嗜热酸性生淀粉α-淀粉酶Gt-amy中结构域C的环化重排及其对Gt-amy催化性能的影响

为确定嗜热酸性生淀粉α-淀粉酶Gt-amy中结构域C对其催化性能的影响,本研究采用基于蛋白质分子结构的环化重排方法对Gt-amy的结构域C进行分子改造,获得Gt-amy环化重排突变体,并比较了突变体的生淀粉吸附率、生淀粉降解率、酶比活力以及动力学常数.与Gt-amy相比,生淀粉吸附能力提高的Gt-amy环化重排突变体,其生淀粉降解能力也得到提高;生淀粉吸附能力降低的Gt-amy环化重排突变体,其生淀粉降解能力也降低.即Gt-amy的生淀粉降解能力和生淀粉吸附能力之间存在正相关性.其中,环化重排突变体Gt-amy-S498的生淀粉吸附率由78.86%提高至93.14%,生淀粉降解率由65.80%提高至90.93%.本研究证实环化重排突变可以作为一种工程学手段改变蛋白质的结构与功能,为提高酶类蛋白质的催化性能提供了思路.

D407A/D430A双位点突变对嗜热酸性生淀粉α-淀粉酶GTamy酶学性质的影响

旨在研究位于嗜热酸性生淀粉α-淀粉酶GTamy的结构域A与生淀粉结合域RSBD间的Ca^(2+)结合位点对其酶学性质的影响,构建了GTamy的Ca^(2+)结合位点突变体GTamy D407A/D430A,并比较了GTamy与突变体GTamy D407A/D430A的酶学性质。结果表明,与GTamy相比,突变体的高温活性和热稳定性明显降低。突变体于80℃的比活力由1 756.75 U/mg降低至1 484.48 U/mg;80℃的半衰期由3 h降低至2.5 h。以可溶性淀粉为底物时,突变体的底物结合能力不变,反应速率为GTamy的79%;以玉米淀粉为底物时,突变体的底物吸附率为GTamy的67.9%,底物降解率为GTamy的59.3%。该Ca^(2+)结合位点可能通过改变RSBD和催化活性中心的分子结构来影响GTamy的高温活性和热稳定性。本研究表明该Ca^(2+)结合位点有利于GTamy维持高温活性和热稳定性。

嗜热酸性生淀粉α-淀粉酶Gt-amy的C末端结构域功能及生淀粉结合位点分析

为确定嗜热酸性生淀粉α-淀粉酶Gt-amy中参与生淀粉结合的结构区域,对Gt-amy的C末端结构域(C-terminal domain,CTD)进行缺失突变,并比较Gt-amy及CTD缺失突变体Gt-amy-T的酶学性质。在与Gt-amy相同的条件下,Gt-amy-T不能结合和降解玉米淀粉,CTD可有效结合玉米淀粉。以可溶性淀粉为底物,Gt-amy-T的kcat值约为Gt-amy的77.9%。CTD的系统进化关系分析显示CTD不是典型的淀粉结合结构域,但是本研究证实CTD属于生淀粉结合结构域,在Gt-amy结合并降解生淀粉中发挥重要作用,并且CTD有利于Gt-amy发挥可溶性淀粉酶活力。此外,本研究将CTD中Tyr残基定点突变为Ala残基,通过比较CTD与突变体的玉米淀粉结合能力以及Gt-amy与Gt-amy W501A/W514A的玉米淀粉结合率和降解率,确定CTD中W501和W514可能是生淀粉结合位点。

组成型分泌表达嗜热酸性生淀粉α-淀粉酶的重组粪肠球菌的构建

该研究以高效组成型启动子P10替换pSIP401-gtamyhds中诱导型启动子,构建重组载体pSIP401Z-gtamyhds,并以其为基础框架,分别导入粪肠球菌(Enterococcus faecalis)来源的8种信号肽(s1~s8),采用电转化法将信号肽筛选载体转入具有优良益生特性的粪肠球菌EXW27,构建组成型分泌表达嗜热酸性生淀粉α-淀粉酶Gt-amy的重组粪肠球菌。以发酵上清液中α-淀粉酶活性为评价指标,对8种信号肽进行筛选,并对嗜热酸性生淀粉α-淀粉酶Gt-amy进行分离纯化和酶学性质研究。结果表明,信号肽s7对嗜热酸性生淀粉α-淀粉酶Gt-amy的分泌效率最高,发酵上清液中α-淀粉酶活性达到310 U/m L。重组Gt-amy的最适反应pH值为5.0,在pH 4.0~8.0范围内具有较好的稳定性,最适反应温度为80℃,于80℃的半衰期为3 h,在40℃条件下反应3 h,对玉米淀粉的降解率可达55.8%。

地衣芽孢杆菌生麦芽糖α-淀粉酶的基因克隆与鉴定

以地衣芽孢杆菌ATCC14580基因组DNA为模板，PCR扩增出1．7kb大小的麦芽糖淀粉酶基因amyM，克隆入表达载体pET-28a（＋），转化Escherichia．coliBL21（DE3），经IPTG诱导，测定麦芽糖淀粉酶酶活性．结果表明，麦芽糖淀粉酶基因amyM获得了活性表达，酶活力为3．797U／mL，SDS—PAGE电泳结果显示出相对分子质量约为67×10^3的特异性蛋白质条带．酶学性质分析表明，重组麦芽糖淀粉酶的最适反应温度为45℃，最适反应pH为6．5，在45℃以下的中低温环境保持稳定，且能在比较宽的pH范围内（pH4．5～8．5）稳定．

生淀粉水解α-淀粉酶AmyZ1热稳定性提升的分子改造

生淀粉水解α-淀粉酶是一种具有生淀粉水解能力的α-淀粉酶,能够在淀粉糊化温度以下降解生淀粉颗粒,适用于淀粉冷水解。ΑmyZ1是来源于海洋微生物的生淀粉水解酶,其催化底物转化应用时热稳定性不足。与热稳定α-淀粉酶结构进行比对分析,发现AmyZ1在结构域B区域多了一个柔性loop区。通过同源建模,确定突变位点,构建缺失柔性区的突变酶AmyZ1∶ΔTG,并对突变酶的酶学性质、Ca^(2+)依赖以及水解高浓度玉米生淀粉的能力进行分析。AmyZ1∶ΔTG最适作用温度为45℃,在35℃~55℃范围内酶活力维持在60%以上。在30℃和35℃条件下,半衰期可分别达到18 h和15 h,是出发酶AmyZ1的10倍和15倍。突变酶AmyZ1∶ΔTG蛋白结构的柔性降低,导致AmyZ1∶ΔTG的催化效率有所降低。与出发酶AmyZ1相比,AmyZ1∶ΔTG的催化性能受外源Ca^(2+)的影响较小,酶活力对外源Ca^(2+)的依赖有所降低,但Ca^(2+)有助于提升其热稳定性。此外,在35℃的作用条件下,AmyZ1∶ΔTG对300 g/L玉米生淀粉的水解率为40%,接近于出发酶AmyZ1。

独活寄生汤对大鼠类风湿关节炎的治疗效果及对血清IL-1β、TNF-α和MMPs的影响

目的研究独活寄生汤对大鼠类风湿关节炎(rheumatoid arthritis, RA)的治疗效果及对血清IL-1β、TNF-α和MMPs的影响。方法通过向雄性SD大鼠足跖面皮下注射牛Ⅱ型胶原建立大鼠胶原性关节炎(collagen-induced arthritis, CIA)模型。将雄性SD大鼠随机分为对照组(Control),模型组(Model),Model+独活寄生汤低剂量组(Model+L),Model+独活寄生汤中剂量组(Model+M),Model+独活寄生汤高剂量组(Model+H),于CIA造模21 d进行对应处理,持续36 d。测定不同实验组大鼠的体质量变化、足肿胀情况及关节炎指数评分,通过试剂盒检测抗氧化酶肝组织中SOD、CAT、GSH及氧化指标MDA活性,ELISA检测血清IL-1β、TNF-α的水平,Western Blot法分析血清MMPs的表达情况。结果 CIA模型组大鼠出现明显的RA症状,包括关节炎评分高、足肿胀、肝脏SOD、CAT、GSH活性降低及MDA活性升高,血清IL-1β、TNF-α和MMPs表达增加(均P<0.05)。独活寄生汤可呈浓度依赖性显著改善上述变化(均P<0.05),高剂量独活寄生汤对调节CIA大鼠体质量、足肿胀情况及关节炎指数评分作用明显。且血清IL-1β、TNF-α、MMP-1、MMP-3和MMP-13蛋白表达水平在高剂量处理的CIA大鼠中减少至正常大鼠相似的水平。结论独活寄生汤具有减轻RA症状、抑制促炎因子的表达及抗氧化作用,作为治疗RA的药物具有进一步研究的价值。

Preparation of semi-solid 7075 aluminum alloy slurry by serpentine pouring channel

The semi-solid slurry of 7075 aluminum alloy was prepared by a serpentine pouring channel （SCP）. Influences of pouring temperature and the number of turns on the microstructure of semi-solid 7075 alloy slurry were investigated. The results demonstrated that the semi-solid 7075 aluminum alloy slurry with satisfied quality could be generated by a serpentine pouring channel when the pouring temperature was in the range of 680-700 ℃. At a given pouring temperature, the equivalent size of the primaryα（Al） grains decreased and the shape factor increased with the increase of the number of turns. During the slurry preparation of semi-solid 7075 aluminum alloy, the flow direction of alloy melt changed many times when it flowed in a curved and closed serpentine channel. With the effect of“stirring”in it , the primary nuclei gradually evolved into spherical and near-spherical grains.

Influence of serpentine channel pouring process parameters on semi-solid A356 aluminum alloy slurry

Semi-solid A356 aluminum alloy slurry was prepared by using serpentine channel pouring process, and the influences of the channel diameters and pouring temperatures on the semi-solid A356 aluminum alloy slurry were investigated. The experimental results show that when the channel diameter is 20 and 25 mm, respectively, and the pouring temperature is 640-680 ℃, the average diameter of primary α（Al） grains in the prepared A356 aluminum alloy slurry is 50-75 and 55-78 μm, respectively, and the average shape factor of primary α（Al） grains is 0.89-0.76 and 0.86-0.72, respectively. With the decline in the pouring temperature, the microstructure of semi-solid A356 aluminum alloy slurry is more desirable and a serpentine channel with smaller diameter is also advantageous to the microstructure imProvement. During the preparation of semi-solid A356 aluminum alloy slurry, a large number of nuclei can be produced by the chilling effect of the serpentine channel, and owing to the combined effect of the chilled nuclei separation and melt self-stirring, primary α（Al） nuclei can be multiplied and spheroidized finally.

Preparation of semi-solid A380 aluminum alloy slurry by serpentine channel

The semi-solid slurry of A380 aluminum alloy was prepared by the serpentine channel. The effects of pouring temperature, curve number and curve diameter of the serpentine channel on the microstructure of the semi-solid A380 aluminum alloy slurry were investigated. The results show that the satisfactory semi-solid A380 aluminum alloy slurry could be obtained when the pouring temperature ranged from 630 to 650 °C. Under the same conditions, increasing the curve number or reducing the curve diameter of the serpentine channel would decrease the average diameter and increase the shape factor of the primary α（Al） grains. The ＂self-stirring＂ of the alloy melt in the serpentine channel was beneficial to the ripening of the dendrites and the spheroidizing of the primary α（Al） grains.

Microstructure evolution of semi-solid 7075 Al alloy slurry during temperature homogenization treatment

Semi-solid 7075 Al slurry was prepared by inverted cone-shaped pouring channel process （ICSPC） and temperature homogenization （TH） treatment was combined to make the slurry uniform and have a controllable solid fraction suitable for the follow-up rheocasting. The influence of cooling rate on the microstructure evolution of primary α（Al） during TH treatment was investigated. The results show that as the cooling rate of the slurry after being prepared reduces, the growth of primaryα（Al） in the slurry tends to be nearly spherical and the uniformity of the organization is also enhanced. This may be due to the fact that lower cooling rate plays an important role in achieving the uniformity of temperature and composition in the remaining liquid, which is crucial to the formation of the spherical and homogeneous microstructure. However, a too low cooling rate will lead to a significant increase in grain growth time, which makes too coarse grains and more particles coalesce, so a certain abnormal growth of grain appears and the shape factor decreases slightly.

Kinetic nucleation of primary α(Al) dendrites in Al-7%Si-Mg cast alloys with Ce and Sr additions

Nucleation of dendritic primaryα（Al） phase with addition of element Ce and Sr in hypoeutectic Al-7%Si-Mg cast alloy was investigated by using differential scanning calorimetry （DSC） and scanning electron microscopy. DSC results were used to calculate the activation energy and nucleation work of primaryα（Al） phase. The results show that the values of activation energy and nucleation work are decreased and the nucleation frequency is increased with the additions of Ce and Sr to the alloys. Moreover, the grain size of dendriticα（Al） phase is well refined, and the nucleation temperatures of primaryα（Al） dendrites are decreased with the additions of Ce and Sr. The effects of elements Ce and Sr additions on kinetic nucleation of primary α（Al） phases were also discussed in hypoeutectic Al-7%Si-Mg cast alloy.

DFT Study of the Effect of Temperature on ZnO Adsorbed on α-AI_2 O_3(0001)Surface

The adsorption and the growth of ZnO on α-Al2O3（0001） surface at various temperatures were theoretically calculated by using a plane wave pseudopotentials （USP） method based on density functional theory.The average adsorption energy of ZnO at 400, 600 and 800 ℃ is 4.16±0.08, 4.25±0.11 and 4.05±0.23 eV respectively. Temperature has a remarkable effect on the structure of the surface and the interface of ZnO/α-Al2O3（0001）. It is found that the Zn-hexagonal symmetry deflexion does not appear during the adsorption growth of ZnO at 400 ℃, and that the ZnO[10^-10] is parallel with the [10^-10] of the α-Al2O3（0001）, which is favorable for forming ZnO film with the Zn-terminated surface. It is observed from simulation that there are two kinds of surface structures in the adsorption of ZnO at 600 ℃： one is the ZnO surface that has the Zn-terminated structure, and whose [10^-10] parallels the [10^-10] of the substrate surface, and the other is the ZnO[10^-10] //sapphire [11-20] with the O-terminated surface. The energy barrier of the phase transition between these two different surface structures is about 1.6 eV, and the latter is more stable. Therefore,the suitable temperature for the thin film growth of ZnO on sapphire is about 600 ℃, and it facilitates the formation of wurtzite structure containing Zn-O-Zn-O-Zn-O double-layers as a growth unit-cell. At 600 ℃, the average bond length of Zn-O is 0.190±0.01 nm, and the ELF value indicates that the bond of （substrate）-O-Zn-O has a distinct covalent character, whereas the （Zn）O-Al （substrate） shows a clear character of ionic bond. However, at a temperature of 800 ℃, the dissociation of Al and O atoms on the surface of the α-Al2O3（0001） leads to a disordered surface and interface structure. Thus, the Zn-hexagonal symmetry structure of the ZnO film is not observed under this condition.

Effect of heat treatment processing on microstructure and tensile properties of Ti-6Al-4V-10Nb alloy

Effects of heat treatment processing on the microstructure and mechanical properties of Ti-6Al-4V-10Nb alloy were investigated. The microstructures were investigated by SEM, TEM and XRD, and the mechanical properties were evaluated by tensile tests at room and elevated temperatures. The results indicate that the lath-like and globular primary α phase, secondary α phase and β phase are obtained after forging and heat treatment processing. The size of secondary α phase is much smaller than that of primary α phase. After heat treatment, the volume fraction of primary α phase is decreased, and that of secondary α phase is increased. With the increase of solution temperature, the volume fraction of primary α phase is gradually decreased, and that of secondary α phase is obviously increased. The yield strength and tensile strength of Ti-6Al-4V-10Nb alloy are significantly enhanced with the solution temperature increasing.

The role of endotoxin,TNF-α,and IL-6 in inducing the state of growth hormone insensitivity

AIM: Critical illnesses such as sepsis, trauma, and burns cause a growth hormone insensitivity, which leads to an increased negative nitrogen balance. Endotoxin is generously released into blood under these conditions and stimulates the production of proinflammatory cytokines such as TNF-alpha, IL-6, and IL-1, which may play a very important role in inducing the growth hormone insensitivity. The objective of this current study was to investigate the role of endotoxin, TNF-alpha and IL-6 in inducing the growth hormone insensitivity at the receptor and post-receptor levels. METHODS: Spague-Dawley rats were injected with endotoxin, TNF-alpha, and IL-6, respectively and part of rats injected with endotoxin was treated with exogenous somatotropin simultaneously. All rats were killed at different time points. The expression of IGF-I, GHR, SOCS-3 and beta-actin mRNA in the liver was detected by RT-PCR and the GH levels were measured by radioimmunoassay, the levels of TNF-alpha and IL-6 were detected by ELISA. RESULTS: There was no significant difference in serous GH levels between experimental group and control rats after endotoxin injection, however, liver IGF-I mRNA expression had been obviously down-regulated in endotoxemic rats. Liver GHR mRNA expression also had a predominant down-regulation after endotoxin injection. The lowest regulation of liver IGF-I mRNA expression occurred at 12h after LPS injection, being decreased by 53% compared with control rats. For GHR mRNA expression, the lowest expression occurred at 8h and had a 81% decrease. Although SOCS-3 mRNA was weakly expressed in control rats, it was strongly up-regulated after LPS injection and had a 7.84 times increase compared with control rats. Exogenous GH could enhance IGF-I mRNA expression in control rats, but it did fail to prevent the decline in IGF-I mRNA expression in endotoxemic rats. Endotoxin stimulated the production of TNF-alpha and IL-6, and the elevated IL-6 levels was shown a positive correlation with increased SOCS-3 mRNA expression. The liver GHR mRNA expression was obviously down-regulated after TNF-alpha iv injection and had a 40% decrease at 8h, but the liver SOCS-3 mRNA expression was the 4.94 times up-regulation occurred at 40 min after IL-6 injection. CONCLUSION: The growth hormone insensitivity could be induced by LPS injection, which was associated with down-regulated GHR mRNA expression at receptor level and with up-regulated SOCS-3 mRNA expression at post-receptor level. The in vivo biological activities of LPS were mediated by TNF-alpha and IL-6 indirectly, and TNF-alpha and IL-6 may exert their effects on the receptor and post-receptor levels respectively.

Interferon-alpha restrains growth and invasive potential of hepatocellular carcinoma induced by hepatitis B virus X protein

AIM: To investigate the effects of interferon-alpha (IFN-α) to restrain the growth and invasive potential of hepatocellular carcinoma (HCC) induced by hepatitis B virus (HBV) X protein. METHODS: The pcDNA3.1-HBx plasmid was transfected into Chang cells by Lipofectamine in vitro, and Chang/HBx was co-cultured with IFN-α. Cell survival growth curve and clonogenicity assay were used to test the growth potential of Chang/pcDNA3.1, Chang/HBx and IFN-α-Chang/HBx in vitro. Growth assay in nude mice was used to detect the growth potential of Chang/ pcDNA3.1, Chang/HBx and IFN-α-Chang/HBx in vivo. Wound healing and transwell migration assays were used to detect the invasive ability of Chang/pcDNA3.1, Chang/HBx and IFN-α-Chang/HBx. RESULTS: Compared with CCL13 cells transfected with pcDNA3.1, CCL13 with stable expression of hepatitis B virus X protein showed the characteristics of malignant cells with high capability of growth and invasion by detecting their growth curves, colony forming efficiency, wound healing , transwell migration assays and growth assays in nude mice. Its capability of growth and invasion could be controlled by IFN-α. CONCLUSION: IFN-α can restrain the growth and invasive potential of HCC cells induced by HBx protein, which has provided an experimental basis for IFN-α therapy of HCC.

Preparation of semi-solid A356 Al-alloy slurry by introducing grain process

A preparation technology of semi-solid metal slurry introducing grain process （IGP） was developed. The effects of processing parameters on the microstructures of the semi-solid A356 Al-alloy slurries were investigated, and the formation mechanism and morphology controlling of the spherical primary a（A1） grains were discussed. The results show that when the preparing slurry is 4 kg, IG size is 10 mm, dosage is 3.5% and dumping temperature （DT） is 611-617 ℃, the mean diameter of the primary a（A1） grains in the semi-solid slurries can reach 40-75 μm and the shape factor can reach 0.82-0.89. When the IG size is 10 mm, DT is 613℃and dosage is 2%-4%, the mean diameter can reach 45-82 μm and the shape factors can reach 0.78-0.88. With decreasing DT or increasing dosage properly, the primary α（A1） grain morphology is better. When QR=QA and Rh=Rc, as long as the DT is suitable, excellent semi-solid slurry can be produced. As a result of the IG melting, a large amount of dendritic fragments can become the direct source of the primary a（A1） grains. Meanwhile, many undercooled areas are formed, where abundant primary α（A1） grains are multiplied by heterogeneous nucleation.

Separation of α-Tocopherol with a Two-Feed Simulated Moving Bed

A two-feed simulated moving bed（SMB） was proposed to utilize the stationary phase more effectively.Zone I is disconnected with zone Ⅱ and zone Ⅲ is disconnected with zone IV.Two feeds are added to zones I and Ⅲ,while two desorbents are added to zones Ⅱ and IV separately.The investigation on the movement of concentra-tion bands indicated that the tail of the strong retained solute and the front of the weak retained solute were eluted from zones I and Ⅲ orderly,so the streams from zones I and Ⅲ could be cut into different fractions to give the products with high purity.The two-feed SMB was used to separate α-tocopherol from its homologue mixture suc-cessfully,and could double the productivity if both feeds were identical to the feed in the conventional four-zone SMB.The solvent consumption could be reduced by reusing the middle fraction without solutes.

Effects of α-tocopheryl acetate supplementation in preslaughter diet on antioxidant enzyme activities and fillet quality of commercial-size Sparus macrocephalus

This study examined the effects of dietary α-tocopheryl acetate supplementation on antioxidant enzyme activities and fillet quality in commercial-size Sparus macrocephalus. Three hundred fish [main initial weight （350±12） g] were divided into three groups （E250, E500 and El000） and reared in 9 cages. The fish were fed for 8 weeks with three diets containing different levels of dietary α-tocopheryl acetate （289, 553, 1069 mg/kg）. Over the experimental period, fish were fed to satiation and reached a final mean weight of （465±28） g without significant body weight difference and proximate composition difference. Fillet α-tocopherol was significantly （P〈0.05） different between groups, reaching levels of 14.2, 22.1, 30.9 pg/mg fillet for groups E250, E500 and El000, respectively. Total serum superoxide dismutase （SOD） activity increased significantly （P〈0.05） in fish fed the diets high in α-tocopheryl acetate, but serum glutathione peroxidase （GPX） activity was unaffected. In storage on ice, fillets of fish fed the diets high in α-tocopheryl acetate exhibited significantly lower （P〈0.05） levels of oxidation. These results suggested that increased dietary α-tocopheryl acetate could increase its flesh deposition, increase the activity of SOD and prevent lipid peroxidation ofSparus macrocephalus fillets in retail storage on ice.

Effects of interferon-alpha on expression of hepatic stellate cell and transforming growth factor-pi and a-smooth muscle actin in rats with hepatic fibrosis

AIM:To investigate the effect of interferon-a (IFN-α) on preventing or reversing hepatic fibrosis in rat experimental model induced by CCI4. METHODS: One hundred and ten Sprague-Dawley rats were divided into five groups: group A (normal controls, n = 18), group B (fibrotic model controls, n = 22), group C (IFN-α prevention, n = 22) initially treated with intramuscular injection of IFN-a in saline daily at the doses of 1× 105 U for 6 wk, group D (IFN-a treatment, n = 24) treated with intra-muscular injection of IFN-a in saline daily at the doses of 1×105 U for 6 wk after the first 6 wk, group E (0.9% sodium chloride treatment control, n = 24) treated with intra-muscular injection of 0.01 mL/kg daily for 6 wk after the first 6 wk. At the end of the experiment, all rats of each group were killed. Samples of the liver obtained by biopsy were subjected to histological, immunohistochemical and electron microscopic studies for the expressions of transforming growth factor-pi (TGF- μ41) and α-smooth muscle actin (α-SMA). RESULTS: The expressions of TGF-pl, the number of activated hepatic stellate cells and a-SMA in hepatic tissue of group C were significantly less than those of group B (P<0.01). The degree of fibrosis score in group B was also significantly less than that of group C under light microscope (P<0.01). CONCLUSION: IFN-a can inhibit the production of TGF-pl, decrease HSC activation and stimulate its apoptosis.