Identification and optimal degradation conditions for cellulase-degrading enzyme of a methanol-utilizing and cellulase-producing bacterium

采用刚果红染色法,从废弃矿山周边土壤中筛选出一株产纤维素酶的甲醇利用细菌,命名为xt - 04.形态特征、生理试验及16S rDNA序列和gyrB序列分析表明,该菌株属于Bacillus methylotrophicus.为提高该菌所产纤维素酶的降解能力,首先通过单因子实验考察了底物CMC -Na浓度、反应温度及缓冲液pH值对纤维素酶活力的影响;然后采用响应面分析法对影响纤维素酶活力的3个单因子进行了优化.结果表明,单因素实验得出的适宜反应温度、缓冲液pH和底物浓度分别为70℃、5.0和2％ (20 mg/mL);响应面法得出的最高酶活力条件:反应温度、pH和底物浓度分别为66.1℃、4.81和19.01mg/mL.在最优条件下,酶活力达到17.85 U/mL,比优化前的酶活力12.84 U/mL提高了39.01％.因此,鉴于这种纤维素酶能耐受较高温度和酸性条件,该菌株所产纤维素酶可能在工业中具有良好的应用前景.     

产纤维素酶菌株的筛选、鉴定及产酶条件的优化

采集新疆吐鲁番地区土样,从中分离筛选1株产纤维素酶菌株C-8;经形态观察、生理生化及16S rRNA序列分析,初步鉴定为芽孢杆菌属(Bacillus sp.)。该菌株所产纤维素酶的最适合作用pH和温度分别为9.0、40℃,且具有良好的pH稳定性和温度稳定性。为了提高C-8菌株产纤维素酶能力,利用响应面法对其发酵产酶条件进行优化。采用Plackett-Burman设计筛选出影响其产酶条件的3个主效应因素,最陡爬坡试验逼近最大响应值区域,利用Box-Behnken响应面分析法优化发酵产酶条件。结果表明,起始pH、CMC-Na%和培养温度为主要影响因素。通过三因素三水平的响应面分析得到最优条件为起始pH8.0、CMCNa%2.5%、培养温度28℃。在此条件下纤维素酶活可达193.89 U/mL,与优化前相比,酶活提高2.35倍。     

一株产纤维素酶的甲醇利用细菌的鉴定及其纤维素降解条件优化

采用刚果红染色法,从废弃矿山周边土壤中筛选出一株产纤维素酶的甲醇利用细菌,命名为xt-04。形态特征、生理试验及16SrDNA序列和gyrB序列分析表明,该菌株属于Bacillusmethylotrophicus。为提高该菌所产纤维素酶的降解能力,首先通过单因子实验考察了底物CMC—Na浓度、反应温度及缓冲液pH值对纤维素酶活力的影响;然后采用响应面分析法对影响纤维素酶活力的3个单因子进行了优化。结果表明,单因素实验得出的适宜反应温度、缓冲液pH和底物浓度分别为70℃、5．0和2％（20mg／mL）;响应面法得出的最高酶活力条件：反应温度、pH和底物浓度分别为66．1℃、4．81和19．01mg／mL。在最优条件下,酶活力达到17．85U／mL,比优化前的酶活力12．84U／mL提高了39．01％。因此,鉴于这种纤维素酶能耐受较高温度和酸性条件,该菌株所产纤维素酶可能在工业中具有良好的应用前景。     

响应面法优化灰色链霉菌产纤维素酶的工艺研究

以灰色链霉菌为原料,在单因素试验的基础上,采用响应面法试验,优化灰色链霉菌产纤维素酶活性的发酵条件。结果表明,单因素试验灰色链霉菌产纤维素酶活性的最适发酵条件:碳源为CMC-Na,氮源为明胶,温度为28℃,pH为7.0,转速为130 r/min。响应面法试验优化灰色链霉菌产纤维素酶活性最佳发酵条件为:温度27.7℃,pH值6.9,转数130.3 r/min,在此优化条件下,灰色链霉菌产纤维素平均酶活性为6.103 U/mL(n=3),与模型的预测值(6.217 U/mL)比较接近,误差为1.83%,证明了该响应面模型具有可靠性。     

超声法提取虎杖色素及其稳定性的研究

用乙醇作为提取溶剂,以提取得率和色价为评价指标,通过单因素实验,探讨料液比、提取温度、超声功率、提取时间、乙醇体积分数对色素提取效果的影响;通过正交实验优化提取条件,并对提取的色素进行了稳定性考察。实验结果表明:虎杖色素的最佳提取工艺条件为乙醇体积分数80%,料液比1∶30,超声温度25℃,超声功率50 W,超声时间20 min,其中料液比为显著影响因素;虎杖色素对热、光、蔗糖、NaCl、维生素C(Vc)等因素的影响较稳定,而pH、H2O2、柠檬酸对虎杖色素稳定性影响较大。     

爱尔兰帚霉产低温纤维素酶的酶学性质和发酵工艺

本文旨在对爱尔兰帚霉E71702M菌株产低温纤维素酶的酶学性质进行初步研究,并获得其最优发酵条件,为低温纤维素酶开发利用提供参考。通过单因素试验确定温度、pH及金属离子对纤维素酶活力的影响;以Plackett-Burman试验设计从8个培养条件中筛选出影响产酶的3个主要因素,即麸皮含量、装瓶量和起始pH;运用Box-Behnken试验设计及响应面分析法确定这3个主要因素的最优发酵条件。对酶学性质的初步研究表明,该酶最适pH为3.0,最适反应温度为30℃,在0℃时残余酶活力为最适反应温度时的56.3%。响应面分析得到最优发酵条件为:麸皮含量8.79g/L、装瓶量40.93m L、初始pH 4.01。通过优化,纤维素酶活力由0.6338IU/m L提高到1.7386IU/m L,提高了174%。     

烟曲霉A-16产纤维素酶工艺优化及酶学特性北大核心CSCD

分离筛选高效降解稻草的菌株,研究菌株产纤维素酶工艺条件及酶学性质。采用刚果红染色法从腐败木质下的土壤中分离筛选到一株产纤维素酶菌株,结合菌株的形态特征和18S rDNA序列同源性比较进行鉴定;通过单因素试验和响应面分析法确定菌株最适产酶条件,并对纤维素酶的稳定性进行研究。分离纯化得到的菌株命名为烟曲霉(Aspergillus fumigatus A-16);响应面实验结果表明,最优产纤维素酶工艺参数为:稻草粉添加量7 g/100 mL,pH 6.0,温度65℃,发酵时间5 d;在此最优条件下,该菌产生的羧甲基纤维素酶(CMCase)和滤纸酶(FPA)活力分别为2 954.76 U/mL和1 086.37 U/mL;其总活力较优化前提高了26.4%。该纤维素酶的适宜反应温度为70℃,适宜pH 6.0。在80℃热处理90 min条件下酶活力可保持在80%以上,说明该酶热稳定性较好。同时,在pH 5.0-7.0范围内比较稳定,放置1 d后可保持70%以上的酶活力。该研究可为利用富含纤维素的生物质原料开发洁净能源及食品级葡萄糖资源提供了基础支撑。     

1株褐藻胶降解菌的筛选、鉴定及产酶条件优化

为获得可产生褐藻胶裂解酶并高效降解褐藻胶的菌株,以海藻酸钠为唯一碳源配制培养基,以透明圈法进行初筛,DNS法复筛,从海洋生物中筛选得到1株高酶活力褐藻胶降解菌株B12,经16S rDNA序列分析、生理生化试验、电镜观察,确定该菌为弧菌属(Vibrio sp.)。通过单因素试验及响应面优化试验对影响菌株生长和产酶条件的5个因素(发酵初始pH值、发酵温度、NaCl质量浓度、接种量和装液量)进行优化。得到该菌株最佳产酶条件:pH 6.52,发酵温度28.2℃,NaCl质量浓度20.1 g/L,接种量2.1%,装液量59.5 mL。在最佳发酵条件下,B12菌株酶活力可达91.68 U/mL,相比于优化前提高了38.5%。菌株开始产酶时间提前6 h, 4℃冷藏酶活力稳定性较好。     

响应面法优化黑土豆色素提取工艺研究

以黑土豆为原料提取色素,在乙醇浓度、回流温度、回流时间和料液比四个单因素实验基础上,通过响应面法优化提取条件,利用 Box-Benhnken 设计模型,研究四个自变量之间的影响.结果表明,得到最佳提取工艺为回流温度47℃,回流时间47．5 min,料液比1∶90,乙醇浓度42．5％.在此条件下,理论上测得黑土豆中色素的吸光度值为0．413,实验测得黑土豆中色素的吸光度值为0．416,理论值与实验值的相对误差为0．726％.该提取工艺简便、具有较高的重现性和可行性.     

交替假单胞菌LP621菌株产右旋糖苷酶的培养条件优化

从江苏连云港海域分离和筛选到1株产右旋糖苷酶的海洋细菌交替假单胞菌Pseudoalteromonas tetraodonis LP621,通过单因素试验和正交试验对该菌株产右旋糖苷酶培养条件进行优化。单因素试验结果表明,最佳培养时间为24 h,最适产酶温度为25℃;产酶pH范围为5.0~11.0,最适产酶pH为6.0;产酶NaCl浓度范围为1%~10%,NaCl浓度为4%时产酶较高;装液量在25%。麦芽糖、胰蛋白胨和酵母膏促进产酶。利用响应面方法对LP621产右旋糖苷酶的发酵条件进行优化。选择培养基pH、时间、麦芽糖浓度和装液量4因素进行优化,结果为pH 7.07,发酵时间21.94 h,麦芽糖浓度0.42%,装液量为21.88%,酶活为270.1U/mL。     

Isolation and Characterization of Carotenoid Pigments of Micrococcus roseus

In addition to canthaxanthin, seven pigment fractions were isolated from Micrococcus roseus. They were purified by solvent partitioning and by column and thin-layer chromatography. Visible absorption spectra, chromatographic behavior, and partition coefficients of the pigments and derivatives prepared from the pigments were used in characterizing them. Both alpha- and beta-carotene derivatives were present. The structure of one pigment was suggested as phoenicoxanthin (3-hydroxy-4,4'-diketo-beta-carotene). Four other pigments were tentatively characterized as a dihydroxy-3,4-dehydro-alpha-carotene, a dihydroxy-alpha-carotene, a diketo-alpha-carotene, and a polyhydroxy-beta-carotene. Two pigments were isolated in trace amounts and could not be characterized. All the pigments studied were isolated as mixtures of cis-trans isomers and all except the diketo-alpha-carotene were isolated as esters from M. roseus. Quantitation of the pigments showed that canthaxanthin (4,4'-diketo-beta-carotene) represented 85% of the pigment recovered from extracts. Three of the other pigments contributed a significant proportion of the remaining pigments, whereas the other four were present in only small amounts. beta-Carotene derivatives comprised 96% and alpha-carotene derivatives 4% of the pigments recovered from extracts.     

Isolation and Identification of Echinenone from Micrococcus roseus

An orange carotenoid from Micrococcus roseus was purified by solvent partitioning followed by column and thin-layer chromatography. Absorption spectra, chromatographic mobility, and partition coefficient suggested that the pigment was echinenone (4-keto-beta-carotene). Reduction yielded a pigment with the spectral and polar properties of isocryptoxanthin (4-hydroxy-beta-carotene), the expected product. The orange pigment and its reduction product co-chromatographed with the respective authentic pigments, confirming the original pigment as echinenone. To our knowledge echinenone has not been identified previously as a bacterial pigment.     

Isolation and Identification of Canthaxanthin from Micrococcus roseus

Cooney, J. J. (University of Dayton, Dayton, Ohio), H. W. Marks, Jr., and Anne M. Smith. Isolation and identification of canthaxanthin from Micrococcus roseus. J. Bacteriol. 92:342-345. 1966.-The principal colored carotenoid of Micrococcus roseus was purified by solvent partitioning followed by column and thin-layer chromatography. Absorption spectra, partition coefficients, and infrared spectra suggested that the pigment was a diketo derivative of beta-carotene. The pigment was subjected to reduction, and the reduced pigment was subsequently dehydrated. Spectral data and partition coefficients of these derivatives indicated that the original pigment was canthaxanthin (4',4'-diketo-beta-carotene). The pigment was an all-trans isomer; it does not exist as an ester in M. roseus. Canthaxanthin has not previously been identified as a bacterial pigment.     

红龙草红色素稳定性的研究

分析了pH值、温度、光、过氰化氢、亚硫酸钠、Vc、葡萄糖、蔗糖和苯甲酸钠等对红龙草（Altemanthera dentate ‘Ruliginosa’）红色素稳定性的影响。结果表明,红龙草红色素对热的耐受性较强,但耐光照和耐氧化性较差,且还原剂亚硫酸钠对其也有微弱的影响;在不同的pH值条件下,其吸收峰没有改变,最大吸收波长为530nm;Vc和蔗糖对该色素没有破坏作用,并有一定的护色效果;葡萄糖和苯甲酸钠对该色素也无明显影响。     

紫红丝膜菌子实体色素的提取及其稳定性研究

采用单因素和正交试验,研究紫红丝膜菌子实体色素的提取条件及其稳定性。色素最佳提取条件为以80%乙醇60℃下提取2h,影响程度为乙醇体积分数>浸提时间>浸提温度。色素对自然光、紫外光不稳定;酸性条件下为紫红色,碱性为堇紫色;对Fe3+不稳定;对氧化剂不稳定,对还原剂稳定;对常用食品添加剂相对稳定;温度的变化对色素影响甚微。     

The major carotenoid pigment of a psychrotrophic Micrococcus roseus strain: purification, structure, and interaction with synthetic membranes.

The major carotenoid pigment of a psychrotrophic Micrococcus roseus strain was purified to homogeneity from methanol extracts of dried cells by reverse-phase liquid chromatography and was designated P-3. On the basis of the UV-visible, infrared, mass, and 1H nuclear magnetic resonance spectra of P-3, it was identified as bisdehydro-beta-carotene-2-carboxylic acid. The pigment interacted with synthetic membranes of phosphatidylcholine and dimyristoyl phosphatidylcholine and stabilized the membranes. These results also indicate that P-3 is different from canthaxanthin, the major carotenoid pigment from a mesophilic M. roseus strain.     

大肠杆菌异源表达的indigoidine与靛蓝的稳定性比较

【背景】Indigoidine是一种来源于微生物的无毒天然蓝色素。【目的】比较Indigoidine和靛蓝的色素稳定性,进而评价Indigoidine的色素稳定性。【方法】构建重组菌株Escherichia coli DH5α/p28s异源表达Indigoidine,考察可见光、紫外线、pH、温度、氧化还原剂、食品添加剂和金属离子对其与商品级靛蓝色素稳定性的影响。【结果】以N,N-二甲基甲酰胺为溶剂,Indigoidine和靛蓝都对可见光、紫外线敏感;2种色素在pH1.0-11.0时稳定,强碱性pH对色素破坏作用很大;Indigoidine抗Vc还原能力强于靛蓝,氧化剂可不同程度地降低2种色素的保存率;2种色素热稳定性不佳,在75℃以下时Indigoidine的色素稳定性优于靛蓝;食品添加剂中的柠檬酸和苯甲酸分别对Indigoidine和靛蓝均具有显著的护色效果;对这2种色素,Ca²⁺、Mg²⁺均具有护色效果,Na⁺、K⁺、Li⁺总体上没有明显的破坏作用,而Zn²⁺、Al³⁺、Cu²⁺、Fe²⁺、Fe³⁺则具有显著的破坏作用。【结论】Indigoidine色素稳定性明显优于靛蓝,具有广阔的开发应用前景。     

Effect of pH and nitrogen source on pigment production by Monascus purpureus

The effect of pH and nitrogen source on pigment production by Monascus purpureus 192F using glucose as the carbon and energy source, was studied in pH-controlled, batch fermentor cultures using HPLC analysis to determine individual pigment concentrations. A maximum of four pigments were detected in fungal extracts. These were the yellow pigments monascin and ankaflavin, the orange rubropunctatin and the red pigment monascorubramine. Monascorubramine was present as the major product in all instances. Fungal growth and ankaflavin synthesis were favoured at low pH (pH 4.0), whereas production of the other pigments was relatively independent of pH. The nature of the nitrogen source affected fungal growth and pigment production, independent of pH. Ammonium and peptone as nitrogen sources gave superior growth and pigment concentrations compared to nitrate. Ankaflavin was not detected in nitrate cultures. The highest red pigment production was obtained using a glucose-peptone medium at pH 6.5, due to the secretion of red pigments into the medium under these conditions. Correspondence to: M. R. Johns     

Spectral tuning of deep red cone pigments

Visual pigments are G-protein-coupled receptors that provide a critical interface between organisms and their external environment. Natural selection has generated vertebrate pigments that absorb light from the far-UV (360 nm) to the deep red (630 nm) while using a single chromophore, in either the A1 (11- cis-retinal) or A2 (11- cis-3,4-dehydroretinal) form. The fact that a single chromophore can be manipulated to have an absorption maximum across such an extended spectral region is remarkable. The mechanisms of wavelength regulation remain to be fully revealed, and one of the least well-understood mechanisms is that associated with the deep red pigments. We investigate theoretically the hypothesis that deep red cone pigments select a 6- s- trans conformation of the retinal chromophore ring geometry. This conformation is in contrast to the 6- s- cis ring geometry observed in rhodopsin and, through model chromophore studies, the vast majority of visual pigments. Nomographic spectral analysis of 294 A1 and A2 cone pigment literature absorption maxima indicates that the selection of a 6- s- trans geometry red shifts M/LWS A1 pigments by approximately 1500 cm (-1) ( approximately 50 nm) and A2 pigments by approximately 2700 cm (-1) ( approximately 100 nm). The homology models of seven cone pigments indicate that the deep red cone pigments select 6- s- trans chromophore conformations primarily via electrostatic steering. Our results reveal that the generation of a 6- s- trans conformation not only achieves a significant red shift but also provides enhanced stability of the chromophore within the deep red cone pigment binding sites.     

Plackett-Burman Design与均匀设计法优化玫瑰色微球菌固定化脱氮的性能

【目的】采用Plackett-Burman Design (PBD)与均匀设计(Uniform Design, UD) 优化玫瑰色微球菌(Micrococcus roseus)固定化脱氮的条件,以提高其脱氮性能。【方法】选择聚乙烯醇(Polyvinyl alcohol,PVA)与海藻酸钠(Sodium alginate,SA)作为固定化细胞材料,研究其成球方式与性能,应用PBD对9个影响脱氮效果的因素进行显著性检验,筛选出PVA、SA、温度、菌液接种量、菌球量5个显著性因素(P<0.05)。采用UD对5个显著因素进行条件优化,优化数据通过Minitab与Matlab软件进行逐步回归分析,并建立二阶多项式模型。【结果】最优脱氮条件为PVA 9.6%、SA 2.2%、温度33.4 °C、菌液接种量8%、菌球量500个/100 mL,连续脱氮72 h,最高脱氮率达到60.9%。【结论】PBD与UD的联合使用能够在显著因子筛选与寻优中发挥重要的作用。连续脱氮试验表明,玫瑰色微球菌去除氨氮主要通过同化、硝化与反硝化途径。