津巴布韦烟叶中淀粉酶和蛋白酶产生菌的分离及鉴定

目的:从津巴布韦烟叶中分离产蛋白酶菌和产淀粉酶能力最高的菌株,并对其进行鉴定。方法:采用淀粉富集培养基和酪蛋白富集培养基分别分离津巴布韦烟叶中的产淀粉酶和产蛋白酶菌株,通过生理生化实验和16SrRNA序列分析鉴定分离的菌株。结果:产蛋白酶菌株菌体不透明、表面有褶皱,蛋白酶酶活为52.10±0.13 U/mL;产淀粉酶菌株菌体表面呈黏状,淀粉酶酶活为3.69±0.07 U/mL;产蛋白酶与产淀粉酶的2株菌均与枯草芽孢杆菌的16S rRNA序列有100%的相似性,结合生理生化指标初步鉴定为枯草芽孢杆菌。结论:获得的2株菌在降解烟叶的蛋白质和淀粉过程中可能起重要作用。     

枯草杆菌SOD高产菌株的诱变选育及产酶条件研究

本实验采用低能氮离子注入技术对枯草芽孢杆菌(Bacillus subtilis)进行辐照诱变处理,选育出一株SOD高产菌株(编号为BsB8)。其生物量略高于出发菌株、SOD产量达3439．3U／g湿菌体,为实验出发菌的1．88倍。该菌株最佳产酶条件为：起始pH为8．2,装液量为150ml／500ml,接种量为1．5％;添加Mn^2 的培养基可显著提高SOD酶活。     

枯草芽孢杆菌产β-甘露聚糖酶固体发酵条件的优化

芽孢杆菌是产甘露聚糖酶的优良菌株,首次研究芽孢杆菌固体发酵条件的优化。以天然麸皮作为基本原料,研究利用枯草芽孢杆菌WY34固体发酵生产β-片露聚糖酶的发酵条件。最佳固体发酵培养条件为：麸皮5g,初始水分含量71％,初始pH7．0,接种量为2mL,1％Tween-80,0.4g魔芋粉,培养温度50℃。在最适条件下培养5d,甘露聚糖酶酶活高达7,650U／g干基,是未优化前酶活的2．78倍。     

碱性过氧化氢酶高产菌的筛选、鉴定及发酵条件优化

从纺织厂的车间污水分离获得菌株WSHDZ-01,其产过氧化氢酶酶活为600U/mL,酶活的pH范围为5.0~12.0。根据对该过氧化氢酶生产菌的形态和生理生化特征的分析和16SrDNA序列分析,鉴定该菌为枯草芽孢杆菌。通过对该菌株发酵培养基中碳氮源的优化,其最佳碳源为10g/L的葡萄糖,氮源为5g/L的NaNO3,在此条件下产酶达3258U/mL。此外,该菌株所产过氧化氢酶的最适反应pH为11.0,最适温度为55℃,在pH11.0和50℃下保持15min后,剩余酶活仍达98%以上。     

亚麻微生物脱胶优势菌的选育及其应用

从亚麻种植土壤与沤麻水中分离出96株能分解果胶的菌株。通过初筛和复筛获得4株果胶酶高产菌株。其中,在果胶平板培养基上生长速度快、产果胶酶活力高的12号菌株被确定为优势菌,经鉴定其为枯草芽孢杆菌。最适脱胶条件为：麻水比1：15,pH7．5,温度32～33℃,预培养的优势菌接种量3％。结果表明,采用优势菌的脱胶周期比对照缩短50％左右,而且麻的纤维质量明显得以改善。     

红树林微生物DH-2胞外蛋白酶的性质及产酶条件优化

从大亚湾红树林土壤样品中分离得到产蛋白酶菌株,鉴定所产胞外蛋白酶的酶学性质以及菌株的最佳发酵培养条件。采用平板透明圈法筛选菌株,福林酚显色法测定蛋白酶的酶活,通过单因素和正交试验确定其最佳发酵培养基以及发酵条件。从壤样品中分离得到一株产蛋白酶的枯草芽孢杆菌DH-2,该菌株分泌的蛋白酶最适反应pH和温度分别为8.0和65℃,50℃保温处理60 min后,剩余酶活仍保留80%以上。该蛋白酶对多种金属离子、有机溶剂及表面活性剂均有较好的耐受性。确定该菌株产蛋白酶的最适条件:1%(m/V,下同)可溶性淀粉,1%胰蛋白胨、1%NaCl,初始pH 5.5及7%的接种量,40℃培养36 h。在最适条件下测得其发酵液的酶活为236.30 U/mL,约为初筛时的酶活的8倍。该蛋白酶具有较为广阔的作用温度和pH范围,金属离子、有机溶剂及表面活性剂耐受性好,酶的性质比较稳定。     

α-葡萄糖苷酶菌株的选育及发酵条件的研究

从2株芽胞杆菌中通过筛选获得了一株产α-葡萄糖苷酶活力较高的嗜热脂肪芽孢杆菌U2,以嗜热芽孢杆菌U2为菌种,优化发酵培养基后,在温度45℃、初始pH6.8、转速200r/min和10%接种量条件下,发酵20h,菌株U2产酶水平可达到2.62U/mL,比出发菌提高了4倍。     

枯草芽孢杆菌产β-甘露聚糖酶固体发酵条件的优化*

芽孢杆菌是产甘露聚糖酶的优良菌株,首次研究芽孢杆菌固体发酵条件的优化。以天然麸皮作为基本原料,研究利用枯草芽孢杆菌WY34固体发酵生产β-甘露聚糖酶的发酵条件。最佳固体发酵培养条件为:麸皮5 g,初始水分含量71%,初始pH 7.0,接种量为2 mL,1%Tween-80,0.4 g魔芋粉,培养温度50℃。在最适条件下培养5 d,甘露聚糖酶酶活高达7,650 U/g干基,是未优化前酶活的2.78倍。     

家蚕肠道枯草杆菌产蛋白酶的发酵条件与酶学性质

肠道微生物分泌的蛋白酶可促进家蚕对桑叶养分的消化吸收,枯草芽孢杆菌是家蚕肠道内一种重要的产蛋白酶菌株。为提高枯草芽孢杆菌蛋白酶的高效利用,对该菌株适宜发酵条件及酶学性质进行了研究。结果表明：各因素对枯草芽孢杆菌产酶活性影响的大小顺序依次为：pH值〉培养温度〉培养时间〉装液量;最适的产酶条件为：pH=7,培养温度：30 ℃,培养时间：36 h;对枯草芽孢杆菌产蛋白酶进行初步提纯后并研究得出该酶反应的最适pH 10.0,最适反应温度为：60 ℃;该酶为碱性蛋白酶、不耐高温、不耐酸,但在35 ℃条件下热稳定性较好。     

葡萄糖醛酸木聚糖酶在枯草芽孢杆菌中的表达及发酵条件优化

为了研究葡萄糖醛酸木聚糖酶在枯草芽孢杆菌中异源表达,笔者从枯草芽孢杆菌中克隆得到带有自身信号肽的葡萄糖醛酸木聚糖酶基因,将其构建到大肠杆菌-枯草芽孢杆菌穿梭质粒中,转化入枯草芽孢杆菌WB800,得到重组菌。通过发酵条件以及培养基成分优化,重组菌中葡萄糖醛酸木聚糖酶酶活达到76.0 U/mL,约为优化前产酶量的5.4倍。葡萄糖醛酸木聚糖酶在枯草芽孢杆菌中实现高效异源表达,为其进一步的实际应用奠定了基础。     

适于罗布麻生物脱胶的果胶酶产生菌分离筛选与表征

在青海柴达木盆地沤制的罗布麻表皮中分离得到两株适于罗布麻脱胶的高产果胶酶菌株,经形态、生理生化指标鉴证和16SrDNA菌种鉴定,确定一株为新的果胶酶产生菌琼氏不动杆菌（Acinetobacter junii）,一株为枯草芽孢杆菌（Bacillus subtilis）。水解圈实验得出前者H／C值（H／C为水解圈直径H与菌落直径C之比）为5,后者为3;经酶活力测定,在37℃下,琼氏不动杆菌（Acinetobacter junii）在11h达到产酶高峰（酶活力为103．2IU／mL）,枯草芽孢杆菌（Bacillus subtilis）培养至9h达到产酶高峰（酶活力为91．6IU／mL）,但前者最高酶活力比后者高12．5％;经脱胶实验得出两菌残胶率分别为18．47％和17．31％,对罗布麻生物脱胶有较好的适用性。     

Production of bioemulsifier by Bacillus subtilis, Alcaligenes faecalis and Enterobacter species in liquid culture

Three bacterial strains isolated from waste crude oil were selected due to their capacity of growing in the presence of hydrocarbons and production of bioemulsifier. The genetic identification (PCR of the 16S rDNA gene using fD1 and rD1 primers) of these strains showed their affiliation to Bacillus subtilis, Alcaligenes faecalis and Enterobacter sp. These strains were able to emulsify n-octane, toluene, xylene, mineral oils and crude oil, look promising for bioremediation application. Finally, chemical composition, emulsifying activity and surfactant activity of the biopolymers produced by the selected strains were studies under different culture conditions. Our results showed that chemical and functional properties of the bioemulsifiers were affected by the carbon source added to the growth media.     

产碱性木聚糖酶菌株的筛选及酶学性质

从青海盐碱湖土壤中筛选到25株产碱性木聚糖酶的菌株, 其中编号为QH14的菌株产酶量达648.79 U/mL, 纯化后比活可达1148.56 U/mg。16 SrDNA鉴定表明菌株QH14属于短小芽孢杆菌, 命名为Bacillus sp. QH14。从该菌株的基因组中克隆获得了碱性木聚糖酶编码基因XynQH14, 并在大肠杆菌E.coliBL21(DE3)中获得重组表达。通过Ni-NTA亲和层析分离纯化后的重组QH14木聚糖酶比活达700.47 U/mg。该碱性木聚糖酶的酶促反应最适温度为60℃, 最适pH为9.2; 55℃处理1h仍保持50%的活力; 在pH7.0~11条件下37℃处理酶液24 h后均保持80%以上的活力, 且在pH11缓冲溶液中50℃处理24 h仍保持31.02%的酶活, 显示了该碱性木聚糖酶较好的热稳定性和碱稳定, 提示该碱性木聚糖酶在制浆造纸、纺织等行业的应用潜力。     

产碱性木聚糖酶菌株的筛选及酶学性质

从青海盐碱湖土壤中筛选到25株产碱性木聚糖酶的菌株,其中编号为QH14的菌株产酶量达648.79U/mL,纯化后比活可达1148.56 U/mg。16 SrDNA鉴定表明菌株QH14属于短小芽孢杆菌,命名为Bacillus sp.QH14。从该菌株的基因组中克隆获得了碱性木聚糖酶编码基因XynQH14,并在大肠杆菌E.coliBL21(DE3)中获得重组表达。通过Ni-NTA亲和层析分离纯化后的重组QH14木聚糖酶比活达700.47 U/mg。该碱性木聚糖酶的酶促反应最适温度为60℃,最适pH为9.2;55℃处理1h仍保持50%的活力;在pH7.0~11条件下37℃处理酶液24 h后均保持80%以上的活力,且在pH11缓冲溶液中50℃处理24 h仍保持31.02%的酶活,显示了该碱性木聚糖酶较好的热稳定性和碱稳定,提示该碱性木聚糖酶在制浆造纸、纺织等行业的应用潜力。     

一株以葡萄糖为底物产2,3-丁二醇微生物菌株的筛选及鉴定

从自然界中筛选出一批以葡萄糖为底物发酵产2,3-丁二醇的菌株,经初步发酵测定发酵液中2,3-丁二醇含量,其中菌株6-7的2,3-丁二醇产量最高达49.6g/L。对其进行常规生理生化鉴定实验,并结合16SrDNA序列分析,比对结果表明,菌株6-7与Bacillus subtilis strain BIHB332相似性达99%。在细菌分类学上属于枯草芽孢杆菌属,将其命名为Bacillussubtilis6-7。其特点是属于环境友好和食品安全型菌株,因此,利用Bacillus subtilis6-7生产2,3-丁二醇具有良好的工业应用价值。     

Phylogeny of gamma-polyglutamic acid-producing Bacillus strains isolated from a fermented locust bean product manufactured in West Africa

Twenty-five Bacillus strains capable of producing gamma-polyglutamic acid (PGA) were isolated from fermented locust bean products manufactured in the savanna area of Ghana. To clarify the phylogeny of these PGA-producing strains, phylogenetic analyses based on sequences of 16S rDNA, rpoB (RNA polymerase beta-subunit) and fus (elongation factor G) genes were performed. A phylogenetic tree based on 16S rDNA indicated that ten isolates were clustered in the same group of Bacillus subtilis. Another ten isolates were located in the cluster of B. amyloliquefaciens, and the remaining isolates were identified as B. pumilus (three isolates) and B. licheniformis (two isolates), respectively. Phylogenetic trees based on the partial sequences of rpoB and fus genes were similar to the phylogeny based on 16S rDNA sequences. Thirty-four strains in 27 species belonging to the genus Bacillus and its neighbors were also investigated for PGA production. It was found that PGA was produced by B. amyloliquefaciens NBRC 14141 and NBRC 15535(T), B. atrophaeus NBRC 15539(T), B. licheniformis NBRC 12107, B. mojavensis NBRC 15718(T), B. pumilus NBRC 12094, B. subtilis NBRC 16449, and Lysinibacillus sphaericus NBRC 3525. Except for L. sphaericus, the above Bacillus species are very closely related in phylogeny, indicating that PGA-producing Bacillus strains constitute a cluster.     

Antibacterial and Anticancer activity of ε -poly-L-lysine (ε -PL) produced by a marine Bacillus subtilis sp

A marine Bacillus subtilis SDNS was isolated from sea water in Alexandria and identified using 16S rDNA sequence analysis. The bacterium produced a compound active against a number of gram negativeve bacteria. Moreover, the anticancer activity of this bacterium was tested against three different human cell lines (Hela S3, HepG2 and CaCo). The highest inhibition activity was recorded against Hela S3 cell line (77.2%), while almost no activity was recorded towards CaCo cell line. HPLC and TLC analyses supported evidence that Bacillus subtilis SDNS product is ?;-poly-L-lysine. To achieve maximum production, Plackett-Burman experimental design was applied. A 1.5 fold increase was observed when Bacillus subtilis SDNS was grown in optimized medium composed of g/l: (NH(4) )(2) SO(4) , 15; K(2) HPO(4) , 0.3; KH(2) PO(4) , 2; MgSO(4) · 7 H(2) O, 1; ZnSO(4) · 7 H(2) O, 0; FeSO(4) · 7 H(2) O, 0.03; glucose, 25; yeast extract, 1, pH 6.8. Under optimized culture condition, a product value of 76.3 mg/l could be obtained. According to available literature, this is the first announcement for the production of ?;-poly-L-lysine (?;-PL) by a member of genus Bacillus. (? 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).     

一株细薄星芒海绵细菌的抗菌活性与分类学研究

采用平板涂布法从我国南海细薄星芒海绵分离得到一株细菌A72,以大肠埃希氏菌、金黄色葡萄球菌、枯草芽孢杆菌、荧光假单胞菌、黑曲霉、白假丝酵母、宛氏拟青霉7种指标菌对A72的抗菌活性进行了研究,同时采用形态学观察、生理生化分析与16S rDNA同源性与系统发育分析进行种属鉴定。研究发现A72对于枯草芽孢杆菌等具有显著的活性,初步确认A72为粪产碱杆菌。     

A combined treatment using ethylmethane sulfonate and ultraviolet light to compare amylase production by three Bacillus sp. isolates

Abstract

In this study, three Bacillus sp.-producing amylase enzymes were isolated from soil samples and identified using 16S rDNA sequence analysis. Amylase production and total protein productions were spectrophotometrically measured. The following media were tested to increase enzyme production: LB medium and molasses. Three Bacillus sp. were identified as follows: Bacillus subtilis subtilis, Bacillus thuringiensis, and Bacillus cereus. Amylase production levels were in the range of 10?U/mL, whereas total protein production levels were at 15?mg/mL. Higher amylase activity was found in the Bacillus subtilis isolate. Ethylmethane sulfonate (EMS) and ultraviolet (UV) mutagenesis in combination were applied to compare amylase production. Amylase activity was increased to around 58% in the treatment with 0.03?mL of EMS and UV when compared to the control group. A pilot scale bioreactor with a total working volume of 10 liters was used to produce amylase by B. subtilis subtilis. In conclusion, B. subtilis subtilis can be used to produce amylase enzyme for various industrial purposes, and, for the first time, the amylase activities of B. subtilis can be enhanced with EMS and UV treatment.