Bacillus subtilis 168中亚硝酸盐还原酶基因的敲除及表型鉴定

该研究首先通过同源重组方式将Bacillus subtilis 168菌株nir基因位点置换为两端具有同向排列的loxP序列选择性标记基因,再运用Cre重组酶将两个loxP间的全部序列切除,成功敲除了Bacillus subtilis 168菌株亚硝酸盐还原酶(nitrite reductase,NiR)的3个亚基基因nasB、nasD和nasE。其中,敲除nasD基因的B.subtilis 168菌株在不同生长时期均未分解培养基中亚硝酸盐,表明该nasD基因缺陷型菌株NiR活性已被完全消除,不再具备降解亚硝酸盐的能力。

Medium factors affecting extracellular protease activity by <i>Bacillus</i>sp. HTS 102—A novel wild strain isolated from Portuguese merino wool

The synthesis of an extracellular protease by Bacillus sp. HTS102—a wild strain recently isolated from the wool of Portuguese Merino ewes, was optimized. This protease is thermostable and particularly resistant to harsh environmental conditions—and appears to bear a unique ability to hydrolyze keratin-rich solid materials. Following a preliminary screening for the most relevant medium factors involved in processing, a fractional factorial design (2VI6-1) was applied to ascertain the effects of six relevant parameters—viz. yeast extract concentration, peptone level, inoculum size, stirring rate, temperature and pH. The concentrations of yeast extract and peptone, as well as the incubation temperature and pH were found to play significant roles;and the 2-factor interaction between yeast extract level and pH was also significant. A 2.2-fold increase in the overall level of protease synthesis was eventually attained, with the improved medium relative to the basal medium—which is noteworthy when compared with competing proteases and previous optimization efforts.

解淀粉芽孢杆菌SQ-2对水稻的促生作用

【目的】对实验室分离筛选得到的一株解淀粉芽孢杆菌SQ-2进行促生特性研究,确定该菌株对水稻促生的浓度范围与作用机制,并分析接种菌株前后的土壤菌群结构。【方法】利用钼蓝比色法与固氮酶试剂盒对菌株SQ-2的溶磷能力及固氮酶活性进行检测。将10^(2)、10^(4)、10^(6)、10^(8)和3×10^(8)CFU/mL的菌悬液接种至水培与土培水稻中,分别培养14 d和20 d后测定其水培、土培水稻的根茎干鲜重、茎长与茎粗。采用苯酚-次氯酸钠比色法、茚三酮检测法与3,5-二硝基水杨酸比色法测定土培水稻土壤中脲酶、蛋白酶与蔗糖酶的活性。利用pH计电位法检测土壤pH值,用对应试剂盒检测土壤速效氮磷钾含量及酸性磷酸酶活性,并对接种3×10^(8)CFU/mL组的土培水稻土壤进行根际细菌群落结构分析。【结果】菌株SQ-2对磷酸三钙的溶解量为229.63 mg/L,固氮酶活性为55.07 U/L。与对照相比,接种菌悬液浓度在10^(4)CFU/mL时,水培水稻根的干、鲜重增长最为显著;在接种浓度为3×10^(8)CFU/mL时,土培水稻根茎的干、鲜重增长最为显著。随着接种菌悬液浓度的升高,上述土壤酶活性与速效氮磷钾浓度都有着不同程度的增加,而接种菌株3×10^(8)CFU/mL的土壤pH值则由原来的7.83降至7.26。接种菌株SQ-2改变了水稻根际土壤中的菌落构成,显著提高了土壤α多样性的Ace、Chao、Sobs与Shannon指数。【结论】解淀粉芽孢杆菌SQ-2对土培、水培水稻均有不同程度的促生效果。在土培实验中,能够通过提高土壤酶活性、速效氮磷钾水平及改变土壤菌群结构来起到促生作用,为细菌菌肥的研发提供了新的菌株资源。

有机溶剂对苏云金芽孢杆菌(Bacillus thuringiensis)几丁质酶的影响

以苏云金芽孢杆菌(Bt)的发酵液为材料,经硫酸铵分级分离、DEAE-32离子交换柱层析分离,获得部分纯化的Bt几丁质酶(EC3. 2. 1. 14)制剂.研究了几种有机溶剂对Bt几丁质酶的影响.结果表明,甲醇、丙三醇、甲醛和戊二醛对几丁质酶有抑制作用;乙醇、丙醇、乙二醇和二甲亚砜在低浓度时对酶有激活作用,随着浓度的升高表现出抑制作用;二氧六环的浓度低于 5%时,对酶的影响不明显,而高于 5%时,对酶则有激活作用;丙酮对酶有激活作用. 图 1参

贝莱斯芽胞杆菌GZ8-6对马铃薯根际土壤酶活性的影响及促生作用

为探究生防菌对马铃薯根系土壤酶活性及马铃薯生长的影响。分别以马铃薯疮痂病Streptomyces scabies X-1菌液、LB液体培养基以及生防菌贝莱斯芽胞杆菌Bacillus velezensis GZ8-6发酵液对马铃薯苗进行灌根处理,测定和比较不同时间段的土壤酶活性和马铃薯生长相关指标。结果表明:X-1和贝莱斯芽胞杆菌GZ8-6处理后,马铃薯根际土壤中脲酶、蔗糖酶、过氧化氢酶和纤维素酶的活性明显高于病原菌处理和LB液体培养基处理。X-1和GZ8-6处理后第30、60天,土壤蔗糖酶活性达到高峰,分别比病原菌处理高1.70倍和2.71倍,土壤脲酶活性在施用后第10、20天较病原菌处理分别提升了52.53%和59.48%;土壤纤维素酶和土壤过氧化氢酶活性在马铃薯生育期内呈上升-下降-上升-下降的趋势。经X-1和GZ8-6处理后马铃薯的侧根数、茎粗、地下部鲜重等各项生长指标都优于病原菌处理和培养基处理,处理后60 d株高和地下部鲜重分别较病原菌处理提高了34.65%和124.79%,茎粗较LB液体培养基处理平均增加0.53 cm。因此,生防菌处理不仅对马铃薯有促生作用,同时还能提高土壤关键酶活性。

贝莱斯芽孢杆菌对桃采后软腐病的防治及对果实诱导抗性的影响

为明确贝莱斯芽孢杆菌Q-84对桃果实软腐病的生防效果,本试验以“映霜红”桃为材料,研究Q-84菌悬液对桃采后软腐病的抑制效果及对果实品质的影响,并对果实过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、几丁质酶(CHI)和脂氧合酶(LOX)4种防御相关酶活性进行测定。结果显示,贝莱斯芽孢杆菌Q-84菌悬液处理可显著抑制桃果实软腐病,抑制果实LOX活性,诱导POD、PAL、CHI活性增加,提高果实抗病和抗氧化能力,减缓桃硬度下降,对桃果实色泽及可溶性固形物和可滴定酸含量无不良影响。表明贝莱斯芽孢杆菌对桃果实软腐病具有较好的防治效果,在桃果实采后生物防腐保鲜领域具有重要应用价值。

1株野生高效纤维素降解菌筛选、鉴定其酶活力测定

为从腐熟的土壤中分离出产纤维素酶的细菌,并进一步研究该菌株的纤维素降解能力。采用羧甲基纤维素(CMC)-刚果红染色法,筛选出1株野生纤维素降解菌ZG2-3,采用16S rRNA分子鉴定其种类,同时从促腐剂中筛选出4株商品纤维素降解菌,比较野生纤维素降解菌与商品纤维素降解菌的酶活差异。结果表明:从娄底市珠山公园桂花树林下腐熟的土壤中采集分离,通过羧甲基纤维素(CMC)-刚果红染色法,筛选得到了纤维素降解能力较高的1株细菌ZG2-3。经鉴定ZG2-3为枯草芽孢杆菌Bacillus subtilis。ZG2-3的羧甲基纤维素酶(CMCase)活力和滤纸酶(FPA)活力分别为0.054 IU·mL^(-1)和0.02 IU·mL^(-1),其中ZG2-3的CMCase活力显著高于商品纤维素降解菌。

生物炭基微生物菌剂修复重金属污染土壤研究

综述了生物炭基微生物菌剂对植物吸收重金属以及对土壤微生物影响的研究进展,分析了微生物修复机理及限制因素,重点归纳了其影响机制,包括促进植物生长,减少植物对重金属的吸收、改善土壤理化性质(有机质、pH等)、改变重金属形态、降低土壤重金属有效性、改变土壤微生物群落组成等。探究了此领域存在的问题及今后的发展方向,以期为生物炭基微生物菌剂修复重金属污染土壤研究提供理论依据。

Bacillus subtilis PTS-394对番茄根系及根围微生态的影响

为了明确枯草芽孢杆菌(Bacillus subtilis)PTS-394对番茄根系及根围微生态的影响,本研究采用生理生化方法测定了番茄根系活力和根围土壤酶活性,并测定了番茄根冠比及根系周围可培养微生物的数量。结果表明,枯草芽孢杆菌PTS-394能够显著促进番茄根系生长,促进生长率达25%;同时PTS-394还能够提高根系活力,在PTS-394处理后第12 h、24 h,根系活力均比对照显著增高,增高比例分别为17.0%和25.6%,随后根系活力和对照基本持平并保持一致。枯草芽孢杆菌PTS-394的施用对番茄根围土壤中的放线菌具有显著的促进作用,对真菌具有轻微的抑制作用,而对细菌则无明显作用。此外,PTS-394能够促进番茄根围土壤脲酶和土壤蔗糖酶的活性,但其对2种酶的作用均有时效性,其影响会随着时间的变化而逐渐减弱。

贝莱斯芽孢杆菌QL-2对连作花生土壤肥力和微生物群落的影响

花生是我国重要的油料经济作物,栽种面积大,年产量高。为缓解连年种植花生导致土壤连作障碍对花生生长以及土壤环境的有害影响,将贝莱斯芽孢杆菌(Bacillus velezensis)QL-2微生物菌剂与普通基肥相结合施撒,评价了贝莱斯芽孢杆菌QL-2对花生生长、土壤肥力、酶活性和微生物群落的影响。结果表明:贝莱斯芽孢杆菌QL-2促进了花生的生长,土壤碱解氮、有效磷、速效钾的含量分别提高了43.24%、19.01%、19.29%,土壤脲酶、蔗糖酶、蛋白酶、酸性磷酸酶的活性分别提高了18.42%、120.59%、10.55%、38.37%。此外,贝莱斯芽孢杆菌QL-2影响了土壤微生物群落的组成,对土壤有益微生物如芽孢杆菌(Bacillus)、鞘氨醇单胞菌(Sphingomonas)、褚氏杆菌(Chujaibacter)的富集,对土壤有害微生物如曲霉菌(Aspergillus)、子囊菌(Ascomycota)丰度的降低有着一定的作用。该结果表明贝莱斯芽孢杆菌QL-2能促进花生生长、改善土壤肥力、驱动有益微生物聚集并且缓解了花生土壤连作障碍问题,为花生的绿色生态种植策略提供了理论基础和技术支持。

产纤维素酶菌株的筛选、鉴定及产酶特性的研究

纤维素作为各种植物秸秆的主要组成物质广泛存在于自然界中,纤维素酶是一种催化纤维素降解产生葡萄糖的酶,可通过不同方式减少环境中纤维素的富集。为获得产酶能力高的降解纤维素菌株,利用刚果红平板法,从腐殖土中分离筛选出一株具有高产纤维素酶能力的菌株CTE1-1,经鉴定为芽孢杆菌(Bacillus sp.)。通过培养时间、培养温度、初始pH及NaCl浓度的单因素实验,确定该菌在含1%CMCA-Na的产酶培养基中培养,温度30℃,初始pH 7.0、NaCl浓度1.5%,经108h培养CMCA酶酶活可达42.59 U/mL,经96h培养FPA酶酶活可达45.66U/mL。该菌株所产纤维素酶在pH 6.0、50℃均表现很强的酶活力,Fe^(3+)、K^(+)对该菌所产纤维素酶具有激活能力,Mg^(2+)对该菌所产纤维素酶无影响,Cu^(2+)、Zn^(2+)对其具有一定的抑制能力。该菌株产纤维素酶能力强,可用于纤维素酶制剂的生产。

贝莱斯芽孢杆菌S3-1与蚯蚓粪混合改良剂的制备及其土壤改良效果的研究

天然有机肥在提高土壤的肥力、促进作物生长等方面有着举足轻重的地位.此前的研究中已发现菌株S3-1与蚯蚓粪共同施用时可以显著提高土壤肥力.本研究在此基础上,对实验样地、蚯蚓粪与商品菌肥进行了选择,根据比例制备改良剂.关于改良剂对土壤环境因子的影响等问题,进行了长达270 d的原位修复实验.实验样地分别为草坪地以及商品菌肥和蚯蚓粪处理地,设置了对照组、单独施加蚯蚓粪组、改良剂处理组、S3-1菌液处理组以及商品菌肥平行实验处理组.结果发现:酸碱中性磷酸酶活性提升率依次为25.44%,20.32%和18.33%;真菌数量随着时间周期的增长率分别为9.92%,41.98%和51.65%,细菌数量的增长速率逐从20.70%~36.12%渐变大为36.12%~80.35%.原位修复实验条件下,脲酶和蔗糖酶活性提升率也显著增强.微生物改良剂用更安全更环保的方法,促进了土壤恢复到自然状态,本研究为改良剂的广泛使用性奠定了实践基础.

解淀粉芽孢杆菌产核苷特性初步研究

为了探索解淀粉芽孢杆菌在植物基原料中的自然发酵特性,围绕细菌生长、肌苷与鸟苷产量,优化3种植物基原料的添加浓度,并确定复合植物基原料添加比例,同时考察最优单一和复合植物基原料发酵过程糖耗、有机酸代谢、酶活力变化和相关基因表达差异。实验结果表明,添加50 g·L^(-1)大豆分离蛋白发酵后核苷产量最高,且该组蛋白酶活力最高(252.52 U·mL^(-1));谷朊粉有利于解淀粉芽孢杆菌具备更强的α-淀粉酶活力,但会产生高质量浓度的乙酸,进而抑制菌体生长;添加50 g·L^(-1)植物基原料且谷朊粉和大豆分离蛋白添加比例为2∶1时发酵72 h核苷产量最高。本研究能够为利用植物基原料研发天然发酵调味料提供有益参考。

哈茨木霉与枯草芽孢杆菌对海红果幼苗生长、光合特性和抗氧化酶活性的影响

【目的】探究哈茨木霉和枯草芽孢杆菌接种对海红果幼苗生长、光合特性和抗氧化酶活性的影响。【方法】以海红果幼苗作为试验对象进行盆栽试验,设置施用无菌水(CK)、接种哈茨木霉菌液(TH)、接种枯草芽孢杆菌菌液(BS)、哈茨木霉菌液与枯草芽孢杆菌菌液混合接种(TB)4个处理,分析接种后30 d和60 d对海红果幼苗生长指标,叶片SPAD值、气体交换参数以及抗氧化酶活性和丙二醛(MDA)含量的影响。【结果】TH、BS、TB处理均促进了海红果幼苗的生长、光合特性及抗氧化酶活性,TB处理效果最佳。与CK相比,接种30 d和60 d,TB处理幼苗株高、茎粗、叶片数、地上部干重、根干重、根长分别显著增加16.52%、7.43%、23.81%、13.71%、21.44%、31.06%和16.27%、16.54%、18.06%、9.36%、15.41%、24.18%(P<0.05);幼苗叶片SPAD值显著增加13.38%和10.85%(P<0.05),净光合速率、气孔导度、蒸腾速率、胞间CO_(2)浓度分别显著增加14.08%、14.08%、31.03%、9.84%和11.88%、27.13%、14.95%、10.92%(P<0.05),过氧化物酶、苯丙氨酸解氨酶、超氧化物歧化酶、过氧化氢酶、多酚氧化酶活性分别显著增加20.93%、30.10%、42.00%、21.52%、23.71%和21.21%、22.52%、35.39%、18.18%、18.11%(P<0.05);接种60 d,MDA含量显著下降9.54%(P<0.05)。【结论】哈茨木霉与枯草芽孢杆菌混合接种可以有效促进海红果幼苗生长、提高叶绿素含量和光合速率、增强抗氧化酶活性。

黄河三角洲盐碱地耐盐促生菌株的筛选与功能评价

通过对黄河三角洲盐碱地植物根际土壤的筛选,获得47株耐盐菌株,其中9株具有促生效果。结合产酸、耐碱、耐不同盐碱土的能力,最终获得1株耐盐菌株GJY-3。GJY-3在氯化物盐土、硫酸盐土、重碳酸盐土中的耐受程度分别为5%、5%、1%,酸碱耐受范围为3.15~10.5,无机磷溶解量为16.71 mg/L,吲哚乙酸的合成能力为31.33 mg/L,同时具备固氮、解钾功能。通过生理生化分析和16s rDNA鉴定,确定该菌株为地衣芽孢杆菌。通过盆栽试验发现:GJY-3在氯化钠盐碱土的促生效果优于硫酸钠盐碱土的;小白菜产量以及土壤脲酶、蔗糖酶、过氧化氢酶活性分别增加17.54%、91.59%、32.24%、5.11%。试验结果表明,GJY-3可用作盐碱地土壤改良菌株。

Purification and Characterization of Three Alkaline Endopolygalacturonases from a Newly Isolated Bacillus gibsonii

A newly isolated Bacillus gibsonii,designated as S-2(CGMCC1215),was cultivated for production of alkaline pectinases utilizing sugar beet pulp as growth substrate.Purification of three alkaline endopolygalacturonases(endoPGs)from the crude pectinases extract was carried out by ultra-filtration,ammonium sulphate fractionation and ion-exchange chromatography,and their enzyme activities characterized.The three purified alkaline endoPGs,designated as S-I,S-II,and S-III,had a molecular weight about38kDa as determined by SDS-PAGE.The Km value and optimal temperature for optimal enzyme activities of S-I,S-II and S-III were1.2mg/mL and60℃,0.9mg/mL and55℃,1.1mg/mL and60℃,respectively.Their best performances were given at an optimal pH10.5,and sodium polygalacturonate was found to be the best substrate.The isoelectric points of S-I,S-II and S-III were5.4,7.4,and8.2,respectively.Surfactants of Tween-80and Tween-20and metal ions such as Mg2+and Ca2+stimulated the activity of S-I,S-II and S-III,whereas S-III was inhibited by Ca2+,and Mn2+and Zn2+ions inhibited the activity of the three enzymes.

Mechanism of the Effect of Bacillus coagulans on Production Performance of Laying Hens in Late Laying Period

[Objective]The paper was to investigate the mechanism of Bacillus coagulans preparation in improving production performance of laying hens in late period of laying.[Method]A total of 648 individuals of"Jingfen 1"laying hens(66 weeks of age)in late period of laying were randomly divided into four groups,six replicates each group,and each group had 27 individuals of laying hens.The laying hens in control group were fed with basal diet,and those in experimental groupsⅠ,Ⅱ and Ⅲ were fed with the basal diets added with 3.33×10^6,1×10^7,3.33×10^7 CFU/g B.coagulans,respectively.The pretrial lasted one week,and the formal test lasted eight weeks.[Result]Compared with the control group,the laying rate in group Ⅰ increased significantly,and the feed-gain ratio in experimental group Ⅱ decreased noticeably while the spleen index increased remarkably;the alkaline phosphatase activities and blood calcium content in experimental groups Ⅰ,Ⅱ and Ⅲ increased significantly(P<0.05),while the triglyceride content decreased remarkably(P<0.05);the urea content in experimental groups Ⅰ and Ⅱ decreased obviously(P<0.05).Adding B.coagulans significantly increased the specific activity of amylase,lipase and protease in various intestinal mucosa of small intestine(P<0.05);adding B.coagulans significantly increased the content of follicle-stimulating hormone(FSH)and estradiol(E2),and significantly increased the mRNA expression of follicle stimulating hormone receptor gene(FSHR).[Conclusion]B.coagulans preparation could significantly improve the production performance of laying hens in late period of laying;appropriately enhance the immune capacity of laying hens;improve the serum biochemical indicators;increase the activity of digestive enzymes in the small intestine;and promote the release of gonadal hormone and the mRNA expression of FSHR gene in the ovary.

碱性纤维素酶高产菌株Bacillus sp.CY1-3的诱变选育

以一株产单组分羧甲基纤维素酶芽孢杆菌Bacillus sp.CY1-3为出发菌,经紫外线和亚硝基胍复合诱变处理后,其纤维素酶产量达到了53.86 U/mL,是出发菌株的4倍.研究发现最优的紫外辐射时间和亚硝基胍浓度分别是4min和0.06%.

Ultraviolet Mutagenesis of Bacillus licheniformis LCB-8 and Screening of High-Yielding Strain

[ Objective ] To enhance amylase, protease and cellulase activities of Bacillus licheniformis LCB-8 by ultraviolet mutagenasis. [ Method ] Ultraviolet light was used to mutate this strain. The activities of amylase, protease and cellulase were determined to get high-yielding strains. [ Resalt] The best time for ultraviolet mutation was 30 s, and the mortality rate reached 72.4%. After primary screening and rascraening, two better strains were obtained. After mutagenasis, the activities of amylase, protease and cellulase of DY-97 strain were enhanced by 150%, 78% and 17% respectively. After mutagenesis, the activities of cellulase, amylase and protease of DY-34 strain were enhanced by 31%, 46% and 64% respectively. [ Conclusion] The DY-97 strain is suitable for fermentation of feed rich in protein and amylum, whereas the DY-34 strain is suitable for fermentation of feed rich in cellulose.

Characterization of Amylase from Some Aspergillus and Bacillus Species Associated with Cassava Waste Peels

Cassava peels are generated as waste on soils during cassava processing in many tropical countries. This work set out to isolate some microorganisms associated with cassava peel degradation and characterize amylase enzymes responsible for the degradation under some physiological conditions. A total of 30 bacteria was isolated from the peels with Bacillus species occurring the most (46.5%) and Enterobacter species (13.3%) being the next. Frequencies of fungal isolations was Rhizopus sp. (35%);Aspergillus niger (25%);Aspegillus flavus (20%) and Penicillium species (20%). Bacillus cereus, Bacillus substilis Bacillus pumillus, Aspergillus niger and Apergillus flavus were selected and screened for their abilities to produce amylase. Amylase activity was highest at day 4 for B. substilis (39.4 units/ml) and A. flavus (66.1 units/ml);at day 3 for B. cereus (55.6 units/ml) and A. niger (44.6 units/ ml). While maximum amylase activity was obtained at day 6 for B. pumilus (80.2 units/ml). Optimum pH for amylases from the two fungal isolate was 6.0 (A. niger = 53.5 units/ml and A. flavus = 65.4 units/ml). While optimum pH for B.cereus (51.7 units/ ml) and B. pumilus (44.6 units/ml) was 6.5 and for B. substilis (56.1 units/ml) at pH 7.0. Amylase activities increased from 20°C to 40°C for amylase from Bacillus sp. and 20°C to 50°C for amylase from the Aspergillus sp. after which there was a decline in activities as temperature increased to 80°C. Effect of heating duration (at 70°C for 5 minutes) on the amylase showed that A. niger has the highest activity of 127 units/ml. Effect of substrate concentration on amylase activity showed that amylase form A. flavus had the highest activity of 72.2 units/ml at 0.4% substrate concentration. The implications of the findings were discussed.