一株产耐热普鲁兰酶菌株Anoxybacillus sp. LM14-2分离鉴定及酶学性质研究

从云南轮马热泉下游淤泥中筛选得到了一株产耐热普鲁兰酶菌株LM14-2。根据形态特征及16S rRNA序列同源性分析,初步判定为Anoxybacillus sp.LM14-2。该菌株发酵上清液中有耐热普鲁兰酶积累,其反应最适pH值为6.0,最适温度为70℃。利用染色体步移技术获得了完整的普鲁兰酶编码基因(HQ660582),经序列相似性进一步分析,确定该蛋白与I型普鲁兰酶保守区b相吻合。通常的普鲁兰酶在高温下很快失活,难以满足淀粉加工,洗涤剂等相关工业的需求,而该新型的耐热普鲁兰酶的作用温度广泛,热稳定性较好,65℃保温55 h后达到其半衰期,具有广阔的开发应用前景。

Characterization of Lipases from Geobacillus stearothermophilus and Anoxybacillus flavithermuscell Lysates

The aim of this study was to characterize lipases from two thermophilic bacteria, Geobacillus stearothermophilus (GS) and Anoxybacillus flavithermus (AF) in heat treated cell lysates. The pH optimum, pH stability, temperature stability and substrate kinetics and specificity of the lipases were determined. Optimum activity of the lipase from GS (LGS) was observed at pH 7.5, and the optimum activity of the lipase from AF (LAF) was at pH 8.0. LGS was stable up to 70°C after 12 hrs while LAF was stable up to 90°C after 12 hrs. Both enzymes were stable at a pH range of 6 to 8 over 12 h at 4°C. LGS had a highest Vmax value of 22 mM·min-1·mg-1 with p-nitrophenyl acetate while the lowest Km was 0.8 mM with p-nitrophenyl laurate. The highest Vmax of LAF was 2.5 mM·min-1·mg-1 with p-nitrophenyl myristate, and the lowest Km was 0.4 mM with p-nitrophenyl octanoate. LGS preferentially hydrolyzed p-nitrophenyl acetate and p-nitrophenyl octanoate while LAF preferentially hydrolyzed p-nitrophenyl myristate and p-nitrophenyldodecanoate. Lipases from both GS and AF showed characteristics that would be beneficial in food processing.

甲苯胁迫下有机溶剂耐受菌Anoxybacillus flavithermus ssp. yunnanesis E13~T膜脂肪酸的变化

【目的】探讨目前唯一具有有机溶剂耐受性的嗜热细菌新物种Anoxybacillus flavithermus ssp.yunnanesis E13T甲苯胁迫下膜脂肪酸的变化。【方法】在不同条件下培养菌株E13T,收集细胞,提取脂肪酸,采用气相色谱-质谱(GC-MS)对脂肪酸进行定量测定分析。【结果】0.3%(V/V)甲苯胁迫下生长时,菌株E13T是在从延滞期进入初始生长的时刻显著上调饱和直链脂肪酸含量,然后随着菌体的生长,饱和直链脂肪酸的含量持续减少;在100%甲苯幸存实验中,菌株E13T的饱和直链脂肪酸的增加幅度更为显著。【结论】与常温下的有机溶剂耐受菌一样,A.flavithermus ssp.yunnanesis E13T也是通过调节细胞膜上的脂肪酸,促使细胞膜变硬以抵御甲苯毒性。但是它是通过调节饱和直链脂肪酸,而不是像常温下的有机溶剂耐受菌那样调节不饱和脂肪酸。

氡温泉中耐辐照嗜热菌的分离及其特性研究

氡温泉水样经8kGy60Coγ射线预处理后,从中分离获得1株耐60 Coγ射线和UV辐照的嗜热菌株R4-33。经形态观察、生理生化试验、脂肪酸分析、(G+C)mol%含量和16S rDNA序列测定,结果表明,菌株R4-33为杆状,革兰氏阴性菌,无鞭毛,形成末端芽孢;最适生长温度60℃,最适生长pH 7.5;能以葡萄糖、麦芽糖和海藻糖等作为唯一碳源生长,水解酪素和淀粉;过氧化氢酶阳性;对青霉素、新霉素、红霉素、万古霉素、链霉素、庆大霉素、丁胺卡拉和氨苄青霉素敏感;细胞主要脂肪酸为C14∶1(48.4%)和C15∶1(15.2%);基因组DNA中(G+C)mo1%含量58.2%。经比对菌株R4-33的16SrDNA序列与Anoxybacillus属菌株有较高的同源性,其中与Anoxybacillus gonensis菌株的相似性高达99.5%。经R4-33菌株生理生化试验及16S rDNA序列分析,该株菌归属于Anoxybacillus属,暂命名为Anoxybacillus sp.R4-33。耐辐射细胞存活试验结果显示菌株R4-33对UV的耐辐射能力达396J/m2,对60Coγ射线耐辐照能力为14kGy,表明其对UV和γ射线辐照具有较高耐受性。

耐热α-淀粉酶的筛选、基因克隆和酶学性质分析

【目的】筛选耐热α-淀粉酶并实现异源表达,同时分析其酶学性质特征。【方法】以M9培养基加上可溶性淀粉作为选择性分离培养基,从腾冲火山温泉土壤中筛选到产淀粉酶的菌株Anoxybacillus sp.GXS-3。根据淀粉酶氨基酸保守序列,设计引物进行PCR扩增,然后对目标序列进行步移扩增,获得淀粉酶基因AmyGX。将AmyGX与表达载体pQE30连接,导入大肠杆菌M15中表达,对重组酶进行分离纯化和酶学性质分析。【结果】AmyGX基因长1 515 bp,编码505个氨基酸残基,前23个氨基酸残基为信号肽序列;重组质粒pQE30-AmyGX编码的蛋白分子量为58.04kDa,对可溶性淀粉催化水解反应的最适温度为60℃,最适pH值为8.0,V_(max)、K_m值分别为0.19U/mg、3.14mg/mL,热半失活温度T_(50)^(30)值为65.2℃;Zn^(2+)、Cu^(2+)、Co^(2+)、Fe^(3+)、Ba^(2+)对该酶具有明显的抑制作用,Na^+、K^+对该酶有激活作用,Mg^(2+)、Ca^(2+)的影响则不明显。【结论】AmyGX是一种中等耐温碱性酶,在造纸、洗涤剂生产和有毒废弃物去除等方面具有潜在的应用前景。

一株产木糖异构酶嗜热菌的分离鉴定及其培养条件优化

从云南腾冲温泉水样中分离到1株产木糖异构酶活性较高的嗜热菌,根据菌株的形态特征和16SrD-NA序列,鉴定为Anoxybacillus flavithermus WL。采用单因素优化实验及正交设计,确定该菌株在木糖添加量15g/L、接种量3%、初始pH8.0、65℃的条件下发酵24h,达到最高酶活13.6U/mL。该菌株木糖异构酶粗酶液的最适反应温度为90℃。

Isolation and molecular identification of cellulolytic bacteria from Dig Rostam hot spring and study of their cellulase activity

Cellulose is the main structural component of lignocellulosic wastes that can be converted to sugars and biofuels by cellulase.Due to wide applications of this enzyme in various industries around the world,cellulase is considered as the third industrial enzyme.The ability of thermophilic bacteria in the production of heat-stable cellulases has made them valuable tools in biotechnology.The aim of this study was isolation and molecular identification of cellulolytic thermophile bacteria from Dig Rostam hot spring and investigating their cellulase activity.Samples were taken from water and sediments of this hot spring,and cellulolytic bacteria were enriched in media containing cellulose as the only carbon source.The bacteria were incubated at 60℃,and single colonies were then isolated on solid media.Congo red assay was used as a quick test for the qualitative screening of cellulase activity.According to these qualitative results,four colonies named CDB1,CDB2,CDB3,and CDB4 were isolated,and their growth curve and some other characteristics were determined by biochemical assays.Moreover,endoglucanase,exoglucanase,and FPase activities of the isolates were investigated quantitatively.Results indicated that CDB1 exhibited the highest endoglucanase(0.096 U/mL)and exoglucanase(0.156 U/mL)activities among other isolates.16S rDNA partial sequencing indicated that CDB1 had 99%similarity to the genus Anoxybacillus,and the other isolates showed the highest similarity to the genus Geobacillus.The cellulase gene of CDB1 isolate with the highest cellulase activity was also cloned,and its sequence is reported for the first time.Further studies on this thermophilic enzyme might be useful for industrial applications.