高产天冬氨酸酶的大肠杆菌细胞的固定化

用聚乙烯醇凝胶包埋具有高活力天冬氨酸酶的大肠杆菌(Escherichia coli)No.1细胞。该酶的表现活力高达1638 00u／g湿细胞,酶活力的回收率为97．5％。固定化细胞和游离细胞天冬氨酸酶的最适pH均为8．0,最适温度分别为40—45℃和40—55℃。二价金属离子Mn2+、Mg2+、Ca2+和Fe2+对热钝化的天冬氨酸酶活力具有保护作用。在37—45℃下,两种细胞的热稳定性相同。二者在pH6．0的柠檬酸缓冲液中比较稳定。固定化细胞在1mol／L、pH8．0的底物溶液(内含Mn2+1mmol／L)中于4℃冰箱保存6个月,天冬氨酸酶的活力保持不变。用固定化细胞柱连续生产L-天冬氨酸,底物转化为产物的转化率达95％以上;产物的总 收率为91．1％。固定化细胞柱连续运转40天,天冬氨酸酶活力仍保持最初酶活力的90％。     

海因酶产生菌SHNU01的包埋固定化研究

对从土壤中筛选得到的高选择性产D-海因酶菌株SHNU01的性质进行了研究并探讨了用PVA-硼酸法进行包埋固定的效果。研究结果显示:固定化后的细胞与游离细胞相比,最适反应温度由游离细胞的37℃上升为47℃,在此温度下固定化细胞的酶活可达到游离细胞的3倍;连续反应7批后,固定化细胞活力为初始活力的80%。固定化细胞在操作稳定性、贮存稳定性等方面较游离细胞也有较大提高。     

Eupergit C250L固定化D-海因酶及其催化性质

D-海因酶是海因酶法制备D-氨基酸的关键酶。利用Burkholderic cepecia1003菌发酵产酶,所得海因酶纯化后,以Eupergit C250L为载体进行共价固定化。分别考察了酶液蛋白浓度、固定化时间对蛋白固定量和酶活回收率的影响以及固定化前后海因酶催化性质的变化。结果表明:较高的酶液蛋白浓度和较长的固定化时间均有助于改善海因酶的固定化效果;固定化可显著提高海因酶的最适作用温度,但对其最适作用pH影响不大;固定化后海因酶对D,L-BH和MH的米氏常数均有较大幅度的降低。固定化酶反应器的实验表明:40℃下,底物(D,L-BH)1.0 g.L-1,体积流速1.0 mL.min-1,经21 h转化,产物N-Phe质量浓度可达0.47 g.L-1,转化率达43.21%。     

米曲霉F-81产中性蛋白酶的固定化条件及其特性

以海藻酸钠为载体,戊二醛为交联剂固定化米曲霉F-81产中性蛋白酶,研究了固定化条件及固定化酶的性质。结果表明,固定化的最佳条件为:固定化时间1 h、海澡酸钠浓度4%、戊二醛浓度9%、CaCl2浓度0.7 mol/L。在此条件下固定化的中性蛋白酶活力为游离酶活力的68%。固定化酶的最适作用温度为65℃,最适作用pH值为7.0。60℃下酶稳定性较好,80℃下处理60 min,粗酶中几乎检测不到酶活力;中性蛋白酶pH稳定范围为6.5-9.5。Km值为24.83 mg/mL,最大反应速率Vmax为0.043 12 mg/min。     

D-阿洛酮糖3-差向异构酶在枯草芽孢杆菌中的高效表达及固定化细胞研究

将来源于Clostridium cellulolyticum H10的DPEase基因在食品级表达系统Bacillus subtilis中进行产酶研究,在3L发酵罐中高密度发酵最终酶活可达495U/ml,得到高表达量的DPEase酶液。通过硅藻土-海藻酸钠(吸附包埋法)对重组细胞进行固定化研究,结果表明,当海藻酸钠浓度为2%、细胞包埋量为50g/L、CaCl_2浓度为2%、硅藻土浓度为1%时,固定化细胞酶活回收率可达64%,固定化细胞与游离细胞相比最适pH不变,最适温度提高5℃,热稳定性明显提高,连续反应7个批次后转化率仍然为28%,仍保持81%的残余酶活,具有很高的工业应用价值。     

聚苯乙烯树脂固定化D-海因酶的初步研究

D-海因酶广泛用于D-氨基酸的制备研究和生产中,目前已有许多固定化D-海因酶及含D-海因酶细胞的研究报道。尝试了不同功能基团的聚苯乙烯树脂进行D-海因酶的固定化,结果表明功能基为伯氨基和仲氨基效果较好,并选取聚苯乙烯树脂D92进行了固定化D-海因酶的研究。采用该树脂制备固定化酶的最优条件是：酶质量浓度6mg／mL、温度25℃、固化时间12h。所得固定化酶的最适作用温度45℃,最适作用pH为8.5,且作用温度及适宜pH较广,Km为游离酶的1,8倍,且储存稳定性、操作稳定性较好。45℃下半衰期为11d。     

地衣芽孢杆菌固态发酵麻疯树饼粕产蛋白酶及其酶学性质

利用地衣芽孢杆菌作为出发菌株,以提油后的麻疯树饼粕作为培养基,采用固态发酵方式生产蛋白酶。控制培养基湿度为125%,添加10%的乳糖和5%的蛋白胨,30°C条件下发酵3d,蛋白酶产量达到最大值(7465U/g)。酶学性质研究表明,蛋白酶最适作用pH为5?6,最适催化温度为55°C,最大催化速度Vmax为0.0324μmol/(s·mg),Km值为0.0531mmol/L。有机溶剂对酶活力有明显促进作用,10%(V/V)甲醇和5%(V/V)乙醇可以使酶活力分别提高13.55%和70.9%。Mg2+可以使蛋白酶活力提高42.54%,而Hg2+却使酶彻底失活。     

海藻酸钠固定化重组川芎咖啡酸-3-O-甲基转移酶

利用IPTG诱导含有川芎咖啡酸-3-O-甲基转移酶(LCCOMT)的大肠杆菌工程菌E.coli BL21(DE3)/pET28a-LCCOMT,经Ni~(2+)亲和层析、质谱鉴定获得纯化的LCCOMT。采用海藻酸钙凝胶包埋固定LCCOMT,单因素实验考察最佳固定化条件对固定化酶活力的影响,并确定了固定化酶的最适温度、pH值、Km、Vmax与反应批次等酶学性质。确定的条件分别为海藻酸钠质量分数1.5%,CaCl_2浓度2.5 g/L,固定化时间2 h,载体与酶质量比40 000∶1。通过正交实验确定最终固定化条件为CaCl_2浓度2.5 g/L,海藻酸钠质量分数2.0%,固定化时间1 h,载体与酶质量分数35 000∶1时,固定化效果最好,此时相对酶活力为75.43%。固定化酶的最适催化温度为37℃、最适pH值为7.5,较游离酶分别增加0℃和0.5;Km、Vmax分别增高0.40和0.74;实验确定固定化酶的半衰期,连续使用6次,酶活力仍保留50%。     

恶臭假单胞菌完整细胞的二氢嘧啶酶的性质

由恶臭假单胞菌(Pseudomonas putida)73104产的二氢嘧啶酶对5-苯基海因特异性较高,但也可以水解5-甲硫乙基海因和5-甲基海因;不能水解5-取代基上具有正、负电荷的海因衍生物。酶作用的最适pH因底物不同而有差异,但最适反应温度却相同。 Ag+、Cu2+和Zn2+及金属离子螯合剂对酶活力有强烈抑制作用。将细胞用EDTA处理后约有70％酶活力损失,再加入金属离子,只有Fe2+能将酶活力恢复到原来活力的90％以上,其它金属离子无此作用,证明Fe2+是酶反应所必需的。静息细胞和新鲜细胞在弱酸或弱碱条件下,于40℃以下保温均可使细胞酶活力提高2—3倍,且保温的菌悬液的离心上清液几乎无酶活力;而无细胞提取液保温后无酶活力提高现象,说明在上述条件下,保温可改善细胞膜对底物的通透性,提高酶作用效率。氧能抑制酶反应,主要是因为氧与底物形成可逆结合复合物所致。游离细胞的二氢嘧啶酶对5-苯基海因、5-甲硫乙基海因和5-甲基海因的Km值分别为1.61×10-2、2.08×10-2和1.61×10-2M。     

产青霉素酰化酶的大肠杆菌AS1.76的固定化

用在有机溶剂中成型的琼脂凝胶包埋,结合戊二醛处理的方法,制备产青霉素酰化酶的大肠杆菌AS1.76固定化细胞,其细胞含量可达50%以上。固定化细胞水解青霉素 G 钾盐的最适 pH 为8.0,比原细胞约高0.3pH 单位;最适温度与原细胞一样,均为45℃。固定化后,Hg2+对酶抑制作用降低,但酶对Fe3+、Cu2+等金属离子敏感性增加。在无底物情况下,与原细胞相比,固定化细胞对 pH 和热的稳定性增加;4℃保存14个月无活力损失。固定化细胞装柱,在pH7.7,37℃下连续裂解青霉素 G,转化率达95%以上。155天无明显酶活力损失。     

Tagatose production by immobilized recombinant Escherichia coli cells containing Geobacillus stearothermophilus l-arabinose isomerase mutant in a packed-bed bioreactor

Using immobilized recombinant Escherichia coli cells containing Geobacillus stearothermophilus l-arabinose isomerase mutant (Gali 152), we found that the galactose isomerization reaction was maximal at 70 degrees C and pH 7.0. Manganese ion enhanced galactose isomerization to tagatose. The immobilized cells were most stable at 60 degrees C and pH 7.0. The cell and substrate concentrations and dilution rate were optimal at 34 g/L, 300 g/L, and 0.05 h(-1), respectively. Under the optimum conditions, the immobilized cell reactor with Mn2+ produced an average of 59 g/L tagatose with a productivity of 2.9 g/L.h and a conversion yield of 19.5% for the first 20 days. The operational stability of immobilized cells with Mn2+ was demonstrated, and their half-life for tagatose production was 34 days. Tagatose production was compared for free and immobilized enzymes and free and immobilized cells using the same mass of cells. Immobilized cells produced the highest tagatose concentration, indicating that cell immobilization was more efficient for tagatose production than enzyme immobilization.     

Effects of cations on guanylate cyclase of sea urchin sperm.

Mn2+ and to some degree Fe2+, but not Mg+, Ca2+, ba2+, Sr2+, Co2+, Ni2+, La3+, or Fe3+ were able to serve as effective metal cofactors for sea urchin sperm guanylate cyclase. The apparent Michaelis constant for Mn2+ in the presence of 0.25 mM MnGTP was 0.23 mM. In the presence of a fixed free mn2+ concentration, variation in mngTP resulted in sigmoid velocity-substrate plots and in reciprocal plots that were concave upward. These positive cooperative patterns were observed at both pH 7.0 and 7.8 and in the presence or absence of Triton X-100. When Mn2+ and GTP were equimolar, Ca2+, Ba2+, Sr2+, and Mg2+ increased apparent guanylate cyclase activity. This increase in enzyme activity at least could be accounted for partially by an increase in free Mn2+ concentration caused by the complex formation of GTP with the added metals. However, even at relatively low GTP concentrations and with Mn2+ concentrations in excess of GTP, Ca2+, Sr2+, and Ba2+ significantly increased guanosine 3':5'-monophosphate production. As the total GTP concentration was increased, the degree of stimulation in the presence of Ca2+ decreased, despite maintenance of a fixed total concentration of Ca2+ and a fixed free concentration of Mn2+, suggesting that the concentration of CaGTP and MnGTP were determining factors in the observed response. The concave upward reciprocal plots of velocity against MnGTP concentration were changed to linear plots in the presence of CaGTP or SrGTP. These results suggest that sea urchin sperm guanylate cyclase contains multiple nucleotide binding sites and that stimulation of guanosine 3':5'-monophosphate synthesis by Ca2+, Sr2+, and perhaps other metals may reflect interaction of a metal-GTP complex with enzyme as either an effector or a substrate.     

Effect of bivalent cations on rat liver cytosol casein (glycogen synthase) kinases 1 and 2. Influence of the protein substrate.

Rat liver cytosol casein kinases 1 and 2 were stimulated by free Mg2+, but the optimal concentration of cation varied with both the casein kinase and the protein substrate used. Mn2+, but neither Ca2+ nor Zn2+, could efficiently substitute for Mg2+ in forming the bivalent-cation-ATP complex used as substrate, but free Mn2+ was inhibitory. The magnitude of these effects depended on the type of casein kinase and the protein substrate used. These results support the idea that, besides the effects of Mg2+ as a component of the Mg-ATP complex, or through interaction with the protein substrate, free Mg2+ is an allosteric effector of both casein kinases.     

Substitution studies of the second divalent metal cation requirement of protein tyrosine kinase CSK

In addition to a magnesium ion needed to form the ATP-Mg complex, we have previously determined that at least one more free Mg2+ ion is essential for the activation of the protein tyrosine kinase, Csk [Sun, G., and Budde, R. J. A. (1997) Biochemistry 36, 2139-2146]. In this paper, we report that several divalent metal cations, such as Mn2+, Co2+, Ni2+, and Zn2+ bind to the second Mg2+-binding site of Csk with up to 13200-fold higher affinity than Mg2+. This finding enabled us to substitute the free Mg2+ at this site with Mn2+, Co2+, Ni2+, or Zn2+ while keeping ATP saturated with Mg2+ to study the role of the free metal cation in Csk catalysis. Substitution by these divalent metal cations resulted in varied levels of Csk activity, with Mn2+ even more effective than Mg2+. Co2+ and Ni2+ supports reduced levels of Csk activity compared to Mg2+. Zn2+ has the highest affinity for the second Mg2+-binding site of Csk at 0.65 microM, but supports no kinase activity, acting as a dead-end inhibitor. The inhibition by Zn2+ is reversible and competitive against free Mg2+, noncompetitive against ATP-Mg, and mixed against the phosphate accepting substrate, polyE4Y, significantly increasing the affinity for this substrate. Substitution of the free Mg2+ with Mn2+, Co2+, or Ni2+ also results in lower Km values for the peptide substrate. These results suggest that the divalent metal activator is an important element in determining the affinity between Csk and the phosphate-accepting substrate.     

Kinetic effects of ATP, divalent metal ions and pH on chicken liver mevalonate 5-diphosphate decarboxylase

The activity of chicken liver mevalonate 5-diphosphate decarboxylase was measured over a wide range of Mg2+ and ATP concentrations. It was found that free ATP activated the enzyme, whereas free Mg2+ had no effect on the enzyme activity. Computed analyses of free species concentrations and pH studies indicated that MgATP2- is the true substrate. The relative efficiencies of Mg2+, Mn2+, Cd2+, and Zn2+ as activating metal ions were evaluated in terms of V/Km for the corresponding (metal-ATP)2- complexes, and the relative ratios were: Mn2+ 100, Cd2+ 37, Mg2+ 14, Zn2+ 1.7. Inhibitory effects were demonstrated for all free divalent cations tested, except for Mg2+, and were in the order Zn2+ greater than Cd2+ greater than Mn2+.     

Interaction of metal ions with lupin seed conglutin gamma

Various metal ions were capable of aggregating and precipitating conglutin gamma, an oligomeric glycoprotein purified from Lupinus albus seeds, at neutral pH values. The most effective metal ions, at 60-fold molar excess to the protein, were Zn2+, Hg2+ and Cu2+; a lower influence on the physical status of conglutin gamma was observed with Cr3+, Fe3+, Co2+, Ni2+, Cd2+, Sn2+, and Pb2+, while Mg2+, Ca2+ and Mn2+ had no effect at all. The insolubilisation of the protein with Zn2+, which is fully reversible, strictly depended on both metal concentration and pH. with middle points of the sharp transitions at three-fold molar excess and pH 6.5, respectively. Conglutin gamma is also fully retained on a metal affinity chromatography column at which Zn2+ and Ni2+ were complexed. A drop of pH below 6.0 and the use of chelating agents, such as EDTA and imidazole, fully desorbed the protein. A slightly lower binding to immobilised Cu2+ and Co2+ and no binding with Mg2+, Cd2+ and Mn2+ were observed. The role of the numerous histidine residues of conglutin gamma in the binding of Zn2+ is discussed.     

Calcium-requirement and its pH dependency of lysophosphoinositide-specific phospholipase C in porcine platelet membranes

The lysophosphoinositide-specific phospholipase C (lysoPI-PLase C) in porcine platelet membranes had an optimal pH of 9.2 and the activity at a physiological pH of 7.3 was 20% of the maximum in the absence of added divalent metals (Murase, S. et al. (1985) J. Biol. Chem. 260, 262). The activity was completely inhibited by 1 mM EGTA in the assay mixture but was restored by addition of excess Ca2+ or Mn2+, indicating that this is a metalloenzyme. However, membranes pretreated with 1 mM EGTA and washed with buffer retained full activity at a free Ca2+ concentration of 5 nM and no stimulation was observed by added Ca2+ at pH 9.2. In contrast to the results obtained at pH 9.2, addition of Ca2+ stimulated lysoPI-PLase C activity severalfold at pH 7.3, apparently by shifting down the optimal pH and broadening the pH profile. The effect of Ca2+ at pH 7.3 was to enhance Vmax with no significant change in Km value. The stimulatory effect of Ca2+ at pH 7.3 alone did not appear to be of physiological significance since millimolar concentrations of Ca2+ were necessary to reach the maximum activity. However, a shift in pH had a profound effect on the Ca2+-dependency of the activity. A rise in 2 pH units increased the apparent affinity for Ca2+ 10,000-fold. These results indicate that the alkalinization and the rise in free Ca2+ concentration known to occur in stimulated platelets could synergistically provide conditions under which the lysoPI-PLase C exerts its activity when the substrate lysoPI is generated by phospholipase A.     

游离及固定化果糖基转移酶部分酶学性质的比较研究

 从诱变、筛选的米曲霉GX0 0 10菌株所产生的果糖基转移酶 ,经过纯化和固定化操作分别制备游离酶和固定化酶 ,对两者的酶学性质进行了比较研究 .结果表明 ,两者在蔗糖转化为蔗果低聚糖的酶促反应中 ,最适pH为 5 5,在pH5 0～ 7 5之间酶活性相对稳定 .游离酶和固定化酶的适宜温度范围分别是 4 5～ 52℃和 4 0～ 55℃ .在 55℃保温 60min ,酶活性保存率分别是 61 6%和 87 5% .固定化酶的热稳定性提高 .0 1mmol LHg2 +和 1mmol LAg+能完全抑制游离酶的活性 ,但只能部分抑制固定化酶的活性 ,1mmol L的Ti2 +能完全抑制两者的活性 .以蔗糖为底物时 ,游离酶的米氏常数Km=2 15mmol L ,而固定化酶Km =386mmol L .游离酶只能使用一次 ,固定化酶反复使用 54次后 ,剩余活力为 55 2 % .用 55% (W V)蔗糖溶液与固定化酶在pH5 0 ,4 6℃下作用 12h ,可获得61 5% (总低聚糖 总糖 )产物 ,其中蔗果五糖含量达到 7 2 % .     

流感嗜血杆菌ATCC49247来源的IgA酶理化性质及其对低糖基化IgA1的分解作用

【背景】近年来,能够特异性切割人IgA1分子的IgA蛋白酶(IgA酶)被认为是治疗IgA肾病(IgAN)的潜在药物,但各种物理化学因素均可能影响其生物学活性。【目的】研究流感嗜血杆菌(Haemophilus influenzae) ATCC49247 IgA酶的理化性质,确定IgA酶最适的作用条件后观察IgA酶对低糖基化IgA1的作用。【方法】从细菌培养液中分离纯化IgA酶,SDS-PAGE电泳后银染法分别检测多种理化条件下IgA酶的水解活性及其对低糖基化IgA1的消化作用。【结果】H. influenzae ATCC49247IgA酶可耐受的反应温度较广,最适温度为50°C;在高于60°C的环境中,IgA酶便不可逆地丧失了稳定性;IgA酶在pH 6.0-9.0的环境下能够保持完整催化活性;1 mmol/L的PMSF和高于10 mmol/L的SDS能够强烈地抑制IgA酶的活性,DTT和EDTA对IgA酶活性无明显影响;所有浓度的Al~(3+)、Fe~(3+)和高浓度的Cu~(2+)、Zn~(2+)、Fe~(2+)对IgA酶均表现出强烈的抑制作用,同时Co~(2+)、Mn~(2+)、Ca~(2+)、Ni~(2+)和Mg~(2+)都对IgA酶活性没有明显影响。选择了最适宜的IgA酶作用条件,发现IgA酶能够有效降解低糖基化IgA1底物。【结论】确定IgA酶最适宜的作用条件能保持良好的酶活性,更好地发挥了IgA酶对低糖基化IgA1的降解作用,为IgA酶的临床研究和药用价值的进一步开发提供一定的依据。     

黑颈鹤粪便分离菌Arthrobacter sp. GN14 的α-半乳糖苷酶基因克隆、表达与酶学特性

【目的】克隆高原唯一珍惜鹤类——黑颈鹤粪便分离菌Arthrobacter sp.GN14的α-半乳糖苷酶基因agaAGN14,并对该酶进行序列分析、系统发育分析和重组酶的酶学特性分析。【方法】利用简并PCR和GCTAIL-PCR方法获得agaAGN14全长,并对其氨基酸序列(AgaAGN14)进行比对和neighbor-joining系统发育树的构建。将agaAGN14重组到载体pET-28a(+)中并转化到Escherichia coli BL21(DE3)中异源表达。利用组氨酸标签纯化重组α-半乳糖苷酶rAgaAGN14并进行酶学性质分析。【结果】agaAGN14全长2109 bp,GC含量66.8%,编码702个氨基酸(77.5 kDa)。AgaAGN14与数据库中序列的最高一致性为53.7%,与其余胃肠道环境α-半乳糖苷酶的一致性<43%。系统发育分析将AgaAGN14聚于具有催化域KWD和SDXXDXXXR的α-半乳糖苷酶分支,与土壤微生物来源α-半乳糖苷酶距离相对较近,而与其余胃肠道环境α-半乳糖苷酶距离相对较远。rAgaAGN14可水解pNPG、棉籽糖、密二糖、水苏糖、菜粕和棉籽粕,表观最适pH为6.5,在pH 6.0-pH 9.0的范围内稳定并维持50%以上的酶活性。rAgaAGN14的表观最适温度为45℃,在10℃、20℃和37℃内稳定并分别具有约28%、30%和80%的酶活。在45℃pH 6.5条件下,rAgaAGN14对pNPG的Km、Vmax和kcat分别为0.41 mmol/L、18.28μmol/min/mg和25.36 s-1。rAgaAGN14受Ag+、Hg2+及SDS抑制,受K+、Ca2+、Mn2+、Fe3+、Ni2+、Cu2+和β-mercaptoethanol部分抑制,受Co2+、Pb2+、Zn2+、Mg2+、Na+和EDTA的影响较小。【结论】首次报道从黑颈鹤粪便中分离到Arthrobacter菌,并对该属细菌α-半乳糖苷酶进行序列分析、系统发育分析、异源表达和重组酶的酶学特性分析。rAgaAGN14序列较新颖,其酶学特性可能是同时适应黑颈鹤肠道环境和高原淡水湿地环境的结果。