Paenibacillus spp.BD3526发酵小麦麸皮生产凝乳酶

对Paenibacillus spp.BD3526凝乳酶的发酵条件和凝乳性能进行了研究,探讨了不同培养基、发酵时间、碳源、氮源和装液量对凝乳酶活力和活菌数的影响,并与商业化的小牛皱胃酶、重组凝乳酶、Rhizomucor miehei来源凝乳酶进行凝乳性能比较。实验结果表明:发酵时间、碳源、氮源和装液量对BD3526凝乳酶活力(milkclotting activity,MCA)和蛋白水解活力(proteolytic activity,PA)均有不同程度的影响。选用小麦麸皮培养基进行发酵至24 h时,MCA达到最大至3 279.76±67.11 SU/m L,凝乳性能强,PA值在整个发酵期间变化不大;添加碳源或氮源会不同程度地降低MCA值;装液量为30 m L时,发酵上清液中菌体浓度最高、MCA达到6 000 SU/m L。与商业化的凝乳酶相比,BD3256凝乳酶在凝乳过程中未使凝乳块产生过度水解,也无乳清析出现象,具有应用到干酪生产中的潜力。

多粘芽孢杆菌(Paenibacillus polymyxa)吸附Cu^(2+)的实验研究

细菌表面往往存在多种化学基团,能够通过吸附作用影响环境流体中金属元素的活动性,从而与表生条件下的元素富集、矿物成核结晶等地球化学过程密不可分。为了深入认识细菌吸附作用的地球化学意义和环境效应,揭示细菌吸附金属离子的热力学行为,选择了多粘芽孢杆菌(Paenibacillus polymyxa)为研究细菌,系统开展了滴定实验和Cu2+吸附实验。通过连续酸滴定方法分析了细菌表面的化学特征,发现多粘芽孢杆菌在pH值为7.54~6.50范围内,表面带负电荷,表现出质子吸附行为;设计开展了Cu2+吸附实验,发现溶液的pH值对Cu2+吸附有一定影响,可能存在Cu2+与细菌表面质子的交换作用;根据Cu2+吸附等温线拟合计算,发现吸附等温线符合Langmuir和Freundlich吸附模型,根据Langmuir模型计算得到每个细胞的Cu2+饱和吸附量高达1.69×10-7mg。

生淀粉酶生产菌株Paenibacillus sp.的筛选和酶的纯化及酶学性质

分离得到1株产生淀粉酶的菌株,通过扩增和测定16S rDNA序列并进行比对,发现是Paenibacillus属的细菌。液体摇瓶发酵结束后,其产生的生淀粉酶比酶活达108.5U/mL。通过饱和(NH4)2SO4沉淀、Sephacryl S-300层析的方法对其所产的生淀粉酶进行分离纯化,得到纯化的酶蛋白比酶活为5112.04U/mg,纯化倍数为14.1,相对分子质量约为1.0×105。该酶以木薯生淀粉为底物时,最适pH5.6,最适温度50℃。金属离子Ca2+、Mg2+对该酶具有激活作用,Zn2+、Cu2+、Fe2+、Ni2+、Mn2+、Co2+和EDTA2-对该酶均具有抑制作用。

Paenibacillus bovis BD3526产胞外多糖的培养条件优化

利用单因素试验和响应面法对Paenibacillus bovis BD3526产胞外多糖发酵条件工艺参数进行优化。结果表明:培养温度、时间、培养基装量与Paenibacillus bovis BD3526胞外多糖产量存在显著的相关性。当麸皮质量浓度为30 g/L时,BD3526产胞外多糖的优选条件为:培养温度29.91℃、培养时间23.96 h、培养基装量59.58m L/250 m L锥形瓶,采取上述优选条件进行培养,理论预测最大胞外多糖产量为11.11 g/L。验证试验实际测得多糖平均产量为10.92 g/L,比理论预测值低1.71%。

一株产琼胶酶土壤细菌Paenibacillus sp. MY03的筛选与分子鉴定

为寻找高产琼胶酶的微生物新资源,促进琼胶低聚糖的研究与开发,本研究自多个土壤样品中进行微生物的分离,借助于卢戈氏碘液染色法进行琼胶酶生产菌的筛选,通过DNS-还原糖测定比较不同微生物的产酶特性,克隆、分析细菌的16S rRNA基因序列进行分子鉴定。最后得到1株胞外琼胶酶活性较高的土壤细菌,鉴定至Paenibacillus属,命名为MY03菌株;分子鉴定表明Paenibacillus sp. MY03是产琼胶酶细菌家族中的新成员。

Paenibacillus spp.BD3526金属蛋白酶对乳蛋白水解位点分析

为了比较具有凝乳功能的Paenibacillus spp.BD3526来源的金属蛋白酶与基因重组凝乳酶对乳蛋白水解位点的差异,采用BD3526金属蛋白酶和凝乳酶对α_(s1)-酪蛋白(casein,CN)、α_(s2)-CN、β-乳球蛋白(lactoglobulin,Lg)和κ-CN进行酶解,分别对不同时间的酶解产物采用高效液相色谱与四极杆飞行时间串联质谱(high performance liquid chromatography of quadrupole time of flight-tandem mass spectrometry,HPLC-Q-TOF-MS/MS)进行分析。结果表明:BD3526金属蛋白酶与凝乳酶在对乳蛋白的水解位点上具有较高的相似性,前者对α_(s1)-CN、α_(s2)-CN、β-Lg和κ-CN水解能力较后者弱,对P1为K(Lys)、R(Arg)且P1'为T(Thr)、F(Phe)和Y(Tyr)间的肽键水解特异性很高,并主要水解K-T(Lys-Thr)、K-F(Lys-Phe)、R-F(Arg-Phe)、R-Y(Arg-Tyr)肽键,水解生成的肽具有血管紧张素转换酶(angiotensin converting enzyme,ACE)抑制、抗菌、免疫调节等功能。

冷泉细菌Paenibacillus algorifonticola产抑菌活性物质提取分离工艺的研究

本文对冷泉细菌——Paenibacillus algorifonticola的代谢产物的提取分离进行了研究。P.algorifonticola可产生抑制白色念珠菌的水溶性物质。大孔树脂D101和XAD7HP串联使用脱除P.algorifonticola发酵液中的色素,树脂与发酵液比例为1∶10(g/mL)为佳。脱色后发酵液中的活性物质可被强酸性阳离子交换树脂FPC22H吸附,0.5 mol/L的氨水可将活性物质洗脱下来,每10 g FPC22H可以吸附120 mL发酵液中的活性物质。本文研究结果为进一步分离纯化P.algorifonticola产抗白色念珠菌物质奠定了基础。

一种来源于Paenibacillus sp. A1的中性β-甘露聚糖酶基因克隆及其酶学性质研究

采用简并PCR和TAIL-PCR技术,从来源于天山冰川土壤的类芽孢杆菌Paenibacillussp.A1菌株基因组中扩增出β-甘露聚糖酶基因编码序列。序列分析表明,该基因全长960bp,编码一个新的糖苷水解酶第5家族的β-甘露聚糖酶。将该基因克隆到原核表达载pET-22b(+)中,经过IPTG诱导表达,酶的胞外表达量达0.62U/mL。酶学特性分析表明其最适pH为6.5,最适温度为55℃,属中性β-甘露聚糖酶,具有较好的底物适应性,适宜用于鱼类等水产动物养殖饲料行业中。

NH_4^+对多黏菌素4E产生菌Paenibacillus polymyxa SIIA-1408代谢过程的影响

目的研究NH_4^+对多黏菌素E产生菌Paenibacillus polymyxa SIIA-1408代谢过程的影响方法通过向Paenibacillus polymyxa SIIA-1408的初始发酵培养基中添加不同浓度的NH_4^+,分析其代谢产物及其产量的变化,对于多黏菌素E以外的目标产物进行分离纯化确定其结构,通过前体的添加和中间代谢产物的验证,确定其可能的代谢途径。结果初始培养基中不添加NH_4^+或添加低浓度的NH_4^+主要产物为(S)-2-羟基-3-苯基丙酸,当NH_4^+的浓度超过一定浓度后主要产物为多黏菌素E。结论在一定浓度范围内,NH_4^+促进Paenibacillus polymyxa SIIA-1408生物合成多黏菌素E,抑制其将苯丙氨酸转化(S)-2-羟基-3-苯基丙酸。

Paenibacillus polymyxaZJ-9混合发酵菊粉和葡萄糖合成R,R-2,3-丁二醇

在7.5 L发酵罐上考察了Paenibacillus polymyxa ZJ-9混合发酵菊粉和葡萄糖合成R,R-2,3-丁二醇的工艺条件。选用菊芋菊粉粗提液为发酵前期底物,分析比较不同初始菊粉浓度下的细胞比生长速率(μ)和产物比合成速率(qp),进而研究了补糖种类和不同补料方式对合成R,R-2,3-丁二醇的影响。结果表明,初始菊粉75.0g/L,当发酵到24 h、31 h时,分别添加15.0 g/L的葡萄糖,发酵效果最佳,44 h时,产物产量达到最高值47.8 g/L,与分批发酵相比,糖转化率由原来的34.9%提高到45.5%,生产强度由原来的0.70 g/(L·h)提高到1.09 g/(L·h),并且副产物乙偶姻、残糖浓度相对较低。

Paenibacillus puldeungensis LK18普鲁兰酶基因的克隆表达及酶学性质研究

本研究根据类芽孢杆菌的普鲁兰酶基因的保守区设计兼并引物,从Paenibacillus puldeungensis LK18中扩增出普鲁兰酶基因(pulA),将其连接至pET-28a(+)载体上,构建出重组表达载体pET-28a(+)-pulA,转入到Escherichia coli BL21(DE3)中,成功地表达了重组普鲁兰酶。结果表明:该普鲁兰酶基因全长1968 bp,编码655个氨基酸。通过Ni柱亲和层析纯化出重组蛋白,测定其比酶活为508.8 U/mg,分子量约为76.95 ku。该重组酶的最适反应温度为45℃,在35℃~40℃下保温120 min后剩余酶活达60%以上;最适作用pH为6.0,在pH 6.0~8.0条件具有较好的稳定性;10 mmol/L的K^+和Mg^2+对该重组普鲁兰酶有激活作用,而Zn^2+、Mn^2+、Ni^2+、Fe^2+、Cu^2+、Co^2+、Ca^2+等对其有不同程度的抑制作用。本研究成功地构建出一株可高效表达普鲁兰酶的重组菌株,具备一定的工业应用价值。

Antagonistic Activities of Volatiles from Four Strains of Bacillus spp. and Paenibacillus spp. Against Soil-Borne Plant Pathogens

The four effective antagonistic Bacillus strains, isolated from the rhizosphere soil of cucumber in a greenhouse, produced antifungal volatiles. These volatiles strongly inhibited the growth of the most tested pathogenic fungi with wide host plants, induced the mycelial morphological abnormalities, and decreased the sclerotoid production of Sclerotinia sclerotiorum in sealed plates. Spores of Botrytis cinerea exposed to these volatiles for 24-48 h in cavity slides cracked and the sporaceous inclusion became brown and effused to the suspension. An interesting phenomenon observed was that all the bacterial volatiles exhibited intense inhibitory activities against the pigment formation of tested pathogenic fungi, including Ascochyta citrullina, Alternaria solani, Alternaria brassicae, and so on. Interactions mediated by microbial volatiles could be widespread in soils, and volatiles may play an important role in reducing disease levels. A phylogenetic analysis based on 16S rDNA sequence placed the four bacteria in three species Paenibacillus polymyxa （BMP-11）, Bacillus subtilis （BL02）, and Bacillus pumilus （BSH-4 and ZB 13）. Through headspace sampling and GC-MS analysis, a rich profile was found from B. subtilis and overlapping volatile patterns could be found among the different species. Studies are under the way to find the possible action mechanisms and to seek the effective application of bacterial volatiles in greenhouse.

产壳聚糖酶海洋Paenibacillus chitinolyticus CLT08的鉴定及酶学性质研究

目前用于制备壳寡糖的壳聚糖酶具有酶活性较低、酸稳定性较差等问题。本研究从连云港海州湾泥样中筛选产壳聚糖酶菌株,并对菌株进行鉴定、酸稳定性和酶学性质研究。结果表明,通过平板透明圈初筛和摇瓶发酵复筛,获得酸稳定性较好的壳聚糖酶产生菌株CLT08,随后利用形态学特征、生理生化测定及16S rDNA序列扩增与分析,鉴定菌CLT08为Paenibacillus chitinolyticus。菌株CLT08产壳聚糖酶最适温度为45℃,最适pH为4.0;Zn^(2+)对酶有显著的激活作用,对Ba^(2+)、Co^(2+)、Fe^(2+)有显著抑制作用。壳聚糖经CLT08壳聚糖酶水解后其聚合度≥6。此研究结果为该酶的进一步研究奠定了基础。

Inhibition mechanisms of secretome proteins from Paenibacillus polymyxa Kp10 and Lactococcus lactis Gh1 against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus

Objective:To determine the inhibition mechanisms of secretome protein extracted from Paenibacillus polymyxa Kp10(Kp10)and Lactococcus lactis Gh1(Gh1)against methicillin-resistant Staphylococcus aureus(MRSA)and vancomycin-resistant Enterococcus(VRE).Methods:The sensitivity and viability of MRSA and VRE treated with secretome proteins of Kp10 and Gh1 were determined using minimal inhibitory concentration,minimum bactericidal concentration,and time-to-kill assays.The morphological changes were observed using scanning electron microscopy and transmission electron microscopy.To elucidate the antimicrobial mechanism of secretome protein of Kp10 and Gh1 against MRSA and VRE,2D gel proteomic analysis using liquid chromatography-mass spectrometry was run by comparing upregulated and downregulated proteins,and the proton motive force study including the efflux of ATP,pH gradient,and the membrane potential study were conducted.Results:MRSA and VRE were sensitive to Kp10 and Gh1 secretome protein extracts and displayed apparent morphological and internal composition changes.Several proteins associated with cellular component functions were either downregulated or upregulated in treated MRSA and VRE by changing the membrane potential gradient.Conclusions:Kp10 and Gh1 secretome proteins reduce the growth of VRE and MRSA by damaging the cell membrane.Cell division,cell wall biosynthesis,and protein synthesis are involved in the inhibition mechanism.

Purification and Characterization of Alkaline Xylanase Secreted from <i>Paenibacillus macquariensis</i>

An alkaline xylanase secreted by Paenibacillus macquariensis RC 1819 has been purified using ammonium sulfate fractionation, ion exchange chromatography using DEAE-cellulose and gel filtration chromatography over Sephadex G-200 and Sephadex G-100. The purified enzyme had the specific activity, 25.2 units/mg protein with birchwood xylan as a substrate. The purified enzyme showed a single protein band over sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme has been found to be 31,000 ± 2000 as determined by using Sephadex G-200 gel filtration chromatography. The subunit molecular weight has also been found to be ~31,000 as determined using SDS-PAGE indicating monomeric enzyme. The enzyme showed optimum activity at pH 8.6 and temperature, 50°C. The Michaelis constant (Km) of the enzyme for birchwood xylan was 2.2 mg/ml as determined using velocity saturation plot. The metal ions viz. Co+2 and Mn+2 stimulated xylanase enzyme activity whereas Hg+2 inhibited the enzyme activity.

Paenibacillus Infection in a Pediatric Patient on ECMO

Paenibacillus is a spore forming gram positive rod that is usually found in the environment. We describe a case of a patient who contracted this organism having never left the hospital after birth. This neonate contracted Paenibacillus septic shock requiring support with venous-arterial extracorporeal membrane oxygenation (ECMO) while still admitted to the hospital after birth. This patient initially presented in severe septic shock due to a Streptococcus agalactiae infection requiring hemodynamic support with ECMO. Following treatment for the Streptococcus agalactiae infection, and while still on ECMO support, the blood culture became positive for Paenibacillus. Given that our patient had never left the hospital after birth, the finding of this organism in the blood is unique. The primary defense against this bacterium is usually the skin. The only invasive procedure this patient had was ECMO cannulation which is done in a sterile fashion. Most species are susceptible to vancomycin, clindamycin, fluoroquinolones, aminoglycosides, carbapenems, penicillin, and cephalosporins. This patient was treated with penicillin G for 14 days for the S. agalactiae infection prior to the blood culture being positive for Paenibacillus. More than a hundred species had been identified in the genus Paenibacillus, however, few found to cause human infection. This case is unique as it is the first pediatric case with a Paenibacillus infection and the first pediatric case where this organism which rarely causes human infection was found in the blood culture of a patient on ECMO.

Inhibitory Activity of <i>Paenibacillus</i>sp. Isolated from Soil in Gotsu City, Shimane Prefecture, Against Xanthomas <i>oryzae</i>pv. <i>oryzae</i>, the Causal Agent of Rice Bacterial Leaf Blight

Microorganism isolates (n = 49) were obtained from the soil samples collected from field in Gotsu city (Kawahira), Shimane. Isolate GT2-E culture inhibited the growth of Xanthomonas oryzae pv. oryzae in disk diffusion method. Rice bacterial leaf blight was suppressed by GT2-E culture in the pre- and post-treated rice leaves. Sequence analysis of 16S rDNA region of the GT2-E isolate indicated that it shared 99% similarity with Paenibacillus polymyxa. The growth of GT2-E on LB medium was observed at 15°C, 28°C, 37°C, and 45°C, but not at 4°C. GT2-E isolate could be grown even in the presence of agrochemicals (Amister, Blasin and Kasumin). Furthermore, the growth of X. oryzae pv. oryzae was inhibited by the culture filtrate of GT2-E isolate in disk diffusion method. However, the inhibitory activity of the culture filtrate was heat-unstable. This result suggested that GT2-E isolate can produce heat-unstable inhibitory compound(s). In conclusion, GT2-E isolate might contribute to the development of a new bactericide and biological agent against rice bacterial leaf blight.

Characterization of sulfide oxidation and optimization of sulfate production by a thermophilic Paenibacillus naphthalenovorans LYH-3 isolated from sewage sludge composting

The sulfur-containing odor emitted from sludge composting could be controlled by sulfide oxidizing bacteria, yet mesophilic strains show inactivation during the thermophilic stage of composting. Aimed to investigate and characterize the thermotolerant bacterium that could oxidize sulfide into sulfate, a heterotrophic strain was isolated from sewage sludge composting and identified as Paenibacillus naphthalenovorans LYH-3. The effects of various environmental factors on sulfide oxidation capacities were studied to optimize the sulfate production, and the highest production rate (27.35%±0.86%) was obtained at pH 7.34, the rotation speed of 161.14 r/min, and the inoculation amount of 5.83%by employing BoxBehnken design. The results of serial sulfide substrates experiments indicated that strain LYH-3 could survive up to 400 mg/L of sulfide with the highest sulfide removal rate (88.79%±0.35%) obtained at 50 mg/L of sulfide. Growth kinetic analysis presented the maximum specific growth rateμm(0.5274 hr-1) after 22 hr cultivation at 50℃. The highest enzyme activities of sulfide quinone oxidoreductase (0.369±0.052 U/mg) and sulfur dioxygenase (0.255±0.014 U/mg) were both obtained at 40℃, and the highest enzyme activity of sulfite acceptor oxidoreductase (1.302±0.035 U/mg) was assessed at 50℃. The results indicated that P. naphthalenovorans possessed a rapid growth rate and efficient sulfide oxidation capacities under thermophilic conditions, promising a potential application in controlling sulfur-containing odors during the thermophilic stage of sludge composting.

一种新型褐藻胶裂解酶及其截短体对酶学性质的影响

褐藻寡糖是褐藻胶的降解产物,因独特的理化性质而具有抗肿瘤、促进生长和调节免疫等功效,受到广泛关注。为了挖掘更高效的褐藻胶裂解酶用于制备褐藻寡糖,对采集于海边土壤中的微生物进行筛选,以获得能够降解褐藻胶的菌株,并从其基因组中克隆表达出褐藻胶裂解酶,考察酶的催化特性。结果发现:从Paenibacillus lautus A2中鉴定出的一种新颖的褐藻胶内切酶AlgC2m,含有439个氨基酸,与PL7家族的AlgL仅有38%的最高序列一致性,其N端的碳水化合物结合模块(CBM32)和C端催化域(CD)通过一个连接子连接。AlgC2m和其截短体AlgC2m-CD降解褐藻酸钠的酶活分别为331.05和82.47 U/μmol,最适温度分别为45和30℃。另外,AlgC2m-CD对聚α-L-古罗糖醛酸(polyG)具有更明显的偏好性,酶解产物除二糖和三糖外,还有聚合度更高的四糖。

马铃薯淀粉废水资源化制备Paenibacillus polymyxa农用菌剂

马铃薯淀粉废水中含有高浓度的淀粉、蛋白质等有机物。利用马铃薯淀粉废水培养植物促生菌,是实现其资源化利用的方法。采用单因素方法结合中心复合设计(CCD)的方法,对废水体积分数(浓度)、培养温度、初始pH这3个因素进行研究,优化马铃薯淀粉废水培养Paenibacillus polymyxa EBL06菌株的适宜生长条件。实验得到马铃薯淀粉废水培养P.polymyxa的最佳条件:废水COD为13.7 g·L^−1,初始pH为7.17,培养温度为31.4℃。该条件下,培养21 h后,微生物活菌数为6.2×10^9 cfu·mL^−1,与模型预测结果基本一致,可以达到《农用微生物菌剂国家标准》(GB 20287-2006)。为了验证该菌剂的应用效果,进行了蔬菜种植实验。结果表明:P.polymyxa菌剂能有效提高小白菜的产量与品质,作物鲜重、干重、株高,以及维生素C含量别提高了68.6%、13.7%、5.6%、41.3%;相比于只施用化肥的组,菌剂同尿素的混施能提高氮肥的利用效率,小白菜植株中维生素含量提高了25.3%、硝酸盐含量减少了15.3%。以上研究结果可为马铃薯淀粉废水的资源化利用,以及P.polymyxa菌剂的应用推广提供参考。