有机磷素的有效性.     

植酸酶及其应用

在以植物性原料为主的畜禽配合饲料中,大部分磷以植酸磷的形式存在,畜禽因缺乏内源性的植酸酶而对其的利用率极低。本文对配合饲料中添加植酸酶进行了研究。