日粮补锌对荷斯坦种公牛精液品质的影响

试验选用６头荷斯坦种公牛,研究了锌对性反射时间、精液品质、精清酶活性及血、精清锌与睾酮浓度的影响。结果表明：添加锌能显著提高精液的鲜精活力、精子密度、冻后精子活力和顶体完整率（Ｐ＜０．０５）;性反射时间明显减少（Ｐ＜０．０１）;鲜精精清中ＡＫＰ和ＧＯＴ活性组间差异不显著,但ＬＤＨ活性２组显著高于１组（Ｐ＜０．０５）;冻后精清ＡＫＰ、ＧＯＴ和ＬＤＨ活性组间差异不显著（Ｐ＞０．０５）。添加锌能显著提高血清锌浓度（Ｐ＜０．０１）,但对精清锌含量的影响不明显（Ｐ＞０．０５）。血清睾酮浓度１组显著高于２组（Ｐ＜０．０５）,而精清睾酮浓度组间差异不显著（Ｐ＞０．０５）。     

Effects of Shading and Regaining Natural Light Treatment on Leaf Color and Physiological Changes of Pennisetum setaceum 'Rubrum'北大核心CSCD

To explore the effect of light on the leaf color and physiological changes of Pennisetum setaceum 'Rubrum', the natural light was used as a control, and the shading net with 10% transniittance was used to shading the new leaves until the new leaves fully devloped. Then, remove sun-shade net and using natural light for re-lighting treatmetn. And then the dynamic changes of leaf color parameters and related physiological indexes at different development stages were measured. The results showed that the new leaves of Pennisetum setaceum ' R u b r u m ' were green after shading, and the leaf color parameters L and b values were increased significantly, and the value of a decreased significantly. The contents of chlorophyll a and chlorophyll b were increased significantly, while anthocyanin, flavonoid, soluble sugar content and phenylalnine ammonialyase, chalcone isomerase activities were significantly decreased. The polyphenol oxidase and peroxidase activities were not changed significantly. After the re-lighting treatment, the leaf color gradually turned purple, and the leaf color parameter changed opposite to shading treatmetn. The change of physiological indexes was opposite to the decrease or increase after shading. The PPO and POD activities were not significantly changed, either. © 2019 China Agricultural University. All rights reserved.     

中性条件下2种酶解方法提取蚕蛹蛋白的比较研究

为了寻求经济实用的酶解蚕蛹蛋白的方法,本研究以新鲜和烘干蚕蛹为原料,在中性条件下比较了中性蛋白酶和风味蛋白酶对蚕蛹的水解程度及蛋白收率。结果表明:在中性条件下2种蛋白酶作用后鲜蛹水解蛋白液的蛋白收率和水解度高于干蛹水解蛋白液。风味蛋白酶水解后的蛋白收率高于中性蛋白酶,但水解度小于中性蛋白酶,造成这样的原因可能是由于风味蛋白酶的内切酶活性较低。因此,在实际应用中可尽量采用新鲜蚕蛹为原料,并根据蛋白水解产物不同的需求采用不同的蛋白酶进行处理。     

源于厌氧真菌Orpinomyces sp.PC-2的xynA在毕赤酵母中的高效表达及其酶学性质分析

β-D-1,4-内切木聚糖酶(endo-1,4-β-D-xylanase,xynA,EC.3.2.1.8)简称β-木聚糖酶(β-xylanase),是木聚糖降解酶系中最重要的成员,广泛用于饲料、食品、造纸和生物能源等领域.厌氧真菌Orpinomyces sp.PC-2 xynA具有潜在的开发价值,但是其异源表达的活性较低.本研究根据毕赤酵母(Pichia pastoris)对基因密码子偏好性,对厌氧真菌Orpinomyces sp.PC-2的xynA进行密码子优化.通过全基因合成技术合成优化后的xynAm,并克隆到毕赤酵母表达载体pPIC9K的多克隆位点,构建重组质粒pPIC9K-xynAm,电击转化至毕赤酵母GS115菌株中,以29℃、0.5％甲醇诱导表达,获得重组xynA,并对该酶的酶学特性进行了研究.结果表明,在摇瓶水平条件下,诱导表达108 h时重组β-木聚糖酶活性达到最大值,为612 IU/mL.在10L发酵罐中诱导表达96 h后,xynA的活性为3 515 IU/mL,比活性为2 411 IU/mg;菌体培养湿重、干重和培养上清总蛋白质的浓度分别为324、156和2.25 g/L.非变性十二烷基硫酸钠-聚丙烯酰胺(sodiumdodecyl sulfate-polyacrylamide gel electropheresis,SDS-PAGE)凝胶电泳显示,重组木聚糖酶的分子量约为42.7 kD.酶学性质分析表明,重组β-木聚糖酶的最适反应温度为55℃,最适反应pH 6.0,在温度为30～50℃、pH 4.0～10.6之间该酶具有较好的稳定性,其Km=27.86 mg/mL,Vmax=277.78 mg/(mL· min).底物特异性分析表明,重组xynA可水解低粘度阿拉伯木聚糖、高粘度阿拉伯木聚糖、燕麦木聚糖、桦木木聚糖和4-O-甲基-D-葡萄糖醛酸-D-木聚糖,但不降解大麦(Hordeum vulgare)β-葡聚糖和地衣多糖.10 mmol/L的Co2+和K+对该酶的活性略有激活作用,Mn2+、Fe2+和SDS对该酶活性有一定的抑制作用.本研究为进一步工业化应用来源于厌氧真菌Orpinomyces sp.PC-2的β-内切木聚糖酶奠定了基础.     

来源于水牛瘤胃未培养微生物的内切葡聚糖酶的碱性羧基末端结构域的功能鉴定

本碱性羧基末端结构域（basic C-terminal domain,BTD）是瘤胃细菌的碳水化合物活性酶（carbohydrateactive enzymes）中未知功能的一个结构域。BTD都位于酶或蛋白质的羧基最末端,大小为30～80个氨基酸（aa）,含有较多的碱性氨基酸,一般该结构域等电点都大于10。本工作构建了来源于水牛瘤胃未培养微生物的内切葡聚糖酶C5614-1的BTD缺失酶C5614-1RBTD57（缺失C5614-1羧基末端的57个aa）和C5614-1RBTD40（缺失C5614-1羧基末端的40个aa）以及C67-1的BTD缺失酶C67-1ΔBTD（缺失C67-1羧基末端的42个aa）,酶学特性分析发现BTD缺失酶与野生酶对pH和温度的稳定性是相似的,说明BTD结构域对酶的酶学特性方面贡献不大。尽管缺失了C5614-1的羧基末端57个氨基酸后,缺失酶C5614-1RBTD57对温度和pH的稳定性明显降低,但同时缺失酶对Avicel的结合能力也显著下降,说明该缺失酶是因为该酶的碳水化合物结合组件（carbohydrate binding module,CBM）的部分缺失而导致了酶的稳定性的改变。本文还对BTD可能的功能做了预测。     

腈水合酶产生菌的分离筛选和酶反应条件的优化

从化工废水处理厂的活性污泥中分离和筛选到一株腈水合酶产生菌HUST-1。经初步鉴定,菌株HUST-1属于红球菌属(Rhodococcus sp.)。对菌株(Rhodococcus sp.)HUST-1所产腈水合酶的反应条件进行研究,结果表明,该菌株的最优酶反应条件:丙烯腈加入量为5%,反应温度为28℃,pH值为7.0,反应时间为15min。在最优酶反应条件下,菌株(Rhodococcus sp.)HUST-1的最高比酶活可达119U·mg-1,比优化前提高24.5%。     

Optimization of phosphoric acid catalyzed fractionation and enzymatic digestibility of flax shives

Flax shives are the woody residue left over from processing flax straw into fiber, and are an abundant renewable lignocellulosic material with a potential for the conversion into bioethanol and other value added products. In this study, prior to enzymatic hydrolysis for the liberation of fermentable sugars, such as glucose and xylose, flax shives were treated with concentrated phosphoric acid. In order to optimize the phosphoric acid pretreatment and enzymatic hydrolysis steps, the effects of three process variables on the fractionation of flax shives, and enzymatic digestibility of pretreated flax shives were evaluated. The optimization process employed a central composite design (CCD), where the variables selected were concentration of phosphoric acid (40.8–86.2%), pretreatment time (9.5–110.5 min), and cellulase loading (13.1–71.9 FPU/g cellulose). Using three-variable and five-level CCD, all tested independent variables were identified to have significant effects (P < 0.05) on the digestibility of pretreated flax shives. It was found that the level of phosphoric acid (P < 0.0001) affects the digestibility most significantly when compared with other variables. When the optimization was conducted under a constrain of minimum cellulase loading, the maximum digestibility of 94.8% was predicted when the phosphoric acid concentration, pretreatment time, and cellulase loading were 86.2%, 110.5 min, and 13.1 FPU/g cellulose at 50 °C and 120 h, respectively. Under these conditions, digestibility of pretreated flax shives in the validation study reached a maximum of 93% at 120 h of incubation, showing good agreement with the values from the validation experiment of 93.4%, indicating high accuracy of the CCD procedure. When triticale straw, pine wood, and poplar wood were pretreated and hydrolyzed under optimum conditions obtained from the flax shives experiment, the digestibility reached 98.2, 74.8, and 95.7%, respectively, suggesting that the modest pretreatment process using phosphoric acid is an effective method for perennial plants as well as hard wood.     

Towards an optimal process for gelatinisation and hydrolysis of highly concentrated starch–water mixtures with alpha-amylase from B. licheniformis

The enzymatic hydrolysis of starch is usually carried out with 30–35 w/w% starch in water. Higher substrate concentrations (50–70 w/w%) were reached by using a twin-screw extruder for gelatinisation and for mixing enzyme with gelatinised starch prior to enzymatic hydrolysis in a batch reactor. The aim of this study was to determine which parameters are important for gelatinisation of wheat starch and to investigate the effects of different extrusion conditions on the enzymatic hydrolysis. After extrusion, the degree of gelatinisation was measured. During hydrolysis, the carbohydrate composition, the dextrose equivalent (DE) and the alpha-amylase activity were measured. Gelatinisation measurements showed that mechanical forces lowered the temperature required for complete gelatinisation. During hydrolysis experiments, high DEs were observed even if starch was not completely gelatinised during extrusion. Due to high substrate concentrations, the residual alpha-amylase activity remained high throughout enzymatic hydrolysis, although high temperatures were used. Increased substrate concentrations did not affect the carbohydrate composition of the product. Furthermore, the time required for the batch hydrolysis step could be varied by choosing a different enzyme-to-substrate ratio. This article provides a basis for detailed optimisation of this process to develop an industrial-scale process at high substrate concentrations.     

大米蛋白的双酶法水解条件研究

为研究大米蛋白的酶法水解条件,提高大米蛋白的溶解、乳化和发泡性能。通过酶催化反应进程确定酶的加入方式;通过均匀设计实验和Mathematica数学软件确定酶的反应条件。结果表明碱性蛋白酶和复合蛋白酶的共同水解效果高于单一酶制剂;酶催化反应过程中,大米蛋白的溶解性、乳化性和发泡性等指标变化趋势不同,水解度与上述指标之间也没有对应关系。得出的结论是双酶法水解更适于改善大米蛋白的溶解性能;两种酶之间有一定的协同作用,适当控制反应条件可以分别得到溶解、乳化或发泡性能显著的大米蛋白水解物。