固定化木瓜蛋白酶填充床反应器水解丝素的初步应用研究

本研究首先用氯化钙乙醇溶液溶解丝素，测定了丝素蛋白的相对分子质量，然后用尼龙固定化木瓜蛋白酶填充床反应器水解丝素。研究结果表明丝素蛋白的相对分子质量大于66200Da，酶解后丝素溶液的蛋白质含量降低，L-酪氨酸含量提高，丝素溶液稳定性提高。     

木瓜蛋白酶对丝素蛋白的降解作用研究

丝素蛋白具有良好的生物相容性及降解性,被广泛地用于生物材料。本研究以废蚕茧为原料,通过脱胶、溶解、透析、浓缩等步骤制备了丝素蛋白溶液,再用木瓜蛋白酶降解丝素蛋白,利用SDS－聚丙烯酰胺凝胶电泳（SDS－PAGE）进行丝素蛋白多肽的鉴定,探讨pH、温度及酶活等因素对木瓜蛋白酶降解丝素蛋白影响。结果表明：在pH8～9、温度60～70℃,木瓜蛋白酶的活性为20～25 U时丝素蛋白水解效果较好,酶的作用时间越长效果越好。本研究为开发丝素蛋白在食品、化妆品中的提供了基础。     

丝素作为固定化酶载体材料的研究进展

酶的固定化在化学、生物医学以及生物感应器等领域应用较广。固定化酶的效果在很大程度上取决于载体结构。丝素作为固定化酶载体材料有其独特的优势。本文综述了国内外以不同的丝素形态作为固定化酶载体材料的研究进展,重点介绍了非纳米丝素固定化酶和纳米丝素固定化酶（丝素纳米颗粒和丝素纳米纤维）的研究进展。     

家蚕丝素固定化葡萄糖异构酶的制备方法及其理化性质

脱胶蚕丝用稀碱溶液处理后制成多孔的碱化丝素 ,分别经物理吸附和戊二醛交联方法制得固定化酶Ⅰ和Ⅱ。每克固定化酶Ⅰ的总活力为 791 0 4U ,活力回收率为 5 2 0 6 % ,活力表现率为 72 2 7% ;每克固定化酶Ⅱ的总活力为 811 39U ,活力回收率为 5 3 6 7% ,活力表现率为 72 97%。蚕丝经高浓度氯化钙溶液溶解、脱盐等处理后制成丝素粉末 ,经吸附后用戊二醛交联固定了葡萄糖异构酶 ,制成固定化酶Ⅲ。每克固定化酶Ⅲ的总活力为 84 4U ,活力回收率为 5 5 2 7% ,活力表现率为 72 4 2 %。经对固定化酶性质的研究表明 :碱化丝素和丝素粉末均能较好地固定葡萄糖异构酶 ;最适温度比游离酶升高了 15℃ ;最适pH没有变化 ,有较强的抗蛋白质变性剂 (8mol/L尿素溶液中的活力在 80 %以上 )能力 ;各固定化酶的Km值在 4× 10 -2 ～ 5× 10 -2 mol/L范围内。实验还发现Co2 + 和Mg2 +离子为固定化酶的激活剂 ,Fe3 + 和Ca2 + 离子为抑制剂。     

丝素作为固定化酶载体的研究进展与应用

本文详细描述了制备丝素固定化酶的各种方法，如包埋法、戊二醛共价交联法以及膜状、粉状、线状三种形状的固定化酶的制作；并就固定化葡萄糖氧化酶、过氧化物酶、脲酶、脂肪酶、果胶酶、过氧化氢酶、糖化酶、超氧化物歧化酶、青霉素酰化酶、木瓜蛋白酶、苯丙氨酸裂解酶、β-葡萄糖苷酶等的研究进展与应用进行了介绍和评价。     

酶解法制备饲用羽毛肽粉

本研究以普通市售羽毛粉为原料,通过正交试验设计,分别研究胰蛋白酶、木瓜蛋白酶、菠萝蛋白酶降解制备饲用羽毛肽粉的条件。综合分析实验结果,建立了3种混合酶降解制备饲用羽毛肽粉的最优工艺:在温度55℃,pH 8,混合酶浓度1/50 g/mL条件下反应24 h,终产品中小分子蛋白含量较无酶组约提高了200%。     

家蚕丝素固定化糖化酶的研究

茧丝经高浓度的氯化钙溶液处理或用稀碱溶液处理后,制成了粉末状丝素和碱化丝素,并以此为载体,用戊二醛为交联剂固定了糖化酶。经对固定化酶性质和动力学参数等的研究表明：丝素能较好地固定糖化酶;每克干粉末状丝素固定化酶的活力约为１８２２ Ｕ, 固定化酶的最适ｐ Ｈ 值比游离酶增加了１０ 到１２５ 个单位,最适温度比游离酶降低了５ ～１０ ℃;固定化酶具有较长的操作半衰期（１４ ～１８ｄ） ;通过米氏常数的测定,粉末状丝素固定化酶的 Ｋｍ １ ＝ ２ ．５３ ×１０ －５ , 碱化丝素固定化酶的 Ｋｍ ２ ＝３１５ ×１０ － ５ 。实验还发现： 在制备 固定化糖化 酶时, 酶的最适 浓度为 ８ ｍ Ｌ／ Ｌ,戊 二醛的 最适浓 度为０３ ％ 。     

家蚕丝素蛋白——一种天然的固定化酶载体

家蚕茧丝素除了用于传统的纺织原料外,在其它各个方面正在得到开发和利用,其中用作固定化酶的载体已得到较为详细的调查和研究.丝素蛋白按其应用要求可以容易地制成各种形状的丝素,如纤维状、粉末状和膜状.目前已有许多有关应用这些形状的丝素作为固定化酶载体的研究报道,其中固定化酶的丝素膜应用分光光度法、红外光谱法、核磁共振和电子自旋共振等方法已被作了详细地研究和分析.这种固定化酶丝素膜已成功地应用于能测定葡萄糖、过氧化氢和尿酸的生物传感器,其中流动注射分析式的葡萄糖和尿酸传感器能快速地测定包括人全血或血清在内的各种生物样品.     

一种新型丝素支架材料的体内降解试验

将制备的冷冻致孔新型丝素支架材料植入兔耳皮下进行体内降解试验,评价丝素支架材料的降解规律以及生物相容性。对植入丝素支架材料的兔耳进行肉眼外观观察、组织切片观察和扫描电子显微镜观察。肉眼外观观察发现植入丝素支架材料部位的皮肤无明显红肿,经过28周左右皮下材料植入部位的突起逐渐消失;组织切片观察发现该丝素支架材料引起的组织反应较弱;扫描电子显微镜在28周时只观察到将近消失的丝素支架材料种植腔,表明材料已基本降解。相比之下28周时丝素膜仍完好,无明显降解。研究结果表明,冷冻致孔新型丝素支架材料有望开发为一种生物相容性优良的可降解吸收性组织工程支架材料。     

消减牛乳酪蛋白致敏性蛋白酶的筛选

以酪蛋白为研究对象，选取复合风味蛋白酶、胰蛋白酶、胰凝乳蛋白酶、碱性蛋白酶、木瓜蛋白酶、菠萝 蛋白酶和中性蛋白酶共7 种食品级蛋白酶，对牛乳酪蛋白进行酶解。通过牛乳过敏患者血清池，评估酶解产物致敏 原性，同时结合水解产物的分子质量分布、水解度、残留表位信息等指标，筛选出最适合用于生产低致敏酪蛋白产 品的蛋白酶。结果表明：在相同条件下经胰凝乳蛋白酶、复合风味蛋白酶处理后酪蛋白与特异性抗体结合能力降低 程度最大，可用于后续低致敏酪蛋白制备的候选蛋白酶；结合T细胞表位预测结果，对酪蛋白水解产物进一步分析 发现，酪蛋白经过胰凝乳蛋白酶酶解后，仅剩余1 条大于9 个氨基酸的T细胞表位肽段LHSMKEGIHAQQK。     

牦牛乳酪蛋白酶解产物二肽基肽酶-Ⅳ抑制活性和特性分析

以牦牛乳酪蛋白为原料,利用碱性蛋白酶、木瓜蛋白酶和胰蛋白酶制备酪蛋白酶解产物,研究其二肽基肽酶-Ⅳ(dipeptidyl peptidase-Ⅳ,DPP-Ⅳ)抑制活性和理化特性.以DPP-Ⅳ抑制率为指标,研究水解时间和蛋白酶的种类对酶解产物DPP-Ⅳ抑制活性的影响,以水解度、三氯乙酸氮溶解指数(trichloroacetic acid-nitrogen soluble index,TCA-NSI)、溶解性和灰分含量为指标,评价酶解产物的理化特性.结果表明:对于相同水解时间点获得的酶解产物,木瓜蛋白酶酶解产物的DPP-Ⅳ抑制活性显著高于其他2种蛋白酶的酶解产物(P＜0.05).利用木瓜蛋白酶水解0.50 h的酶解产物具有最高的DPP-Ⅳ抑制活性(抑制率可达(53.95±1.57)％)、较高的水解程度以及最低的灰分含量.说明利用蛋白酶水解技术能够使牦牛乳酪蛋白中具有DPP-Ⅳ抑制活性的多肽释放,木瓜蛋白酶酶解产物可作为功能性乳基料用于功能性食品的开发.     

牛乳酪蛋白初级抗菌肽粉乳基料的制备

为制备抗菌活性较好的肽粉乳基料,以新鲜牛乳为原料提取酪蛋白,以酪蛋白酶解物对大肠杆菌、沙门氏菌和金黄色葡萄球菌的抑菌圈直径为指标,从木瓜蛋白酶、碱性蛋白酶、中性蛋白酶和胰蛋白酶中筛选出一种蛋白酶水解牛乳酪蛋白,并通过单因素试验和正交试验确定该酶水解牛乳酪蛋白优化水解条件。结果表明：木瓜蛋白酶水解牛乳酪蛋白,其酶解物对3种菌均有较好的抑制作用;优化水解工艺条件为：酶解温度50℃、酶解时间4.5h、加酶量4500U/g、pH5.5、底物质量浓度6g/100mL;牛乳酪蛋白酶解物及初级肽粉乳基料稳定性均较好。     

生物反应器培养BHK-21悬浮细胞增殖伪狂犬病病毒工艺优化

旨在筛选伪狂犬病病毒（PRV）敏感的BHK-21细胞并分析其生长和病毒增殖特性,优化反应器中BHK-21悬浮细胞的培养和病毒增殖条件,建立生物反应器培养BHK-21悬浮细胞增殖PRV工艺。本研究利用响应面和单因素优化法,以细胞生长动力学特性、TCID₅₀病毒滴度等参数为指标,优化1.2 L生物反应器中BHK-21悬浮细胞的最佳培养和增殖病毒条件,在5 L生物反应器中进一步批培养验证。结果显示,筛选获得PRV高敏感的BHK-21-02贴壁细胞和BHK-21-XF02悬浮细胞各1株,BHK-21-XF02悬浮细胞在含3%血清的SLM-BHK低血清培养基和SFM-BHK无血清培养基中均能实现良好的生长和病毒增殖。利用响应面法优化得到1.2 L反应器最佳培养条件为接种密度1.20×10⁶cells·mL^-1、搅拌转速120 r·min^-1、DO值40%,5 L反应器批培养72 h细胞密度可达（7.61±0.18）×10⁶ cells·mL^-1、细胞活率为（96.93±1.18）%。利用单因素法优化得到1.2 L反应器最佳病毒增殖条件为MOI 0.001、培养温度37℃、细胞密度2.0×10⁶cells·mL^-1、搅拌转速80 r·min^-1,5 L反应器批培养接毒后48 h病毒滴度达到最大值（7.13±0.11） lgTCID₅₀·mL^-1。本研究可为PRV疫苗相关研究和规模化生产提供参考。     

Comparing two commercial enzymes to estimate in vitro proteolysis of purified or semi-purified proteins

Two experiments compared the suitability of two commercial enzymes to estimate in vitro proteolysis of different proteins. Experiment 1 compared the proteolytic activity over various incubation times of a microbial enzyme (protease from Streptomyces griseus) with a plant enzyme (papain from Papaya latex) by using either 1.33 (high, H) or 0.4 (low, L) units (U, amount) of each enzyme per mg crude protein (CP) of purified proteins including bovine (BA) or egg albumin (EA). Experiment 2 compared the activity of 0.66 U of each of these enzymes per mg CP of semi-purified proteins including casein, wheat gluten (WG) and maize gluten meal (MG). Each incubation was terminated by adding trichloroacetic acid (TCA) and the TCA soluble supernatant collected to estimate concentration of total amino acids (AA) as the measure of proteolysis of each protein over each time. The data on proteolysis over time were fitted into a non-linear model to derive constants for solubility (a) and rate (c) and extents (a + b) of proteolysis of each food by each amount of each enzyme. All data on proteolysis over time and the derived constants were statistically analysed to study the effect of food, enzyme, amount and their interactions. Significant differences were observed between foods, enzymes, enzyme amount (H vs. L for Experiment 1 only) for the proteolysis at most incubation times in both experiments (p<0.001). The mean proteolysis over all times for BA was 1.6 (SD, 0.52) times greater than EA (p<0.001). While the protease gave about four times (SD, 2.1) more proteolysis than papain (p<0.001), the high amount of enzyme gave only about two times (SD, 0.28) greater proteolysis than that of the low amount (p<0.001). On average, the protease was over three times faster (c) than papain (p<0.001) and high amount was two times faster than the low amount of enzyme (p<0.001). While both purified foods were similar in solubility (p>0.05), they differed in the rate and extent of proteolysis (p<0.001). The BA was degraded about four times faster than EA (p < 0.001). Amongst semi-pure proteins, casein gave the highest but MG the lowest proteolysis at each incubation (p<0.001). However, the magnitude of proteolysis depended upon enzyme, food and hours of incubation. On average, casein was degraded at a much faster rate than WG or MG by both enzymes. It appeared that the protease and not papain can be used to estimate in vitro proteolysis of pure and semi-pure food proteins. However, further studies are needed to standardise the relevant procedures when using protease to estimate proteolysis of ruminant foods.     

盐水灌溉对不同暖季型草坪草蒸散量的影响

草坪蒸散量是指导草坪合理灌溉的重要指标。以7属11种共13份暖季型草坪草品种（种源）为材料,采用盆栽结合盐水（20 g· L^-1 NaCl）灌溉的方法,研究盐胁迫对其蒸散量的影响及与抗盐性的关系。结果表明：对照和盐胁迫条件下,草种间蒸散量均有显著差异。对照条件下,草种间的日均蒸散量变异系数为22.28%,日均蒸散量最高为‘Pan1’巴哈雀稗（Paspalum notatum）（5.71 mm·d^-1）,最低为‘南京’狗牙根（Cynodon dactylon）（2.77 mm·d^-1）。盐水灌溉条件下,草种间的日均蒸散量变异系数为36.05%,明显高于对照,不同草种的日均蒸散量排序为：‘Diamond’沟叶结缕草（Zoysia materlla）（3.87 mm·d^-1）>‘S004’钝叶草（Stenotaphrum secundatum）（2.90 mm·d^-1）>‘兰引3号’结缕草（Zoysia japonica）（2.34 mm·d^-1）>‘Z080’结缕草（2.25 mm·d^-1）>‘Adalayd’海雀稗（P.varginatum）（2.20 mm·d^-1）>‘P011’双穗雀稗（P.distichum）（2.17 mm·d^-1）>‘南京’狗牙根（2.10 mm·d^-1）>‘Tifway’杂交狗牙根（2.00 mm·d^-1）>‘Pan1’巴哈雀稗（1.90 mm·d^-1）>‘B003’野牛草（Buchloe dactyloides）（1.84 mm·d^-1）>‘H001’牛鞭草（Hemarthria sibirica）（1.61 mm·d^-1）>‘E126’假俭草（Eremochloa ophiuroides）（1.57 mm·d^-1）>‘TifBliar’假俭草（1.50 mm·d^-1）。除‘Diamond’沟叶结缕草外,盐水灌溉下参试草种的蒸散量均有一定程度的降低,降幅从15.29%（‘S004’钝叶草）到66.74%（‘Pan1’巴哈雀稗）。相关性分析表明,盐处理下草坪草的蒸散量与草种的抗盐性成正相关。     

Risk factors for medial meniscal injury in association with cranial cruciate ligament rupture

Objectives : To determine the significant risk factors for medial meniscal injury in naturally occurring cranial cruciate ligament rupture and to quantify the risk using multivariate analysis. Methods : A retrospective case control study was performed of dogs that had undergone surgery for cranial cruciate ligament rupture. Data recorded included patient signalment (age, breed and sex), the duration of the lameness, the extent of the cranial cruciate ligament rupture (complete or partial) and the condition of the medial meniscus. Logistic regression was used to analyse the relationship between these variables and tears in the medial meniscus. Results : One hundred and sixty-one of 443 stifles (36·3%) in 366 dogs had a medial meniscal tear. The risk of a medial meniscal tear was increased by 12·9 times in association with complete cranial cruciate ligament rupture (OR 12·9; 95% CI 6·8 to 24·2), by approximately 2·6% for each additional week of lameness (OR 1·026; 95% CI 1·009 to 1·043) and by approximately 1·4% for each additional kilogram of bodyweight (OR 1·014; 95% CI 1·000 to 1·028). Golden retrievers and Rottweilers were at increased risk and West Highland white terriers were at reduced risk of medial meniscal tears compared with Labrador retrievers. Clinical Significance : To minimise the risk of medial meniscal tears, surgical stabilisation should not be unnecessarily delayed.     

柱花草叶肉原生质体提取与瞬时表达体系构建

柱花草(Stylosanthes guianensis)是一种重要的热区豆科牧草,为利用原生质体瞬时表达体系开展柱花草基因功能研究,以20 d龄期‘热研5号’柱花草子叶为试验材料,通过正交试验考察不同因素对原生质体分离及转化效率的影响。结果表明：采用1.5%纤维素酶、1.25%离析酶、0.5 mol·L^-1甘露醇和4 mmol·L^-1MES酶解液组合,酶解时间为16 h时,原生质体的产量和活性均达到最佳,分别为(4.567×10⁶)个·g^-1和92.271%;原生质体浓度为(6×10⁵)个·mL^-1、质粒浓度为0.6μg·μL^-1,PEG-4000浓度为50%、转化时间为5 min时,转化效率最高,可达52.524%。构建了目标蛋白SgRKL1表达载体pA7-BIUTNT::SgRKL1-GFP,利用PEG介导法将其转入柱花草原生质体,激光扫描共聚焦显微镜检测结果显示SgRKL1蛋白定位于细胞膜上,说明所建立的柱花草叶肉原生质体瞬时表...     

Influence of signalment on developing cranial cruciate rupture in dogs in the UK

Objectives : To investigate risk factors associated with cranial cruciate ligament rupture in dogs. Methods : Retrospective case‐control study: medical records of a first‐opinion veterinary practice were searched for dogs diagnosed with cranial cruciate ligament rupture (1995 to 2007). For each case, six unaffected dogs were randomly selected from all dogs presenting that day for comparison. Multi‐variable binary logistic regression was performed to assess the association of variables on likelihood of cruciate rupture. Results : Frequency of cranial cruciate ligament rupture was 1·19% [95% confidence interval (CI) 1·02 to 1.36%]. West Highland white terriers (n=17), Yorkshire terriers (n=14) and Rottweilers (n=11) were at significantly increased risk of cranial cruciate ligament rupture (P≤0·002). Rottweilers were at five times greater risk compared with other pure breeds (OR 5·12, 95% CI 2·281 to 11·494, P<0·001), obesity quadrupled the risk of cranial cruciate ligament rupture (OR 3·756, 95% CI 1·659 to 8·502, P=0·001) and females were twice as likely to suffer cranial cruciate ligament failure compared to males (OR 2·054, 95% CI 1·467 to 2·877, P<0·001). Dogs less than two years old were statistically less likely to sustain cranial cruciate ligament rupture than dogs older than eight years (OR 0·246, 95% CI 0·127 to 0·477, P<0·001). There was no significant difference in median weights (in kilograms) of neutered dogs, compared to their entire counterparts in either the case group (P=0·994) or in the control group (P=0·630). There was also no significant difference in body condition (‐underweight/normal weight/overweight/obese) of neutered versus entire dogs among the cases (P=0·243), or the controls (P=0·211). Clinical Significance : Cranial cruciate ligament rupture is more likely in Rottweilers and in female dogs, older dogs and obese dogs. Following multi‐variable analysis, it was established that neutering was not associated with increased risk of cranial cruciate ligament rupture.     

Genetic structure of milk protein polymorphisms and effects on milk production traits in a local dairy cattle

The milk protein genetic structure of the Reggiana dairy local cattle with remarkable milk quality properties was analysed and compared with the structure of Italian Friesian and Italian Brown cattle. A total of 1138 individual milk samples from the three breeds were typed by isoelectrofocusing. Polymorphism was found at the α_s1‐casein (CSN1S1), β‐casein (CSN2), κ‐casein (CSN3), and β‐lactoglobulin (LGB) loci. High frequencies were observed for some casein alleles in Reggiana (CSN1S1*C, 25%; CSN2*B: 28%) with considerable differences to those observed in a survey carried out 30 years before. A great variation among breeds was observed for casein haplotype frequencies and high linkage disequilibrium was detected at the three polymorphic casein loci CSN1S1‐CSN2‐CSN3. The prevalent haplotypes were CA²B (23%; Reggiana), BA²A (48%; Italian Friesian) and BA²B (51%; Italian Brown). Higher G_ST values between breeds were observed when haplotype frequencies were considered instead of gene frequencies. In the Reggiana cattle, the occurrence of two casein alleles at a rather high frequency (CSN1S1*C, CSN2*B) allowed to estimate the effects of rare casein haplotypes on milk traits. The CA²B haplotype was associated with a significantly higher milk protein and fat content and a significantly higher protein per cent was found also for the BA¹B haplotype.