高效木聚糖降解酶系定制的新进展与挑战

木聚糖是植物细胞壁中含量最丰富的非纤维素多糖,大约占陆地生物质资源的20%-35%。不同物种来源的木聚糖结构因取代方式不同而具有广泛的异质性,这对生物质资源向生物燃料和其他高值产品高效转化提出了重大挑战。因此,需要开发由不同类型酶组成的最佳混合物以有效糖化木聚糖类底物。但是针对特定类型的底物设计高效降解酶系十分困难,应考虑底物的类型、底物的组成和物理性质、多糖的聚合度以及不同降解酶组分的生化性质等。本文从不同植物木聚糖的结构异质性与合成复杂性方面展示了其抗降解屏障,同时介绍了木聚糖主链降解酶系及侧链降解酶系的多样性以及协同降解作用,综述了复杂生境中微生物种群产生的混合酶系、降解菌株产生的高效酶系,以及基于特定木聚糖底物改造并定制简化高效的酶系统。随着不同种类木聚糖精细结构和木聚糖降解酶底物特异性的深入研究,针对特定底物类型进行绿色高效木聚糖酶系定制,加速木聚糖类底物的降解,从而实现木质纤维素资源的绿色高值化利用。     

植物细胞壁多糖合成酶系及真菌降解酶系北大核心CSCD

秸秆类植物细胞壁多糖高效降解转化对我国农业经济的绿色可持续发展具有重要意义,然而植物细胞壁在长期进化过程中形成了复杂结构限制了工业化酶解转化的过程。一方面从植物细胞壁多糖合成酶系的多样性、细胞壁多糖成分的复杂性、超分子结构的异质性等方面综述了形成植物细胞壁抗降解屏障的原因;另一方面从真菌降解植物细胞壁酶系的多样性、不同菌株降解酶组成差异性等分析降解转化植物细胞壁时发挥的不同作用,从而为工业转化合理复配真菌降解酶系,提高秸秆生物质的利用效率提供理论支持。     

海洋褐藻多糖的复杂结构、降解酶系以及生物学功能

海洋大型藻类(包括褐藻、红藻和绿藻)具有生物质资源产量高、生长过程中不占用耕地和淡水资源等优点,是未来生物炼制的优良原料。2021年,中国褐藻产量为190万吨,远高于其他经济藻类。但是与绿藻相比,褐藻所含的褐藻酸盐和红藻所含的3,6-脱水-L-半乳糖等多糖组分不容易发酵,极大地限制了其高值转化的进程。本文针对褐藻多糖的高效降解与高值转化这一研究热点,总结了褐藻的系统发育与褐藻多糖(褐藻胶、岩藻多糖以及昆布多糖)的复杂结构组成,分析了3类海洋多糖降解酶系的家族、空间结构及其特异性识别专一底物的活性架构等特征,并对褐藻多糖降解产物及其衍生寡糖的生物学功能进行了构效分析,以期揭示海洋多糖降解酶系的高效催化机制和特异性识别机理,推动褐藻的高效生物降解转化,为精准定制生物活性寡糖,构建绿色低碳工业化生产工艺提供参考。     

海藻多糖降解酶的性质和作用机理

海藻主要由蓝藻、绿藻、红藻和褐藻四大类组成。世界海洋中估计生长有 80 0 0多种海藻。海藻的生产与它的利用价值有密切关系 ,就褐藻、红藻和绿藻这三大门类来说 ,褐藻和红藻以其种类多、产量丰富和含有用途广泛的褐藻胶、琼胶和卡拉胶等 ,由自然生产逐步为人工养殖所代替。全世界每年约生产5万吨海藻胶 (其中 ,褐藻胶 2 5万吨 ,卡拉胶 1 45万吨、琼胶 0 7万吨 )创值近 4亿美元。这些海藻胶是海藻细胞壁内的主要填充物质 ,约占细胞干重的 2 0 %～ 3 0 % [1] 。近年来 ,各国的科学家大力开展从海洋开发新药物的研究计划。因此 ,对海藻、…     

采用酶液活性测定法快速筛选及构建石油烃高效降解菌系

快速筛选复杂有机物降解微生物混合菌系,在污染物治理过程中具有重要的实践意义.本研究首次尝试利用MicroResp^TM技术分析微生物酶液活性的方法,快速标定高效降解菌及混合菌系的石油烃降解能力,并采用传统的摇瓶培养检测法予以验证.通过微生物胞内、胞外及混合酶液的活性分析,考察了不同酶系(胞外、胞内及混合酶液)、菌系对石油烃分子的降解情况.结果表明: 结合MicroResp^TM技术的酶液活性测定法能够快速检测石油烃代谢酶系的降解能力,其灵敏度好、通量高,与传统的菌株摇瓶培养方法的检测结果基本一致.其中,7株菌株的120种全组合菌系活性测定试验在12 h周期内1次完成.筛选周期由传统摇瓶培养所需的7 d缩短10倍以上.以酶活性测定结果为指导设计的复配菌系具有较高的降解效率,最高石油烃降解率达(56.1±1.6)%.表明本筛选方法精度高、通量高,可用于石油烃降解功能菌系的构建.     

海洋细菌Agarivorans sp.HZ105的琼胶降解酶系

通过克隆得到菌株Agarivorans sp.HZ105中3个琼胶酶基因,长度分别为2 988 bp、1 437 bp和1 362 bp,分别编码琼胶酶HZ1、HZ3和HZ4,分别属于糖苷水解酶GH50、GH118和GH16家族。将这些琼胶酶基因与质粒p ET-32(a)构建重组表达载体,转化大肠杆菌BL21(DE3),实现了琼胶酶基因的重组原核表达,制备了重组酶,研究了琼胶酶的酶解产物。琼胶酶HZ1降解琼脂糖以及高聚合度新琼寡糖(聚合度为8、10、12和14)得到新琼二糖和新琼四糖;琼胶酶HZ3降解琼脂糖的终产物是高聚合度新琼寡糖;琼胶酶HZ4降解琼脂糖和高聚合度新琼寡糖为新琼四糖和新琼六糖。因此推测菌株HZ105主要先用琼胶酶HZ3和HZ4降解琼脂糖为较高聚合度的新琼寡糖,随后这些寡糖被琼胶酶HZ1和HZ2(课题组先前报道的另一个琼胶酶)降解为低聚合度新琼寡糖。首次研究报道了Agarivorans属中能产生4个琼胶酶的细菌菌株及其琼胶降解酶系,丰富了有关细菌降解琼胶酶体系及其中各琼胶酶作用的研究和认识,也有利于菌株HZ105琼胶酶的有效开发应用。     

木聚糖降解酶系基因代谢调控研究进展

木聚糖是半纤维素的主要组成部分,是一类数量很大的再生生物资源,工业利用前景广阔。木聚糖降解需要多种酶的参与,主要有木聚糖酶、木糖苷酶、α-葡萄糖醛酸酶、乙酰木聚糖酯酶、阿拉伯糖酶、阿魏酸酯酶、p-香豆酸酯酶等。主要综述了木聚糖降解酶系基因代谢调控的研究进展,主要包括转录激活因子XlnR、抑制蛋白CreA、不同诱导物、pH值、HAP-CCAAT复合物等对木聚糖降解酶系基因表达的影响,最后探讨了木聚糖降解酶系基因代谢调控存在的问题,并对今后的研究进行了展望。     

一株多糖降解菌的分离、鉴定与琼脂糖降解能力

【目的】筛选海洋来源的多糖降解菌,分析其多糖降解能力并初探机制。【方法】碘液染色法从海泥中初筛琼脂糖降解菌,唯一碳源生长法分析菌株的多糖利用能力,克隆16S rRNA基因以分析系统分类地位。用硫酸铵沉淀法制备胞外粗酶制剂,DNS-还原糖法测定琼胶酶活性,活性染色法分析胞外琼胶酶系的组成特征。分离、纯化琼脂糖的酶解产物,通过TLC测定寡糖Rf值、阳离子质谱测定分子量。【结果】分离到1株能液化琼脂糖的海洋细菌JZB09,鉴定至桃色杆菌属(Persicobacter)。JZB09能利用11种不同的多糖为唯一碳源生长,在利用琼脂糖、纤维素和木聚糖时生长较好。胞外粗酶制剂的琼胶酶活力约77.2U/mg,含有至少2条琼胶酶,大小约45kDa、70kDa。酶制剂降解琼脂糖后的产物是系列新琼寡糖,四糖是主产物,表明β-琼胶酶在胞外琼胶酶系降解琼脂糖时起关键作用。【结论】海洋细菌Persicobacter sp.JZB09是1株多能型多糖降解菌,可分泌β-琼胶酶降解琼脂糖且活性显著,具有潜在开发价值。     

一株海藻多糖降解菌的分离与鉴定

【目的】从腐烂的褐藻中筛选一株海藻多糖降解菌,编号L206,分析其对不同多糖的降解能力。【方法】通过形态观察、生化单因子试验及16S r RNA基因鉴定细菌,DNS法测定海藻多糖降解酶活性等。【结果】海洋细菌L206,革兰氏阴性短杆菌,生长对数期为3-21 h,适宜生长的Na Cl质量浓度为0-3%(质量体积比);通过16S r RNA基因鉴定为白色噬琼胶菌(Agarivorans albus);L206被海带粉诱导至72 h时,综合复合酶活力达到最大,其中淀粉酶活力最高(28.17 U/m L),木聚糖酶次之(23.83 U/m L)。【结论】白色噬琼胶菌L206是一株多能型多糖降解菌,对褐藻多糖有特殊的降解能力,具有潜在开发价值。     

Chimeric aspartic proteinases and active site binding

Two chimeric enzymes were constructed by exchanging domains between porcine pepsinogen and rhizopuspepsinogen in order to examine the contributions of the subsites present on different domains toward enzymatic specificity. Both chimeras exhibited the characteristic features of aspartic proteinases, such as auto-activation at low pH and abrogation of enzymatic activity by pepstatin. The activity of the chimera containing the N-terminal domain of rhizopuspepsinogen and the C-terminal domain of porcine pepsinogen (rhzNppC) could be observed by HPLC after prolonged incubation with the substrates. In contrast, the reciprocal chimera, ppNrhzC, containing the N-terminal domain of porcine pepsinogen and the C-terminal domain of rhizopuspepsinogen exhibited catalytic activity, measurable by a spectrophotometric assay. Kinetic data and inhibitor analyses strongly suggest that interdependency may exist between adjacent subsites contributed by different domains. Therefore, in order to develop an optimal substrate or inhibitor, the effect of adjacent residues of the ligand has to be examined along with the preferences for each subsite.     

Spruce budworm elastase precipitates Bacillus thuringiensis δ-endotoxin by specifically recognizing the C-terminal region

A gut juice protein from Choristoneura fumiferana (spruce budworm) larvae that precipitates certain δ-endotoxins shows a unique specificity for the C-terminal amino acid sequence. Using homolog scanning mutants, we have identified a contiguous region of the Cry1Aa toxin which interacts with the 75-kDa toxin precipitating protein (TPP-75)¹ resulting in precipitation. The contiguous region from Cry1Aa can be transferred to Cry1Ac and results in an identical precipitation reaction. The precipitation reaction occurs rapidly and is unique in that the ratio of precipitating protein to toxin is low (estimated at 0.01), unlike antibody–antigen reactions which exhibit mole ratios close to 1. TPP-75 has been characterized as an elastase-like serine protease. We have taken advantage of this serine protease character and incorporated a radiolabel using an irreversible inhibitor. The radiolabel has allowed us to show the coincidence of the catalytically-inhibited TPP-75 with the toxin in a blotting assay and to follow the degradation of TPP-75 during storage. TPP-75 represents the first evidence that gut juice proteins may selectively attenuate the activity of δ-endotoxins, prior to binding to putative receptors on susceptible cells. TPP-75 should be evaluated as a possible resistance mechanism for those larvae that do not exhibit a receptor-based resistance.     

Structural analysis of the zinc hydroxide–Thr-199–Glu-106 hydrogen-bond network in human carbonic anhydrase II

The singnificance of the zinc hydroxide–Thr-199–Glu-106 hydrogen-bond network in the active site of human carbonic anhydrase II has been examined by X-ray crystallographic analyses of site-specific mutants. Mutants with Ala-199 and Ala-106 or Gln-106 have low catalytic activities, while a mutant with Asp-106 has almost full CO₂ hydration activity. The structures of these four mutants, as well as that of the bicarbonate complex of the mutant with Ala-199, have been determined at 1.7 to 2.2 Å resolution. Removal of the γ atoms of residue 199 leads to distorted tetrahedral geometry at the zine ion, and a catalytically important zinc-bound water molecule has moved towards Glu-106. In the bicarbonate complex of the mutant with Ala-199 one oxygen atom from bicarbonate binds to zinc without displacing this water molecule. Tetrahedral coordination geometries are retained in the mutants at position 106. The mutants with Ala-106 and Gln-106 have a zinc-bound sulfate ion, whereas this sulfate site is only partially occupied in the mutant with Asp-106. The hydrogen-bond network seems to be “reversed” in the mutants with Ala-106 and Gln-106. The network is preserved as in native enzyme in the mutant with Asp-106 but the side chain of Asp-106 is more extended than that of Glu-106 in the native enzyme. These results illustrate the importance of Glu-106 and Thr-199 for controlling the precise coordination geometry of the zinc ion and its ligand preferences with results in an optimal orientation of a zine-bound hydroxide ion for an attack on the CO₂ substrate. © 1993 Wiley-Liss, Inc.     

Arg149 is involved in switching the low affinity, open state of the binding protein AfProX into its high affinity, closed state

The substrate binding protein AfProX from the Archaeoglobus fulgidus ProU ATP binding cassette transporter is highly selective for the compatible solutes glycine betaine (GB) and proline betaine, which confer thermoprotection to this hyperthermophilic archaeon. A detailed mutational analysis of the substrate binding site revealed the contribution of individual amino acids for ligand binding. Replacement of Arg149 by an Ala residue displayed the largest impact on substrate binding. The structure of a mutant AfProX protein (substitution of Tyr111 with Ala) in complex with GB was solved in the open liganded conformation to gain further insight into ligand binding. In this crystal structure, GB is bound differently compared to the GB closed liganded structure of the wild-type AfProX protein. We found that a network of amino acid side chains communicates the presence of GB toward Arg149, which increases ligand affinity and induces domain closure of AfProX. These results were corroborated by molecular dynamics studies and support the view that Arg149 finalizes the high-affinity state of the AfProX substrate binding protein.     

酶法生产L-苯丙氨酸酶转化条件研究

通过单因子和正交试验 ,对酶法生产L 苯丙氨酸的转化条件进行优化 ,在L 苯丙氨酸积累浓度不降低的情况下 ,减少转化时间 ,缩短生产周期 ,得到最适的转化条件 ,提高酶的二次利用率 ,降低成本。