重组水稻几丁质酶的发酵生产、纯化及酶学性质

用7L发酵罐对毕赤酵母(Pichia pastoris)工程菌株NB01进行了重组几丁质酶的诱导表达,通过提高通气量和调整搅拌转速,使溶氧维持在30%以上,NB01可在发酵84 h内达到产酶高峰,酶活最高可达48.0795 U/mL.利用中空纤维超滤膜对发酵液浓缩、提纯,再经过Sephadex G-100柱层析后得到单一组分的重组几丁质酶,酶纯度提高了7.9961倍.重组几丁质酶在60℃以下具有良好的热稳定性,在pH 2～6范围内有较强的耐受力,在4℃条件下贮藏具有较好的稳定性.     

昆虫几丁质酶的分类、功能分析、重组表达及酶学性质的研究进展

昆虫几丁质酶及其类似蛋白在昆虫生长和发育阶段起到重要调节作用。最近几年,运用新的生物技术对昆虫几丁质酶的结构,时空表达,和组织特异性表达以及生物学功能进行了深入研究,取得了许多新的成果。本文就昆虫几丁质酶基因的结构特征,几丁质酶生理功能、重组表达特点、酶活测定方法及影响酶活性因素等方面进行讨论与综述。     

海洋细菌QDC01的鉴定及其几丁质酶基因的克隆与分析

海洋微生物是几丁质酶的重要来源。本研究从青岛海域海蜇(Rhopilema esculenta)体中分离到一株产几丁质酶的细菌QDC01,通过形态、培养特征以及16S rDNA序列同源性分析,将该菌株鉴定为嗜水气单胞菌(Aeromonas hydrophila)。根据已报道的嗜水气单胞菌几丁质酶基因相关序列设计引物,克隆了QDC01几丁质酶基因,命名为ahchi(GenBank:JX863407)。应用生物信息学软件对获得的基因序列及编码的蛋白序列进行分析,结果显示,ahchi开放阅读框(ORF)长2 100 bp,无内含子,其编码的蛋白由699个氨基酸组成,分子量为74.875 kD,等电点为5.81。序列比对和同源性分析结果表明,该基因与嗜水气单胞菌(Aeromonas hydrophila subsp.hydrophila)ATCC 7966T几丁质酶基因(GenBank登录号:CP000462)的相似性最高,为98%;相应的氨基酸序列同源性为98%。系统进化分析以及Ahchi的保守结构域分析表明,Ahchi属于Ⅰ型几丁质酶,糖苷水解酶19家族。采用限制性内切酶(BamHⅠ和HindⅢ)双酶切法构建了原核表达载体pET-30a(+)-ahchi,并经IPTG诱导在大肠杆菌(Escherichia coli)中成功表达。在基础产酶发酵条件下,菌株QDC01所产粗酶液酶活力达0.21 U/mL,采用文献报道的优化产酶发酵条件,菌株QDC01所产粗酶液酶活力可达0.58 U/mL,是前人报道的产几丁质酶嗜水气单胞菌SWCH-6所产酶活力的1.49倍,同时也高于已报道的气单胞菌(Aeromonas sp.)CJ-5的酶活力(0.41 U/mL),为微生物源几丁质酶开发应用提供了又一优良种质资源。     

生物信息学在几丁质酶研究中的应用

几丁质酶在防治病原真菌,抵御农业害虫,参与共生固氮及植物的生长调控等方面都有重要作用。几丁质酶种类较多,在大小、三维结构、理化性质和酶学性质等方面差异较大,因而,采用生物信息学方法快速有效地分析几丁质酶是十分有必要的。对将生物信息学应用到新型几丁质酶基因的发现、几丁质酶的结构分析、几丁质酶的后基因组分析以及在几丁质酶分析中出现的问题等几方面进行了综述。     

山冈单胞菌ZL261几丁质酶基因的克隆及原核表达

分离自西藏色季拉山海拔4 530 m高山草甸土壤的山冈单胞菌(Collimonas pratensis)ZL261具有很高的几丁质酶活性,为了明确其几丁质酶种类及作用特点,依据已报道的食真菌山冈单胞菌(Collimonas fungivorans)Ter331胞外内切几丁质酶基因chiI相关序列设计引物,利用分段扩增的方法得到了菌株ZL261几丁质酶基因,并对其序列和编码蛋白的氨基酸进行分析。结果表明,全长1 338 bp的chiIQ基因编码的几丁质酶ChiIQ由445个氨基酸组成,分子量为46.04 kDa,等电点为5.67。序列比对和同源性分析发现,该基因与食真菌山冈单胞菌Ter331几丁质酶基因chiI序列相似性最高(92%),其氨基酸序列含有几丁质酶GH18家族高度保守序列SXGG和GXDXDXE,因此,ChiIQ属GH18家族。利用双酶切法构建了ZL261几丁质酶基因原核表达载体,并在大肠杆菌BL21中成功诱导表达。本试验结果为后期研究几丁质酶在山冈单胞菌生防应用中的功能以及生防菌ZL261的开发和应用奠定了基础。     

土壤中几丁质酶的研究进展

几丁质酶是影响土壤中氮矿化的一种重要的酶,其分解几丁质控制着氮循环的关键步骤。本文对土壤中几丁质酶的分类、来源、分布、影响因素、催化动力学特性及测定方法进行了综合评述,以期为我国开展土壤几丁质酶的相关研究提供理论参考。     

菌寄生真菌黄蓝状菌(Talaromyces flavus)几丁质酶基因的克隆与生物信息学分析

菌寄生真菌的几丁质酶有很强的降解几丁质能力,在控制植物病害方面起着重要的作用.为克隆和研究菌寄生真菌黄蓝状菌(Talaromyces flavus)几丁质酶基因(tfchi1),本研究根据真菌几丁质酶基因的保守序列设计扩增基因中间片段的简并引物,采用RT-PCR、3'-RACE及5+-TAIL的方法获得了该基因的DNA和mRNA序列(GenBank:GU361769,GU361770).tfchi1长为2 561 bp,具有6个内含子,长度分别为129、78、68、65、53和59 bp,包含1 194bp的ORF,编码一个由397个氨基酸组成的蛋白.推导的tfchi1氨基酸序列以及蛋白质结构生物信息学分析表明,该蛋白具有典型的几丁质酶催化区保守序列SXGGW和DGXDXDWE,属于糖苷水解酶18家族几丁质酶,与柄篮状菌(Talaromyces stipitatus ATCC 10500)几丁质酶(XP_002480365)氨基酸序列同源性为96％,分子量为43.47kD,等电点为4.97.该蛋白无信号肽序列,有15个潜在的N-糖基化位点,Tfchi1的二级结构以无规卷曲和α-螺旋为蛋白的主要结构元件,三级结构中有(α/β)8的圆桶形结构.tfchi1转化毕赤酵母(Pichia pastoris )GS 115,酵母工程菌可分泌具几丁质酶活性的表达产物,重组蛋白的分子量与理论值相符.结果说明,本研究已从T.flavus中正确克隆了1个糖苷水解酶18家族几丁质酶基因.     

几丁质酶在农业方面的应用研究概况

目前,合成类杀虫剂和杀真菌剂的滥用造成了严重的环境污染。而几丁质酶有潜在的抗含几丁质的病原体的特性,因此在农业方面应用前景广泛。综述了在几丁质酶抗植物病原真菌、杀虫、抗细菌和果蔬采后病害的防治等方面的应用研究情况。在此基础上,对几丁质酶在生物农业上的应用前景进行了展望。     

硅对水稻几丁质酶和β-1,3-葡聚糖酶活性的影响及其与抗纹枯病的关系

在溶液培养条件下,以水稻抗病品种91SP和感病品种Lemont为材料,研究施硅和接种纹枯病菌对水稻纹枯病发生情况、外切几丁质酶、内切几丁质酶和β-1,3-葡聚糖酶活性的影响。结果表明,施硅能降低抗病品种91SP的纹枯病病级和病情指数,显著降低感病品种Lemont的病级和病情指数。在未接种纹枯病菌条件下,施硅增加了抗病品种几丁质酶活性,增加了感病品种的几丁质酶活性,但对β-1,3-葡聚糖酶活性影响不大。接种纹枯病菌后,水稻几丁质酶活性被迅速激活后又下降,施硅通过提高抗病品种91SP几丁质酶和β-1,3-葡聚糖酶活性,以及通过提高感病品种Lemont几丁质酶活性来增强对纹枯病的抗性,但感病品种Lemont施硅处理的几丁质酶活性降低幅度小于抗病品种91SP。     

金属离子对苏云金芽孢杆菌几丁质酶活力的影响

几丁质酶(EC3．2．1．14)是细菌、病毒、真菌等微生物、高等植物和昆虫体内普遍合成的一种具有生物催化活性的水解酶类。它能特异地催化水解几丁质的β-1,4-糖苷键生成N-乙酰-D-氨基葡萄糖(NAG)。几丁质酶因为具有水解几丁质破坏围食膜的作用而被作为防治真菌病害和害虫的潜在靶标(蒋红彬等,2000；沙莉等，2003)。几丁质酶能增强苏云金芽孢杆菌(Bt)的杀虫效果，有利于克服或延缓昆虫对Bt的抗性。     

Gene cloning and characterization of a novel recombinant antifungal chitinase from papaya (Carica papaya)

A chitinase cDNA clone (CpCHI, 1002 bp) was isolated from papaya fruit, which encoded a 275 amino acid protein containing a 28 amino acid signal peptide in the N-terminal end. The predicted molecular mass of the mature protein was 26.2 kDa, and its pI value was 6.32. On the basis of its amino acid sequence homology with other plant chitinases, it was classified as a class IV chitinase. An active recombinant CpCHI enzyme was overexpressed in Escherichia coli. The purified recombinant papaya chitinase showed an optimal reaction temperature at 30 degrees C and a broad optimal pH ranging from 5.0 to 9.0. The recombinant enzyme was quite stable, retaining >64% activity for 3 weeks at 30 degrees C. The spore germination of Alternaria brassicicola could be completely inhibited by a 76 nM level of recombinant CpCHI. Recombinant CpCHI also showed antibacterial activity in which 50% of E. coli was inhibited by a 2.5 microM concentration of the enzyme.     

稳定塘藻类生长规律及其影响的中试研究

在兰州构建了自然型稳定塘（自然塘）和人工型稳定塘（人工塘）的中试系统，研究了两种稳定塘中藻类生长状况及藻类生长对稳定塘污染物去除功能的影响。相比于自然塘，人工塘增加了折流式导流墙且悬挂了无纺布载体。结果表明，增设导流墙使得人工塘中水流速度比自然塘增加了2．4倍，这抑制了人工塘中藻类的生长繁殖。自然塘中藻类比人工塘中藻类生长更为旺盛，叶绿素a峰值分别出现在3月和4月，为279．44mg·m^-3和115．65mg·m^-3。藻类光合作用使得稳定塘出水DO和pH都明显高于进水，自然塘藻类生长更为旺盛，因此出水与人工塘出水相比具有更高的DO和pH。更高的pH使得自然塘具有更好的氨氮和总磷去除率，分别为56％和19％。     

里氏木霉木聚糖酶基因Ⅱ在毕赤酵母中的分泌表达及其酶学性质研究

利用RT-PCR技术从高产木聚糖酶菌株T. reesei Rut C-30中成功扩增出其主要木聚糖酶基因XynⅡ,经序列分析证实,在该菌株诱变选育过程中其成熟肽序列有两处碱基发生突变;将不带原基因信号肽的XynⅡ克隆到毕赤酵母高效表达载体pPICZαA上,线性化后经电击转化到毕赤酵母中,经Zeocin及PCR筛选后得到的转化子用1%甲醇诱导。SDS-PAGE证实,重组子实现了分泌表达,且发酵上清中几无杂蛋白;对该重组酶酶学性质分析表明,该酶最适反应温度为60℃,最适反应pH值为6.0,该酶热稳定性较好,在50℃下保温30 min仍能保留其95%以上活性。     

Effect of Oxygen on the Adsorption of Pentachlorophenol by Peat from Water

Pentachlorophenol (PCP), a versatile biocide has been extensively used in industry and agriculture. PCP has been designated as a priority organic pollutant by the United States Environmental Protection Agency (USEPA) and its use in consumer products has been banned since 1984. Extensive use of PCP as a wood preservative has led to soil, surface water and groundwater pollution. Batch adsorption studies using peat at various dissolved oxygen (DO) levels in water showed that higher PCP removals can be attained at elevated DO levels. This effect can be quantified by a comparison of the adsorption capacities of peat from linearized Freundlich isotherms developed for different oxygen levels. An increase of approximately 300% in adsorption was observed for an increase in the DO level from 2 to 26 mg L^-1. Thus, molecular oxygen was found to significantly impact the removals of PCP by peat. The increase in adsorptive capacity of peat was not very significant for an increase in DO level from 2 mg L^-1 to 10 mg L^-1.However a rapid increase in adsorptive capacity was observed for an increase in DO level from 10 to 26 mg L^-1.     

自动增氧型垂直流人工湿地处理农村生活污水试验研究

针对人工湿地中溶解氧浓度不足的问题,采用自动增氧型垂直流人工湿地处理系统进行了农村生活污水脱氮除磷对比试验研究。结果表明,自动增氧型湿地内的DO浓度比非增氧型人工湿地高0.3 mg.L-1左右,TN、NH4＋-N去除率分别达到了67.41%、69.04%,比非增氧型湿地高14.57%、19.79%,但TP去除率与非增氧型湿地差异不显著。说明自动增氧措施对于增加人工湿地中的DO浓度,提高脱氮效率是有效的,但对于除磷效率无显著影响。     

气源及活性剂对曝气滴灌带水气单双向传输均匀性的影响

曝气滴灌过程中水、氧、气传输均匀性是评价曝气灌溉质量的重要指标。活性剂的添加和传输方式的优选对曝气滴灌传输过程中微气泡的存在和溶解氧的保持有重要意义。为提高水气耦合物在滴灌过程中传输的距离和均匀性,该文采用Mazzei 1078文丘里空气射流器进行曝气增氧,以空气和氧气为供试气源,研究活性剂BS1000浓度(0、1、2和4 mg/L)和传输方式(单向和双向)对曝气滴灌下水、氧、气传输特性的影响。结果表明:曝气导致单向传输下流量均匀性略有下降,但可显著提高灌溉水中溶解氧和掺气比例;随着活性剂浓度的增加,掺气比例显著增加(P0.05);活性剂的添加促进了氧气曝气下溶解氧的增加;溶氧均匀性和流量均匀性随着活性剂浓度的增加无显著性变化,但单向传输下4 mg/L BS1000的出气均匀性较未添加活性剂显著降低;双向传输的流量均匀性、溶氧均匀性和出气均匀性分别在95%、96%和67%以上,较单向传输分别平均提高14.00%、4.05%和30.64%(P0.05),是曝气滴灌长程管道传输推荐的布置方式。研究结果为曝气滴灌过程中灌溉技术参数优化和管道的科学布置提供理论依据。     

水稻几丁质酶的原核表达、复性及体外抑菌活性的研究

利用RT-PCR反应,从纹枯菌诱导的水稻叶片cDNA中克隆了1个水稻几丁质酶基因RC7的全长编码序列（ORF）。通过亚克隆方法,RC7与原核表达载体pGEX-4T-1上的GST融合,构建成GST-RC7重组蛋白的原核表达载体pGST-RC7,然后,将其转化到大肠杆菌细胞中。经异丙基-β-D-硫代半乳糖苷（IPTG）诱导,获得了以包涵体形式高效表达的GST-RC7。经过洗涤、溶解（变性）、复性及纯化等处理,包涵体中GST-RC7得到溶解、复性和纯化。纯化的复性GST-RC7蛋白具有几丁质酶活性。对6种重要作物病原真菌进行体外抑菌活性分析的结果表明, RC7可显著抑制水稻纹枯病菌、小麦纹枯菌、小麦赤霉菌、棉花黄萎菌、烟草赤星菌的菌丝生长,说明RC7可作为上述植物病害抗性基因育种的重要基因。     

拟南芥Alpha-dioxygenase2(AtDOX2)在毕赤酵母中的表达、纯化及活性鉴定

为了获得具有生物活性的拟南芥(Arabidopsis thaliana)alpha-dioxygenase2(AtDOX2),将其对应基因AtDOX2编码区克隆到酵母表达载体pPIC9k中,获得重组表达载体pPIC9k-AtDOX2,将线性化的重组载体电击转化入毕赤酵母(Pichia pastoris)表达菌株GS115,经G418筛选、PCR鉴定和甲醇诱导时间优化,获得重组AtDOX2的高效表达菌株GS115/pPIC9k-AtDOX2。SDS-PAGE分析结果显示,0.5%甲醇诱导96h重组蛋白表达量最高,其表达量占胞外总蛋白的15%。重组AtDOX2的表观分子量约为70kD,经Ni-NTA柱亲和层析可获得纯度大于80%的重组蛋白。2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS)法测定结果表明重组蛋白具有过氧化物酶活性,且其活性受Ca2+和Mg2+激活,受EDTA、咪唑和Mn2+抑制;2,4-二硝基苯肼(2,4-DNP)法测定结果显示,重组AtDOX2具有双加氧酶活性,Ca2+对其双加氧酶活性也有激活作用。结果说明利用酵母表达系统获得...     

Comparison of Relationships Between pH, Dissolved Oxygen and Chlorophyll a for Aquaculture and Non-aquaculture Waters

The relationships between pH, dissolved oxygen (DO) and chlorophyll a in aquaculture and non-aquaculture waters are assessed in this paper. The research includes the evaluation of field and experimental studies at the Panjiakou Reservoir (between Aug and Oct 2009) and the review of international data covering two decades. The results indicated that typical eutrophic non-aquaculture water had mean concentrations of chlorophyll a of higher than 10 ??g L^?1, and significant positive correlations were found between pH, DO and chlorophyll a. When the mean concentration of chlorophyll a was less than 10 ??g L^?1, no correlation was found between DO and chlorophyll a for waters with a high exchange rate or heavily organically polluted natural waters. Diurnal variations were found for both pH and DO. A corresponding significant positive correlation was found between both water quality parameters. In general, when the mean concentration of chlorophyll a was less than 10 ??g L^?1 within aquaculture waters of low exchange rate, only a weak or no correlation at all was found between pH, DO and chlorophyll a during summer and autumn. On the other hand, a significant positive correlation between pH and chlorophyll a and a significant positive correlation or no correlation between DO and chlorophyll a were found for aquaculture waters with a high exchange rate during summer and autumn. Strong diurnal variations for both pH and DO were identified. A significant positive linear correlation between pH and DO was found for field enclosure experiments.     

Dynamics of dissolved oxygen (DO) in ponded water of a paddy field

The transfer of heat and dissolved oxygen (DO) through water is important to understand the phenomenon of ponded water in a paddy soil. The heat from solar radiation is absorbed at the soil surface and transferred into the ponded water by convection. This study clarified the dynamics of DO, as well as the role of convection in water in DO transfer in the ponded water of a paddy field. DO concentration in the ponded water of a paddy field was measured in situ in the daytime and during the night. The results were confirmed in lab-scale model experiments. The DO concentration and temperature profiles in the ponded water of a lab-scale paddy field model were investigated under convective and non-convective conditions using solar radiation and infrared radiation, respectively. Under the ponded condition, solar radiation was absorbed at the soil surface whereas infrared radiation was absorbed at the water surface and thereby convective and non-convective conditions were generated, respectively. The diurnal variation in DO concentration was closely related to the intensity of solar radiation. Oxygen generation by micro-algae and its subsequent circulation by convection resulted in uniform DO concentration profiles, with super-saturated values in the ponded water in the daytime. Eventually oxygen was released to the atmosphere by deaeration until DO in water was depleted to the saturated level. During the night the oxygen moved from the atmosphere into the water surface by reaeration which depends on the oxygen deficit related to saturation. The oxygen deficit is caused by the respiration of microorganisms. The oxygen, that moved from the atmosphere to the water surface, was transferred to the soil surface by convection in the water layer. Thus convection plays an important role in the DO transfer in the ponded water of a paddy field. The DO dynamics is correlated with biological processes in the ponded paddy soil.