响应面法优化褐藻胶降解菌Cobetia sp.20发酵培养基

为了提高褐藻胶降解菌株Cobetia sp.20产褐藻胶裂解酶的能力,利用响应面法优化其发酵产褐藻胶裂解酶的培养基。首先利用单因素法分别对发酵培养基中的不同碳源、碳源添加量、不同氮源、氮源添加量以及氯化钠添加量、磷酸二氢钾添加量、硫酸镁添加量和pH进行探究,研究各因素对产酶的影响。在单因素实验的基础上,通过Plackett-Burman试验确定Cobetia sp.20发酵培养基中影响产酶的主要因素。通过响应面试验建立回归方程。研究结果表明,Cobetia sp.20最优发酵培养基配方为褐藻胶15.00 g/L、硫酸铵7.50 g/L、氯化钠15.00 g/L、硫酸镁0.50 g/L、磷酸二氢钾5.30 g/L、硫酸亚铁0.01 g/L、pH值7.58。优化后酶活为142.79 U/mL,比优化前提高了26.36%。褐藻胶裂解酶活的提高,为褐藻胶裂解酶的工业化生产提供了参考。     

多株功能菌种混合培养条件的响应面优化及其生长关系

对3种具有土壤中多种重金属污染修复和病虫害防治功能的光合细菌（Photosynthetic bacteria,PSB,包括Rhodobacter sphaeroides H菌株、Rhodopseudomonas palustris N菌株和Rhodospirillium rubrum M菌株）、枯草芽胞杆菌（Bacillus subtilis, BS）和植物乳酸杆菌（Lactobacillus plantarum, LP）进行混合培养,利用单因素和响应面试验优化培养基配方以及培养条件。确定功能菌混合培养的最佳条件：以光合细菌培养基为基础培养基,外加碳源为10 g/L蔗糖、氮源为1.25 g/L硫酸铵、培养温度为31.5 ℃、初始pH值为7.1、接种量为接种后的初始培养活菌数1.90×10⁹cfu/mL、培养时间为28.0 h。通过功能菌的加性模型法、生长动力学以及混合培养的生长关系分析表明,光合细菌、枯草芽胞杆菌和植物乳酸杆菌的生长均存在互惠互利关系。     

毛霉脂肪酶的研究

从土样中分离筛选到一株脂肪酶菌株—毛霉(Mucor sp.)M₂,其优化后的培养基组成(%);黄豆粉4.0、蔗糖0.5、橄榄油1.0、硫酸铵0.1、磷酸氢二钾0.2硫酸镤0.01、pH自然。产酶最适条件：初始pH6.5、培养温度28℃、培养周期96h.该酶最适作用温度50℃、最适pH8.0、pH稳定范围为7.0～10.0,Fe²⁺、Ca²⁺、Mg²⁺、K⁺对酶有激活作用。     

产脂肪酶重组枯草芽胞杆菌的发酵优化

笔者所在实验室前期筛选到1株产脂肪酶粘质沙雷氏菌,克隆其脂肪酶基因,构建重组枯草芽胞杆菌Bacillus subtilis 168/pMA5-lipA,成功实现了来源于粘质沙雷氏菌的脂肪酶基因在枯草芽胞杆菌中的表达。基于以上工作基础上,对B.subtilis 168/pMA5-lipA进行了摇瓶水平上的产酶发酵优化。首先通过单因素和正交试验确定了有利于产脂肪酶的最佳培养基成分,并对发酵条件进行了优化。结果表明:优化后的培养基组分为蔗糖35 g/L,玉米浆27.5 g/L,(NH4)2SO41.25 g/L,CaCl24 g/L,pH 7.0。在最优发酵培养基的条件下,37℃、160 r/min摇床培养33 h,每毫升发酵液中重组菌脂肪酶酶活可达98.6 U,是优化前的3倍。     

刺孢小克银汉霉菌体氨基酰化酶的性质研究*

首次选育出有较高氨基酰化酶活性的菌株刺孢小克银汉霉(Cunningham ella echinula-ta)9980,并进行液体培养,比较了3种不同培养基中菌体细胞氨基酰化酶活性,考察了几种因素对菌体细胞酶活的影响。结果表明:蛋白胨培养基中菌体细胞酶活最高,达680u/g。菌体细胞酶活最适温度55℃,最适pH7.0,最佳底物浓度为0.2mol/L,缓冲液中的无机离子对酶活有抑制作用,10^-3~10^-4mol/L的Co²⁺对酶活     

保水功能菌枯草芽胞杆菌LX-1固态发酵的工艺优化

γ-聚谷氨酸(γ-PGA)对土壤保水和提高植物抗逆性具有明显作用,为了利用农产品加工下脚料发酵生产含有γ-PGA的保水功能肥料,对福建省微生物研究所环境微生物研究室分离到的产γ-PGA的枯草芽胞杆菌(Bacillus subtilis)LX-1的固态发酵培养基配比进行优化。实验以发酵产物的荧光素双醋酸酯酶(FDA)酶活和增比黏度为指标,在通过单因素实验选定固态发酵料的碳源、氮源、无机盐溶液的最佳配比以及无机盐较佳添加量的基础上,通过拟水平均匀设计U₇* (7⁴试验,得出菌株LX-1固态发酵的最佳培养基配方：V豆粕V麦麸V无机盐溶液=644,其中无机盐溶液的最佳配方为K₂HPO₄·3H₂O 5.24 g/L、MgSO₄·7H₂O 0.78 g/L、NaCl 11.67 g/L。为利用农产品加工下脚料发酵生产含γ-PGA保水肥料提供参考。     

一株海洋来源铜绿假单胞菌Gxun-7角蛋白酶基因的克隆、表达及重组酶酶学性质

【背景】角蛋白酶是一类特异性降解角蛋白的水解酶,在动物饲料、生物肥料、医学、洗涤、制革及环境治理等方面具有重要的应用潜力。【目的】对前期从海洋环境筛选出的一株铜绿假单胞菌Gxun-7的角蛋白酶基因进行克隆、表达,并探究重组酶酶学性质,为角蛋白酶在工业生产中的应用奠定基础。【方法】以铜绿假单胞菌Gxun-7基因组推定的角蛋白酶基因为基础,设计引物克隆获得角蛋白酶基因kp2,构建重组表达质粒pET22b-kp2,并转化到E. coliRosettagamiB (DE3)中进行诱导表达,同时对重组表达菌株的表达条件进行优化。利用镍柱分离纯化重组角蛋白酶并研究其酶学性质。【结果】重组角蛋白酶的分子量约为33 kDa,最适温度和pH值分别为40 ℃和8.0,在温度30-60 ℃和pH 6.5-8.0具有较好的稳定性。金属离子Co²⁺、Cu²⁺和化学试剂十二烷基磺酸钠(sodium dodecyl sulfonate,SDS)、乙二胺四乙酸(ethylenediaminetetraacetic acid,EDTA)、苯甲基磺酰氟(phenylmethylsulfonyl fluoride,PMSF)对酶活力有抑制作用,而Mg²⁺、K⁺、巯基乙醇和二硫苏糖醇(dithiothreitol,DTT)对酶活力有促进作用。重组角蛋白酶具有良好的耐盐性,在12.5%的NaCl作用下相对酶活为87.55%。以酪蛋白为底物时,酶的K_m值为60.92 mg/mL、V_max值为9.70 U/mL。【结论】海洋来源铜绿假单胞菌Gxun-7的重组角蛋白酶具有良好的温度、碱、盐稳定性,可应用于工业生产中。     

谷氨酸棒杆菌H+-ATPase基因失活提高谷氨酸产生量

为了证实在谷氨酸棒杆菌中,利用H⁺-ATPase基因失活构建高产谷氨酸基因工程菌的应用可行性,通过重组PCR技术部分缺失H⁺-ATPaseγ亚基基因序列,采用插入失活方法构建H⁺-ATPase失活的谷氨酸棒杆菌。考察了其谷氨酸产生能力及对生长速率的影响。实验结果表明,H⁺-ATPase失活的谷氨酸棒杆菌在含有100g/L的葡萄糖培养基中摇瓶发酵,其谷氨酸最大累积量为51.6g/L, 比野生菌株提高了42.9%。生长速率研究结果表明,H⁺-ATPase失活的谷氨酸棒杆菌生长速率略低于野生谷氨酸棒杆菌。证实了H⁺-ATPase基因失活对提高谷氨酸产量的作用,为利用H⁺-ATPase基因构建高产谷氨酸基因工程菌株提供了科学依据。     

融合自组装双亲短肽对重组过氧化氢酶酶学性质的影响

[目的]利用融合自组装双亲短肽策略对源自枯草芽孢杆菌(Bacillus subtilis)的过氧化氢酶KatA进行改性,以强化重组过氧化氢酶在工业中的应用适应性。[方法]将自组装双亲短肽S1vw通过连接肽PT-linker融合在KatA的N端,构建重组质粒pHT254-S1vw-PT-katA,将其与携带天然酶基因的pHT254-katA分别转入枯草芽孢杆菌WB800N中进行分泌表达,之后将分离纯化得到的纯酶进行酶学性质研究。[结果]成功构建出工程菌并将胞外粗酶液通过乙醇沉淀、DEAE阴离子交换层析、疏水层析和凝胶过滤层析4步纯化,最终获得电泳纯的重组酶蛋白。酶学性质研究结果显示,融合酶S1vw-PT-KatA和天然酶KatA的最适反应温度均为30℃,最适反应pH值均为11.0。然而,融合酶在pH 12.0下孵育30 min的相对酶活为77.3%,是相同处理条件下天然酶相对酶活的14.9倍,在65℃和70℃下孵育30 min的相对酶活分别为19.8%和17.5%,是相同处理条件下天然酶相对酶活的1.8倍和1.7倍。此外,融合酶在4℃储存14 d后相对酶活为88.6%,而天然酶仅具有44.3%的相对酶活。同时,融合酶的k_cat/K_m提高到天然酶的2.3倍。[结论]融合自组装双亲短肽S1vw提高了重组过氧化氢酶KatA的pH稳定性、温度稳定性、储存稳定性和催化效率,不仅获得了催化效率和应用适应性改良的重组酶蛋白,为针对过氧化氢酶的进一步分子改造提供了策略参考和实验依据,而且促进了其在工业上的规模化制备和应用。     

响应面法优化类芽胞杆菌HB172198产褐藻胶裂解酶发酵培养基

为了提高类芽胞杆菌新种HB172198产褐藻胶裂解酶活力,本研究采用响应面法对该菌株液体发酵培养基进行了优化实验。在单因素实验和Plackett-Burman试验筛选出海藻酸钠、胰蛋白胨、NaCl、MgSO4·7H2O等4个显著影响产酶因素的基础上,通过Box-Behnken设计及响应面法进行回归分析,得出产褐藻胶裂解酶最佳发酵培养基,其成分为:海藻酸钠7.50 g/L、胰蛋白胨13.57 g/L、NaCl 29.75 g/L、MgSO4·7H2O 0.08 g/L。优化条件下该菌株最大酶活性达14.60 U/mL,是优化前的1.87倍。本研究为菌株HB172198产褐藻胶裂解酶的大规模生产和工业应用提供了重要的理论依据。     

Foreign exploration for Scirtothrips perseae Nakahara (Thysanoptera: Thripidae) and associated natural enemies on avocado (Persea americana Miller)

Scirtothrips perseae Nakahara was discovered attacking avocados in California, USA, in 1996. Host plant surveys in California indicated that S. perseae has a highly restricted host range with larvae being found only on avocados, while adults were collected from 11 different plant species. As part of a management program for this pest, a “classical” biological control program was initiated and foreign exploration was conducted to delineate the home range of S. perseae, to survey for associated natural enemies and inventory other species of phytophagous thrips on avocados grown in Mexico, Guatemala, Costa Rica, the Dominican Republic, Trinidad, and Brazil. Foreign exploration efforts indicate that S. perseae occurs on avocados grown at high altitudes (>1500 m) from Uruapan in Mexico south to areas around Guatemala City in Guatemala. In Costa Rica, S. perseae is replaced by an undescribed congener as the dominant phytophagous thrips on avocados grown at high altitudes (>1300 m). No species of Scirtothrips were found on avocados in the Dominican Republic, Trinidad, or Brazil. In total, 2136 phytophagous thrips were collected and identified, representing over 47 identified species from at least 19 genera. The significance of these species records is discussed. Of collected material 4% were potential thrips biological control agents. Natural enemies were dominated by six genera of predatory thrips (Aeolothrips, Aleurodothrips, Franklinothrips, Leptothrips, Scolothrips, and Karnyothrips). One genus each of parasitoid (Ceranisus) and predatory mite (Balaustium) were found. Based on the results of our sampling techniques, prospects for the importation of thrips natural enemies for use in a “classical” biological control program in California against S. perseae are not promising.     

Taxonomic status of the species of the green algal genusNeochloris

Komárek has recently reviewed the various species assigned to the green algal genusNeochloris Starr (Chlorococcales, Chlorococcaceae) and removed those with uninucleate vegetative cells to a new genus,Ettlia. Watanabe & Floyd, unaware ofKomárek's work, also reviewed the species ofNeochloris and distributed them among three genera—Neochloris, Chlorococcopsis gen. nov., andParietochloris gen. nov.—on the basis of details of the covering of the zoospore and the arrangement of the basal bodies of the flagellar apparatus. This paper reconciles these two treatments and makes additional recommendations at the ranks of genus, family, order, and class.     

Die GattungKarschia — Bindeglied zwischen bitunicaten Ascomyceten und lecanoralen Flechtenpilzen?

The genusKarschia, in the earlier sense, including saprophytes and parasites on lichens, has been thought to be a non-lichenized parallel genus of the lichen genusBuellia. Modern workers included it on the one hand inBuellia, on the other hand combined it with bitunicate ascomycetes. It is now proved thatKarschia is heterogeneous and contains but superficially similar members both of the genusBuellia of theLecanorales and of typical or masked bitunicateAscomycetes. Therefore, it can not be regarded as a link betweenLecanorales andDothideales. The type species ofKarschia belongs to theDothideales.

     

Systematic significance of mature embryo of bamboos

The mature embryo of seven species belonging to five genera of Indian bamboos is described. In all these the basic pattern of embryo organisation is same: the scutellar and coleoptilar bundles are not separated by an internode, the epiblast is absent, the lower portion of the scutellum and the coleorhiza are separated by a cleft and the margins of embryonic leaves overlap. The features unique to fleshy fruited bamboos are: presence of a massive scutellum, the juxtaposition of plumule and radicle and the occurrence of a bud in the axil of the coleoptile. The fleshy fruit bearing bamboos should be classified into one group, the tribeMelocanneae. Evidence is provided to recognise additional groups in the subfamilyBambusoideae.     

Phylogeny of cardinalfishes (Teleostei: Gobiiformes: Apogonidae) and the evolution of visceral bioluminescence

The cardinalfishes (Apogonidae) are a diverse clade of small, mostly reef-dwelling fishes, for which a variety of morphological data have not yielded a consistent phylogeny. We use DNA sequence to hypothesize phylogenetic relationships within Apogonidae and among apogonids and other acanthomorph families, to examine patterns of evolution including the distribution of a visceral bioluminescence system. In conformance with previous studies, Apogonidae is placed in a clade with Pempheridae, Kurtidae, Leiognathidae, and Gobioidei. The apogonid genus Pseudamia is recovered outside the remainder of the family, not as sister to the superficially similar genus Gymnapogon. Species sampled from the Caribbean and Western Atlantic (Phaeoptyx, Astrapogon, and some Apogon species) form a clade, as do the larger-bodied Glossamia and Cheilodipterus. Incidence of visceral bioluminescence is found scattered throughout the phylogeny, independently for each group in which it is present. Examination of the fine structure of the visceral bioluminescence system through histology shows that light organs exhibit a range of morphologies, with some composed of complex masses of tubules (Siphamia, Pempheris, Parapriacanthus) and others lacking tubules but containing chambers formed by folds of the visceral epithelium (Acropoma, Archamia, Jaydia, and Rhabdamia). Light organs in Siphamia, Acropoma, Pempheris and Parapriacanthus are distinct from but connected to the gut; those in Archamia, Jaydia, and Rhabdamia are simply portions of the intestinal tract, and are little differentiated from the surrounding tissues. The presence or absence of symbiotic luminescent bacteria does not correlate with light organ structure; the tubular light organs of Siphamia and chambered tubes of Acropoma house bacteria, those in Pempheridae and the other Apogonidae do not.     

Biological control of three floating water weeds,<Emphasis Type="Italic">Eichhornia crassipes,Pistia stratiotes</Emphasis>, and <Emphasis Type="Italic">Salviniamolesta</Emphasis> in the Republic of Congo

Since 1999, four specific weevils (Coleoptera, Curculionidae) were released in the Republic of Congo against three exotic floating water weeds: Neochetina eichhorniae Warner and N. bruchi Hustache against water hyacinth, Neohydronomus affinis Hustache against water lettuce, and Cyrtobagous salviniae Calder and Sands against water fern. Recoveries of exotic weevils were made from all 24 release sites except one, and all four species have established and spread (up to 800 km for water hyacinth weevils). Within a few years of releases, control of water fern and water lettuce was such that fishing and navigation could be resumed, while reductions of water hyacinth populations were only beginning.     

A molecular phylogeny of Hebeloma species from Europe

In order to widen the scope of existing phylogenies of the ectomycorrhizal agaric genus Hebeloma a total of 53 new rDNA ITS sequences from that genus was generated, augmented by sequences retrieved from GenBank, and analysed using Bayesian, strict consensus and neighbour joining methods. The lignicolous Hebelomina neerlandica, Gymnopilus penetrans, and two species of Galerina served as outgroup taxa. Anamika indica, as well as representatives of the genera Hymenogaster and Naucoria, were included to test the monophyly of Hebeloma, which is confirmed by the results. Hebeloma, Naucoria, Hymenogaster and Anamika indica cluster in a strongly supported monophyletic hebelomatoid clade. All trees largely reflect the current infrageneric classification within Hebeloma, and divide the genus into mostly well-supported monophyletic groups surrounding H. crustuliniforme, H. velutipes, H. sacchariolens, H. sinapizans, and H. radicosum, with H. sarcophyllum being shown at an independent position; however this is not well supported. The section Indusiata divides with strong support into three groups, the position of the pleurocystidiate Hebeloma cistophilum suggests the possible existence of a third subsection within sect. Indusiata. Subsection Sacchariolentia is raised to the rank of section.