Optimal production of exopolysaccharide by Bacillus licheniformis KS-17 isolated from kimchi

A strain producing exopolysaccharide (EPS) with strong hydroxyradical scavenging activity and antityrosinase activity has been isolated previously. However, the EPS production rate of the strain (7.3 g/L) was relatively low to utilize it at the industrial level. Therefore, in this study, optimal medium and fermentation conditions were examined to enhance EPS production by Bacillus licheniformis KS-17. Maximum EPS production was obtained in medium with 125 g/L sucrose, 30 g/L ammonium sulfate, and 10 mM calcium chloride without a phosphate source at 37°C for 5 days with the initial pH adjusted to 8.5. Under optimal culture conditions, EPS was produced at up to 27.2 (at 5 days) vs. 12.0 g/L (at 7 days under basal conditions), which was 2.3 times greater and in a shorter time than the production yield possible without optimizing conditions.     

Glucose fermentation kinetics and exopolysaccharide production by ropy Pediococcus damnosus IOEB8801

The metabolic behaviour of ropy Pediococcus damnosus IOEB8801 is examined under various culture conditions and the consequences on EPS production are discussed. EPS synthesis appears like a metabolic leak occurring when growth slows down. The level of the main intracellular enzymatic activities implicated in glycolysis or EPS and cell wall synthesis are not modified by the turning on of EPS synthesis. As a result, an efficient preliminary growth phase is essential for subsequent important EPS production. Thus, it seems that wines in which pH is high, glucose and a nitrogen source are present and which are not agitated at all are more likely to become ropy when P. damnosus IOEB 8801 is present.     

基于定向进化技术提高地衣芽孢杆菌L-天冬酰胺酶活性

为提高地衣芽孢杆菌L-天冬酰胺酶活性,通过定向进化技术对其进行分子改造。经过两轮易错聚合酶链式反应和一轮DNA shuffling,从19 100多个突变株中筛选到突变体S10、S16和S21,其酶比活力较野生型分别提高了106%、74%和43%,且突变酶K_(cat)/K_m都有所增大。其中,突变体S10氨基酸序列发生3个突变,K43E、N67S和I269L。三维模拟结果显示,第43位氨基酸突变为谷氨酸、第67位氨基酸突变为丝氨酸,可能提高了底物亲和力和催化效率,从而提高酶活性。圆二色谱分析表明,相比野生酶,突变酶的α-螺旋数减少、无规则卷曲有所增加,表明其刚性略有降低,柔性有所增加。利用定向进化策略能够有效地提高地衣芽孢杆菌L-天冬酰胺酶活性。     

1株分离于铀矿的可利用玉米秸秆高效产氢的芽孢杆菌

采用二重叠皿隔绝空气培养法从铀矿中分离获得1株可利用玉米秸秆高效产氢的菌株w-14,经生理生化及16S rRNA基因序列分析鉴定为丁酸梭菌(Clostridium butyricum);通过两段发酵技术,研究了用木霉、曲霉菌制备的纤维素酶麸曲酵解玉米秸秆的温度、时间、p H、麸曲用量对底物的糖化效率和w-14菌株利用玉米秸秆酵解物产氢的影响,结果表明:玉米秸秆酵解的麸曲浓度为20 g/L,温度为40℃、时间84 h,p H为5.0时酵解物还原糖为402 mg/g-cs,w-14菌株利用此酵解物发酵累积产氢量为99.7 m L/g-cs;采用正交试验优化出w-14菌株以玉米秸秆酵解物为碳源基质产氢发酵最适条件,并在5 L间歇搅拌深层发酵罐中完成验证,结果表明:初始p H 7.0,种龄16 h,接种量10%,发酵温度37℃,w-14菌株的产氢量达140.24 m L/g-cs,比优化前提高了41.38%。经5 L间歇搅拌深层发酵反应器验证,w-14菌株利用秸秆的最高累积产氢量达149.09 m L/g-cs,氢气浓度达到55.63%。     

Purification and Characterization of a Thermostable alpha-Amylase from Bacillus licheniformis

The heat stability of a bacterial α-amylase is important for industrial starch utilization. Although extensive studies have been done on heat stable α-amylase from various bacterial species little is known about the α-amylases of Bacillus licheniformis. In order to get better understanding of thermostable amylases produced by different strains of B. licheniformis and provide information how to utilize the enzyme in starch processing, studies on purification and characterization of a commercial heat stable bacterial α-amylase from B. licheniformis BLM 1777 are reported.     

Structural and technological characterization of ropy exopolysaccharides produced by Lactobacillus plantarum strains isolated from Tarhana

Food Science and Biotechnology - Exopolysaccharide producing starter cultures enable manufacturing “clean labeled” foods with improved textural and nutritional properties. The...     

以L-苏氨酸为发酵底物的2,5-二甲基吡嗪高产菌株构建

构建一种以L-苏氨酸为发酵底物的高值化学品2,5-二甲基吡嗪(2,5-dimethylpyrazine, 2,5-DMP)生产菌株,为解决L-苏氨酸产能过剩,实现2,5-DMP生物法生产提供可靠思路。通过利用Bacillus subtilis 168(B.subtilis 168)外源表达不同微生物种属来源的L-苏氨酸脱氢酶(L-threonine dehydrogenase, TDH),并比较其利用L-苏氨酸为底物合成2,5-DMP的产量,挑选出2,5-DMP高产菌种,在此基础上进一步外源表达NADH氧化酶(NADH oxidase, NOX),以促进辅因子再生。实验构建了1株高产2,5-DMP的基因工程菌株B.subtilis 168/pMA0911-tdh(E.c)-nox。该菌株以5.83 g/L的L-苏氨酸为底物,发酵24 h后2,5-DMP的产量高达616.04 mg/L,与对照菌株B.subtilis 168/pMA0911相比,产量提高了22.5倍。在TDH过表达的基础上,NOX的参与有利于2,5-DMP产量的提高。该研究首次实现了2,5-DMP高效的生物转化,一方面缓...     

一株地衣芽孢杆菌的分离及在大豆肽发酵中的应用

从土壤中分离得到1株蛋白酶高产菌株,经鉴定为地衣芽孢杆菌。以大豆分离蛋白为原料,用地衣芽孢杆菌来发酵生产大豆多肽,通过对发酵液中大豆蛋白起始浓度、发酵液初始pH值、发酵温度、摇瓶转速、发酵时间等因素进行研究,以多肽得率为指标初步探索了发酵工艺条件,并以大豆多肽含量为指标,采用响应面分析法,对发酵法制备大豆多肽的生产工艺进行了优化。结果表明,在大豆蛋白起始浓度为3%,初始pH值为6.5,发酵培养温度34.0℃,摇瓶转速180r/min,发酵时间为18h～21h的条件下,发酵大豆蛋白所得多肽的含量可达19.2mg/mL,多肽得率达到70%,且所得到的大豆多肽发酵液具有良好的口感。     

嗜热地衣芽孢杆菌植酸酶的原核表达及酶学性质研究

将地衣芽孢杆菌SR01（Bacillus licheniformis SR01）的植酸酶phy基因重组于表达载体p GEX-4T-3中,导入大肠杆菌BL21（Escherichia coli BL21）构建工程菌p GX1143,并得到了高效表达。对其酶学性质的研究表明:该酶最适反应p H为5.0,最适反应温度为55℃,具有广泛的p H稳定性和较好的热稳定性;Co²⁺)、Li⁺、Mg²⁺)和Ba²⁺对酶活性有促进作用,Cu²⁺、Pb²⁺、Fe²⁺、Ag⁺、SDS对酶活性有不同程度的抑制作用,并且Pb²⁺、Fe²⁺和SDS对酶活性的抑制作用较强烈;此外,该酶还具有非常好的抗胰蛋白酶水解能力和部分抗胃蛋白酶水解的能力。      

Evaluation of exopolysaccharide production by Leuconostoc mesenteroides strains isolated from wine

ABSTRACT:  Exopolysaccharide (EPS)-producing lactic acid bacteria are responsible for the alteration of wine and other fermented beverages. The potential to produce EPS was investigated for Leuconostoc mesenteroides strains isolated from Spanish grape must and wine. Most strains were able to produce EPS from sucrose containing media. Based on their EPS-producing phenotype and on their EPS monosaccharide composition, the L. mesenteroides strains analyzed could be arranged in 2 groups. One group comprises mucoid strains producing a glucan polymer, and the other group includes strains producing a fructan polymer. The presence of a glucosyltransferase encoding gene in the glucan producing L. mesenteroides strains was assayed by PCR. Two primer sets, PF1-PF8 and GTFF-GTFR, were used to amplify internal fragment of known glucosyltransferase genes. None of the glucan-producing strains gave a positive amplicon by the primer sets used. Therefore, new tools need to be developed to broaden the range of potentially spoiling agents detected by PCR in fermented beverages.     

地衣芽孢杆菌产碱性蛋白酶发酵条件优化

碱性蛋白酶是一类重要的工业用酶,广泛应用于洗涤、制革、酿造等行业,发挥着重要作用。为提高地衣芽孢杆菌的产酶活力,在单因素实验的基础上,采用3因素3水平的响应面分析法,以蛋白酶活力为响应值,对影响地衣芽孢杆菌20203产碱性蛋白酶的因素进行研究分析,得到了最佳发酵条件为:葡萄糖1.5%,豆粕6%,乳糖4%,磷酸铵1.2%,KCl 0.03%,CaCl2 0.07%,MgSO4 0.02%,吐温80 0.05%,初始pH9.0,培养温度37℃,摇瓶转速300r/min,5d后发酵液酶活为2382u/mL。通过非变性聚丙烯酰胺凝胶电泳的方法得到该蛋白酶的分子量为35ku。     

地衣芽孢杆菌发酵淀粉产乳酸条件的优化

主要研究地衣芽孢杆菌转化淀粉生产乳酸的发酵条件。从北京郊区土壤中筛选到一株可发酵淀粉等糖质原料生产乳酸的地衣芽孢杆菌WX51,通过单因素及正交试验,确定了其最佳培养基组成为可溶性淀粉40 g/L,硫酸铵0.5 g/L,KH2PO4 1.36 g/L,MgSO4.7H2O 0.2g/L,FeSO4.7H2O 0.01g/L,NaCl 2g/L,玉米浆0.5g/L,CaCO3 20g/L;最佳培养条件为:250mL摇瓶装液10%,50℃、200 r/min培养40h、接种量2%。经优化后,该菌乳酸产量由28.4g/L提高为36.5g/L,淀粉的转化率由71.0%提高为91.2%,产酸速率由0.6g/(L.h)提高为0.9g/(L.h)。     

An exopolysaccharide from a probiotic: Biosynthesis dynamics,composition and emulsifying activity

An exopolysaccharide (EPS) was isolated from Bacillus coagulans RK-02. Time course accumulation of EPS was studied with respect to biomass growth. The probiotic bacterium produces an EPS during exponential and stationary growth phases. Growth kinetics of the producer organism was characterized. The probiotic bacterium exhibited high affinity for the growth limiting substrate and hence grows at a higher specific growth rate. Based on HPLC and FTIR analyses, the EPS was found to be heteropolymer composed of galactose, mannose, fucose, glucose and glucosamine. The EPS showed significant emulsifying activities in different vegetable oils/hydrocarbon substrates. To the best of our knowledge, this is the first report on a bacterial EPS with fucose as a constituent sugar and also the first report on an EPS from B. coagulans.     

地衣芽孢杆菌A 产碱性蛋白酶的研究

研究从经过60Co 照射的东海香参水解液中分离出的地衣芽孢杆菌A 产碱性蛋白酶的产率、分离纯化与生化特性。经硫酸铵沉淀、DEAE- Sepharose 离子交换层析和Sephadex G-75 凝胶层析分离纯化后,碱性蛋白酶产率为5.8%;经SDS- PAGE 电泳测得其分子质量为35kD。酶的最适反应温度为50℃,最适pH 值为11,热稳定性较好。该蛋白酶具有一定的耐氧化能力。     

嗜热放线菌海藻糖合成酶在地衣芽孢杆菌中的异源表达

海藻糖合成酶可将麦芽糖异构化为海藻糖,是海藻糖酶法生物转化的中心酶制剂。为了实现海藻糖合成酶在食品安全型表达宿主中高效生产,进而应用于食品级海藻糖的酶法合成,构建了革兰氏阳性菌重组表达质粒并转化入地衣芽孢杆菌。重组质粒携带了来自于嗜热放线菌Thermomonospora curvata海藻糖合成酶的编码基因,在地衣芽孢杆菌木糖异构酶启动子及其阻遏蛋白的介导下表达,胞内海藻糖合成酶发酵活力为12.1 U/m L。考察了不同培养条件对重组菌生长和产酶的影响,结果显示质量分数4%的麦芽糊精和质量分数0.4%的豆饼粉分别为产酶适宜的碳源和氮源;菌体培养10 h后加入终质量浓度为1 g/d L的诱导剂,于37℃诱导12 h后重组菌产酶最高达到23.7 U/m L。     

地衣芽孢杆菌2709 蛋白酶分离纯化研究

对产自地衣芽孢杆菌(B. licheniformis)2709 的碱性蛋白酶通过乙醇沉淀、盐析、DEAE 阴离子交换层析、凝胶层析4 步纯化,最终获得电泳纯的酶。以去酰胺度及酶比活力为指标,对碱性蛋白酶分离纯化条件进行优化。结果发现：提纯酶的比活力达61069U/mg,纯化倍数为38.7,活性回收率为19.3%,去酰胺度为20.9%。并研究该酶的基本酶学特性,结果发现：该酶最适作用pH 值为10.0,最适反应温度为50℃。40℃保温2h 后该酶保持80% 以上的活力,在pH8～11 之间有较高的pH 值稳定性。     

抗氧化胁迫促进地衣芽孢杆菌合成杆菌肽

地衣芽孢杆菌（Bacillus licheniformis）发酵合成杆菌肽过程中存在的氧化胁迫影响了杆菌肽的合成效率。添加0.2%抗坏血酸和0.4%半胱氨酸使杆菌肽效价在摇瓶水平分别比对照提高了20.7%和29.4%。另外,胞内过氧化氢酶活比对照分别降低了68.3%和43.8%。半胱氨酸合成加强工程菌株在10 L罐发酵过程中,峰值生物量（84×109 CFU/mL,26 h）比出发菌株提高了9.0%,杆菌肽效价（1 090 U/mL,32 h）也提高了13.5%。Cys含量（213.3 mg/L,24 h）和过氧化氢酶活力（14～32 h）分别比对照提高了约60%和降低了15%～40%。说明Cys作为前体氨基酸参与杆菌肽合成与参与氧化自由基清除之间存在竞争关系,工程菌株通过增强Cys合成更好地满足了细胞对两者的生理需求,维持了细胞活性、促进了杆菌肽合成效率。     

地衣芽孢杆菌弹性蛋白酶纯化和性质研究

用地衣芽孢杆菌发酵液制备弹性蛋白酶粗酶液,采用硫酸铵分级沉淀和Sephadex凝胶柱层析的方法分离纯化弹性蛋白酶,并对弹性蛋白酶的酶学性质进行研究。结果表明：弹性蛋白酶粗酶液经40%～70%饱和度的硫酸铵纯化后比活力提高到120U/mg,经凝胶柱层析纯化后比活力可达到292U/mg,纯化倍数为12.6,SDS-PAGE法证实弹性蛋白酶分子质量为29.5kD。对酶学性质的研究结果表明：弹性蛋白酶最适反应温度为55℃,最适反应pH值为7.4,以刚果红-弹性蛋白为底物,米氏常数Km为9.67mg/mL。低浓度金属离子Ca2+和K+对酶活力有促进作用,而Mg2+、Mn2+、Zn2+和Al3+对酶活力则有抑制作用。     

嗜热地衣芽孢杆菌SR01葡聚糖酶基因的原核表达及酶学性质研究

从一株嗜热地衣芽孢杆菌SR01中克隆了β-葡聚糖酶的编码基因,并通过原核表达对重组酶的酶学性质进行了研究。结果显示,该酶编码基因ORF包含741bp,编码246个氨基酸,理论分子量为28.03ku,等电点为6.42。在温度30℃、浓度0.05mmol/L条件下IPTG诱导4h,重组蛋白得到显著表达。酶的最适反应温度、pH值分别为55℃、pH6.0~7.0;在温度40~90℃、pH5.0~10.0条件下具有良好的稳定性。Cu2+、Fe2+、Ca2+、Ba2+、Mn2+、EDTA对该酶有不同程度的抑制作用,K+、Na+对该酶起轻微的激活作用。该酶对体外模拟胃肠环境具有较好的耐受性,在模拟胃液中放置90min仍有80%左右的活性,胰液对该酶有一定的激活作用。