湖羊瘤胃微生物GH9家族葡聚糖酶基因IDSGLUC9-25的表达与功能表征

【目的】葡聚糖酶是饲用添加剂的重要成分,本研究旨在从湖羊消化道微生物中挖掘性质优良的GH9家族葡聚糖酶基因,用于研发新型饲用酶制剂。【方法】从湖羊瘤胃微生物cDNA中扩增IDSGLUC9-25基因,在大肠杆菌中进行异源表达,对重组蛋白进行诱导表达和纯化,研究重组蛋白的酶学性质和底物水解模式。【结果】IDSGLUC9-25基因编码527个氨基酸,包含一个CelD_N结构和一个GH9家族催化结构域;重组蛋白rIDSGLUC9-25分子量约为62.7 kDa,最适反应温度和pH分别为40℃和6.0,在30-50℃下活性较高,在pH 4.0-8.0范围内能够保持较高的稳定性,经pH 4.0-8.0缓冲液处理1 h后残余活性均大于90%;底物谱分析表明,rIDSGLUC9-25能催化大麦β-葡聚糖、苔藓地衣多糖、魔芋胶和木葡聚糖,比活性分别为(443.55±24.48)、(65.56±5.98)、(122.37±2.85)和(159.16±7.73) U/mg;利用薄层色谱法(thin layer chromatography, TLC)和高效液相色谱法(high performance liquid chromatography, HPLC)分析水解产物发现,rIDSGLUC9-25降解大麦葡聚糖主要生成纤维三糖(占总还原糖64.19%±1.19%)和纤维四糖(占总还原糖26.24%±0.12%),催化地衣多糖主要生成纤维三糖(占总还原糖78.46%±0.89%)。【结论】本研究报道了一种来自密螺旋体属细菌的内切β-1,4-葡聚糖酶IDSGLUC9-25 (EC 3.2.1.4),能高效催化多糖底物生成纤维三糖和纤维四糖,为研发饲用酶制剂和制备低聚寡糖建立基础。     

两株蓖麻蚕核型多角体病毒DNA同源性的研究

两株不同来源的蓖麻蚕核型多角体病毒(ArscsNPV和ArNPV)经提纯后,使用SDS—苯酚抽提病毒核酸,并使用限制性内切酶EcoRI,BamHI酶解后,用分子杂交方法与缺口平移标记的ArscsNPV-DNA探针杂交,分析了两株蓖麻蚕NPV病毒核酸的同源性。EcoRI酶解的ArNPV-DNA产生8个片段,其中5个片段能与ArscsNPV-DNA探针杂交。BamHI酶解ArNPV-DNA产生7个片段,其中6个片段能与ArscsNPV-DNA探针杂交。结果表明:两株蓖麻蚕NPV之间病毒核酸具有很高的同源性。使用斑点杂交方法分析了ArscsNPV与ArNPV,柞蚕NPV及家蚕NPV之间的核酸同源性,结果表明:ArscsNPV与ArNPV,柞蚕NPV具有同源性。而与家蚕NPV无核酸同源性。     

基于细胞色素c的胞外电子传递过程

电活性微生物具有独特的胞外电子传递功能,在地球化学循环和环境污染修复中起着重要作用。细胞色素c在电活性微生物胞外电子传递过程中扮演了重要角色,不仅参与直接电子传递途径,还参与电子媒介介导的间接电子传递。其电子传递功能不仅对地球环境中铁、锰、碳等元素的循环具有重要作用,还应用于能源生产、废水处理、生物修复等众多领域,具有良好的应用潜力。本文以电活性微生物的2个模式菌属(希瓦氏菌属和地杆菌属)为例,综述了电活性微生物将电子由胞内转移至胞外的方式和途径,详细阐述了细胞色素c在该胞外电子传递过程中的重要作用,总结了细胞色素c介导的胞外电子传递过程所涉及的分析方法,并对微生物胞外电子传递未来的研究方向提出了展望。     

棒状腐败乳杆菌Lc7的生物学特性及益生作用

【目的】对分离自健康成人粪便样本的棒状腐败乳杆菌(Loigolactobacillus coryniformis)Lc7进行分类学鉴定和益生潜力评估。【方法】基于16SrRNA基因和基因组核心基因构建系统发育树,对Lc7进行分类学鉴定;通过耐酸和胆汁酸盐、粘附、抗氧化和抑菌实验,以及溶血、明胶酶活性和抗菌药物敏感性实验,评估Lc7的益生特性。同时,构建小鼠溃疡性结肠炎模型,评估Lc7的体内抗炎潜力。【结果】Lc7鉴定为L. coryniformis,在酸和胆汁酸盐的连续作用下,Lc7的存活率为70.17%。Lc7对HT-29细胞的粘附指数为56.33 CFU/cell,其自聚集和疏水性分别为80%和40%;Lc7对福氏志贺菌和鼠伤寒沙门菌等7个常见致病菌均有较强的抑制能力;对1,1-二苯基-2-苦基肼(1,1-diphenyl-2-picryl-hydrazyl,DPPH)和羟自由基(hydroxyl radicals,·OH)的清除率分别为91.70%和48.53%;Lc7无溶血现象和明胶酶活性,对选取的大多数抗生素均敏感。在小鼠结肠炎实验中,Lc7干预组小鼠结肠长度明显长于模型组(...     

The potato StMKK5-StSIPK module enhances resistance to Phytophthora pathogens through activating the salicylic acid and ethylene signalling pathways

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in plant responses to both biotic and abiotic stress. A screen of a Nicotiana benthamiana cDNA virus-induced gene silencing (VIGS) library for altered plant responses to inoculation with Phytophthora infestans previously identified an NbMKK gene, encoding a clade D MAPKK that we renamed as NbMKK5, which is involved in immunity to P. infestans. To study the role of the potato orthologous gene, referred to as StMKK5, in the response to P. infestans, we transiently overexpressed StMKK5 in N. benthamiana and observed that cell death occurred at 2 days postinfiltration. Silencing of the highly conserved eukaryotic protein SGT1 delayed the StMKK5-induced cell death, whereas silencing of the MAPK-encoding gene NbSIPK completely abolished the cell death response. Further investigations showed that StMKK5 interacts with, and directly phosphorylates, StSIPK. Furthermore, both StMKK5 and StSIPK trigger salicylic acid (SA)- and ethylene (Eth)-related gene expression, and co-expression of the salicylate hydroxylase NahG with the negative regulator of Eth signalling CTR1 hampers StSIPK-triggered cell death. This observation indicates that the cell death triggered by StMKK5-StSIPK is dependent on the combination of SA- and Eth-signalling. By introducing point mutations, we showed that the kinase activity of both StMKK5 and StSIPK is required for triggering cell death. Genetic analysis showed that StMKK5 depends on StSIPK to trigger plant resistance. Thus, our results define a potato StMKK5-SIPK module that positively regulates immunity to P. infestans via activation of both the SA and Eth signalling pathways.     

Avicularin alleviates acute liver failure by regulation of the TLR4/MyD88/NF-κB and Nrf2/HO-1/GPX4 pathways to reduce inflammation and ferroptosis

Acute liver failure (ALF) is an inflammation-mediated hepatocyte death process associated with ferroptosis. Avicularin (AL), a Chinese herbal medicine, exerts anti-inflammatory and antioxidative effects. However, the protective effect of AL and the mechanism on ALF have not been reported. Our in vivo results suggest that AL significantly alleviated lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced hepatic pathological injury, liver enzymes, inflammatory cytokines, reactive oxygen species and iron levels and increased the antioxidant enzyme activities (malondialdehyde and glutathione). Our further in vitro experiments demonstrated that AL suppressed inflammatory response in LPS-stimulated RAW 264.7 cells via blocking the toll-like receptor 4 (TLR4)/myeloid differentiation protein-88 (MyD88)/nuclear factor kappa B (NF-κB) pathway. Moreover, AL attenuated ferroptosis in D-GalN-induced HepG2 cells by activating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1)/glutathione peroxidase 4 (GPX4) pathway. Therefore, AL can alleviate inflammatory response and ferroptosis in LPS/D-GalN-induced ALF, and its protective effects are associated with blocking TLR4/MyD88/NF-κB pathway and activating Nrf2/HO-1/GPX4 pathway. Moreover, AL is a promising therapeutic option for ALF and should be clinically explored.     

酶法生产L-高苯丙氨酸的研究进展

L-高苯丙氨酸(L-homophenylalanine,L-HPA)作为一种重要的非天然氨基酸,是合成治疗高血压的普利类药物等的关键中间体,具有广阔的市场前景。目前L-高苯丙氨酸的合成主要依赖于化学法,但化学合成L-高苯丙氨酸具有原料昂贵、步骤繁琐和污染严重等缺点,限制了广泛应用。因此,国内外研究者对L-高苯丙氨酸的酶法生产进行了深入的研究。本文就目前酶法合成L-高苯丙氨酸的工艺,包括脱氢酶法、转氨酶法、海因酶法和脱羧酶法的研究进展进行了综述,为酶法合成L-高苯丙氨酸提供一定的借鉴,为最终实现L-高苯丙氨酸的酶法工业化生产奠定基础。     

谷氨酸棒杆菌代谢工程高效生产L-缬氨酸北大核心CSCD

L-缬氨酸作为一种支链氨基酸,广泛应用于医药和饲料等领域。本研究借助多种代谢工程策略相结合的方法,构建了生产L-缬氨酸的微生物细胞工厂,实现了L-缬氨酸的高效生产。首先,通过增强糖酵解途径、减弱副产物代谢途径相结合的方式,强化了L-缬氨酸合成前体丙酮酸的供给;其次,针对L-缬氨酸合成路径关键酶—乙酰羟酸合酶进行定点突变,提高了菌株的抗反馈抑制能力,并利用启动子工程策略,优化了路径关键酶的基因表达水平;最后,利用辅因子工程策略,改变了乙酰羟酸还原异构酶和支链氨基酸转氨酶的辅因子偏好性,由偏好NADPH转变为偏好NADH,从而提高了L-缬氨酸的合成能力。在5L发酵罐中,最优谷氨酸棒杆菌工程菌株Corynebacterium glutamicum K020的L-缬氨酸产量、得率和生产强度分别达到了110g/L、0.51g/g和2.29 g/(L·h)。     

不同结香方法对土沉香木质部芳香物质形成的影响*

探究不同结香方法对土沉香木质部芳香物质形成的影响,为创新和优化土沉香结香技术提供理论参考。设置钻孔、火烧孔和黄绿墨耳菌溶液3个结香处理,12个月后取样检测各处理的土沉香木质部组织变色范围、淀粉和可溶性糖含量,并用95%乙醇(色谱纯)提取个处理土沉香样品的醇溶提取物,测定沉香四醇和主要芳香物质成分。结果表明：(1) 黄绿墨耳菌溶液处理的土沉香木质部组织变色范围最大,纵向变色距离最长达120cm,横向变色距离最宽达3.84cm;钻孔和火烧孔处理,纵向变色距离均超过120cm,但横向变色距离最宽仅为0.58cm和1.06cm。(2) 3种结香处理的土沉香木质部组织淀粉含量均显著低于CK (P＜0.05),降幅范围在24.82~55.25%;黄绿墨耳菌溶液和火烧孔处理的土沉香木质部组织可溶性糖含量均显著高于CK (P＜0.05),钻孔与CK差异不显著((P＞0.05)。(3)钻孔、火烧孔和菌液处理沉香醇溶性提取物含量达到11.84%、13.26%和21.08%,显著高于CK (P＜0.05);沉香四醇含量达到0.18%、0.26%和0.42%,对照处理未检测出沉香四醇。(4) 钻孔、火烧孔和黄绿墨耳菌溶液处理的土沉香芳香物质中分别鉴定出29种、33种和36种,主要成分包括色酮及其衍生物、沉香螺醇、愈创木醇、香树烯、苄基丙酮、二氢卡拉酮、枯苏醇、α-檀香醇和壬醛。说明黄绿墨耳菌溶液诱导土沉香木质部组织变色范围最大,淀粉含量消耗和可溶性糖增幅最大,醇溶提取物、沉香四醇含量及其芳香物质积累的效果最优。