一株纤维素降解细菌的筛选、鉴定及产酶条件分析

目的筛选高活性的纤维素降解细菌,并进行初步鉴定和产纤维素酶条件分析。方法采集吉首旗帜山松树林的土壤样品,通过富集培养和刚果红平板染色法筛选分离纤维素降解细菌;通过形态观察、生理生化特性检测和基于16S rRNA基因序列的系统发育分析对分离的菌株进行初步鉴定。利用单因素实验对产纤维素酶条件进行优化。结果分离获得1株高活性纤维素降解细菌JDM11,初步鉴定其为Bacillus velezensis;菌株JMD11产纤维素酶最佳培养温度、最适初始pH和培养时间分别为28℃、7.0～7.5和32h,在该条件下其滤纸酶(FPase)和羧甲基纤维素酶(CMCase)活力分别为260.32U/ml和651.75U/ml。结论菌株JDM11是1株高活性纤维素降解的Bacillus velezensis。     

Construction of a promoter-probe vector for the methanol-utilizing bacterium acetobacter methanolicus MB 58

We report here the construction of a promoter-probe vector, pRS2, which can be utilized in either Acetobacter methanolicus MB 58 or Escherichia coli due to the presence of broad-host-range replicon RSF 1010. The vector provides several unique restriction sites for promoter cloning as well as resistance markers for the selection of transformants. The promoter-probe vector was constructed by inserting an EcoRI-SalI-polylinker fragment of pUC 19 into EcoRI/SalI digested pMK 16. The resulting plasmid, pRS1, was cloned into the unique EcoRI site of the broad-host-range plasmid RSF 1010. The vector was used to clone promoter-containing sequences derived from the A. methanolicus MB 58 chromosome as well as the E. coli lac-promoter.     

The growth dynamics of a methanol-utilizing bacterium L3 in a batch bioreactor

The growth dynamics of a methanol utilizing bacterium, L3, in batch bioreactors were experimentally investigated. Formaldehyde, a key intermediate of methanol oxidation, is indicated to have a significant role in the complex batch growth behavior of L3. The intricate batch growth dynamics of many microorganisms can be elegantly characterized by examining the specific rates of exchange of nutrients and products between the cells and the cellular environment. Following such an analysis, the batch growth of L3 on methanol was characterized by the presence of unbalanced and balanced growth phases. The nature and significance of nutrient and product concentration profiles or semilog-arithmic profiles of nutrient and product exchange rates during balanced and unbalanced growth phases are also outlined.     

Isolation of a homocysteine gamma-lyase-producing bacterium and study of its enzyme production conditions

Aim: To investigate the possibility of finding a new homocysteine (Hcy) γ‐lyase with the desired properties for Hcy measurement in bacteria. Methods and Results: Through a process of enrichment, the Hcy γ‐lyase‐producing bacterium strain N2‐1 was isolated from soil. Based upon its morphological, physiological, and biochemical characteristics, as well as its 16S rDNA sequence and phylogenetic tree analysis, this isolate belongs to the genus Serratia. The effects of pH, aeration, inducers, carbon (C) and nitrogen (N) sources on enzyme production were studied. Methionine, yeast extract, and glucose were selected as the optimal inducer, C and N sources, respectively. Maximum production of Hcy γ‐lyase was obtained when the isolate was cultured at 30°C at pH 6·5 for about 36 h in the optimum medium. Results also showed that this Hcy γ‐lyase has relatively high specificity towards Hcy. Conclusions: Because of its high specificity for Hcy, this bacterial Hcy γ‐lyase has the potential application in Hcy determination. Significance and Impact of the Study: In addition to isolating a bacterium that produces Hcy γ‐lyase suitable for Hcy determination, this study also indicates that the bacterium could be a source for production of Hcy γ‐lyase for clinical applications.     

Cloning of formate dehydrogenase gene from a methanol-utilizing bacterium Mycobacterium vaccae N10

The gene of NAD⁺-dependent formate dehydrogenase (FDH) from Mycobacterium vaccae N10 was cloned into Escherichia coli by hybridization with digoxigenin-labeled DNA probes, which were prepared by amplification of the chromosomal DNA from the bacterium by the polymerase chain reaction with degenerate primers. The primers were designed on the basis of the most conserved parts of known sequences of FDH from different organisms. An open-reading frame of 1200 bp exhibited extremely high sequence similarity to the FDH gene of Pseudomonas sp. 101. The deduced amino acid sequence of FDH from Mycobacterium vaccae N10 (McFDH) was identical to that of Pseudomonas sp. 101 (PsFDH) except for two amino acid residues: isoleucine-35 (threonine in PsFDH) and glutamate-61 (lysine in PsFDH). The physicochemical properties of both enzymes appeared to be closely similar to each other, but the thermostability of McFDH was a little lower than that of PsFDH. To examine the role of the two amino acid residues in the thermostability of the enzymes, glutamate-61 of McFDH was replaced by glutaminyl, prolyl and lysyl residues by site-directed mutagenesis. All the mutant enzymes showed higher thermostability than the wild-type McFDH. The negative charge of glutamate-61 contributes to the stability of the wild-type enzyme being lower than that of PsFDH.     

Isolation of a thermostable uricase-producing bacterium and study on its enzyme production conditions

A uricase-producing bacterium was isolated from soil with a medium containing uric acid as the only carbon source. Based on its morphological and physiological characteristics, as well as 16S rDNA sequence and phylogenetic tree analysis, this new isolate belong to the genus Microbacterium. After heat treatment at 70 °C for 30 min, the uricase retained about 100% of the initial activity. The enzyme activity remained largely unchanged when it was stored in borate buffer at pH 8.5 at 37 °C for 40 days. The effects of different factors on the enzyme production were studied. Maize milk was the best C and N resources, and the uric acid showed to be an inducer for uricase production. When the strain was cultured at 30 °C at pH 7.5 for 30–36 h, the uricase activity peaked at 1.0 U/ml.     

纤维素酶产生菌及其发酵条件优化

通过刚果红染色产脱色圈初筛出能分解纤维素的菌株,再利用DNS法分别测定纤维素酶的酶活,得到分解纤维素能力最强的一株真菌Lv-1。该菌株菌丝为黄绿色,孢子为深绿色,有多分支的无隔菌丝,长孢子囊孢子,用DNS法测定其酶活为62.42 U/m L,后对其进行了产酶条件优化。经单因素试验和正交试验得到该菌的最佳产酶条件为:在以10 g/L羧甲基纤维素钠为碳源,4 g/L蛋白质,2 g/L硫酸铵为氮源,1 g/L硫酸二氢钾为无机盐的最适产酶培养基中,初始p H为6.0,培养温度37℃,装液量100 m L/250 m L,发酵36 h。在此发酵条件下,其产酶活力可达96.898U/m L,试验结果显示,该菌株在产纤维素酶能力上具有显著优势,且菌株Lv-1产酶活力较优化前高出34.478 U/m L,提高了55.24%。     

Methylobacterium soli sp. nov. a methanol-utilizing bacterium isolated from the forest soil

A Gram-negative, pink-pigmented, non-spore-forming rod shaped, methanol-utilizing bacterium, strain YIM 48816(T), was isolated from forest soil collected from Sichuan province, China. Strain YIM 48816(T) can grow at 4-37 °C, pH 5.0-7.0 and 0% NaCl (w/v). Based on 16S rRNA gene sequence similarity studies, it belonged to the genus Methylobacterium, and formed a phyletic line. The 16S rRNA gene sequence similarities were 96.2% to Methylobacterium mesophilicum DSM 1708(T) and 96.0% to Methylobacterium brachiatum DSM 19569(T), and the phylogenetic similarities to all other Methylobacterium species with validly published names were less than 96.0%. The major menaquinones detected were Q-10 (97.14%) and Q-9 (2.86%). The major fatty acids were C18:1 ω7c (80.84%). The DNA G + C content was 66.2 mol%. It is apparent from the genotypic and phenotypic data that strain YIM 48816(T) belongs to a novel species of the genus Methylobacterium, for which the name Methylobacterium soli sp. nov. is proposed. The type strain is YIM 48816(T) (CCTCC AA 208027(T) = KCTC 22810(T)).     

A study of methanol transport/uptake by a methanol-utilizing bacterium L3 in a batch bioreactor

Two novel, nitrogen-limited and oxygen-limited, perturbed transient experiments were performed to examine the presence of active transport of methanol and study the effect on methanol uptake by a methanol-utilizing bacterium, L3, in a batch bioreactor. Transient limitations of both ammonium ions and O(2) in batch cultures were found to cause methanol leakages out of the cells, suggesting the presence of an active transport of methanol in L3. Such experimental results were used to indirectly estimate the intracellular levels of methanol during a batch growth of L3. The results of our analysis indicate that the intracellular methanol level is high and show an increasing trend during the unbalanced phase, but falls to a constant low level in the balanced phase of a typical batch growth(8) of L3. A simple modelling analysis suggests that a four-parameter "pump-and-extrusion" model could be used to adequately describe the transport of methanol between the extra cellular and intracellular phases of batch cultures of L3.     

Anaerobic degradation of toluene by a denitrifying bacterium

A denitrifying bacterium, designated strain T1, that grew with toluene as the sole source of carbon under anaerobic conditions was isolated. The type of agar used in solid media and the toxicity of toluene were determinative factors in the successful isolation of strain T1. Greater than 50% of the toluene carbon was oxidized to CO2, and 29% was assimilated into biomass. The oxidation of toluene to CO2 was stoichiometrically coupled to nitrate reduction and denitrification. Strain T1 was tolerant of and grew on 3 mM toluene after a lag phase. The rate of toluene degradation was 1.8 mumol min-1 liter-1 (56 nmol min-1 mg of protein-1) in a cell suspension. Strain T1 was distinct from other bacteria that oxidize toluene anaerobically, but it may utilize a similar biochemical pathway of oxidation. In addition, o-xylene was transformed to a metabolite in the presence of toluene but did not serve as the sole source of carbon for growth of strain T1. This transformation was dependent on the degradation of toluene.     

Fermentative degradation of triethanolamine by a homoacetogenic bacterium

With triethanolamine as sole source of energy and organic carbon, a strictly anaerobic, gram-positive, rod-shaped bacterium, strain LuTria 3, was isolated from sewage sludge and was assigned to the genus Acetobacterium on the basis of morphological and physiological properties. The G+C content of the DNA was 34.9±1.0 mol %. The new isolate fermented triethanolamine to acetate and ammonia. In cell-free extracts, a triethanolamine-degrading enzyme activity was detected that formed acetaldehyde as reaction product. Triethanolamine cleavage was stimulated 30-fold by added adenosylcobalamin (co-enzyme B₁₂) and inhibited by cyanocobalamin or hydroxocobalamin. Ethanolamine ammonia lyase, acetaldehyde:acceptor oxidoreductase, phosphate acetyltransferase, acetate kinase, and carbon monoxide dehydrogenase were measured in cell-free extracts of this strain. Our results establish that triethanolamine is degraded by a corrinoid-dependent shifting of the terminal hydroxyl group to the subterminal carbon atom, analogous to a diol dehydratase reaction, to form an unstable intermediate that releases acetaldehyde. No anaerobic degradation of triethylamine was observed in similar enrichment assays.Abbreviation NTA nitrilotriacetate     

Growth of a methanol-utilizing yeast

A halophilic thermotolerant yeast species, identified as Hansenula polymorpha Morais et Maia, was isolated from a mixed culture obtained from sea-water from the Arabian Gulf. The species grew on methanol at 25–42°C, pH 3.5–6.7, and in a medium compounded with 75% sea-water. Either thiamin HCl and biotin or yeast extract proved essential for growth. In shake flask studies a depression of the yield was observed when methanol concentration increased; at concentrations in excess of 0.1%, v/v, inhibition of growth also occurred. In a batch culture grown in a 14 l fermenter, the values of T_d, μ and Y_s were found to be 3 h, 0.23 h⁻¹ and 0.38, respectively.     

Identification and characterization of a pigment-producing denitrifying bacterium

Herein, a denitrifying bacterium that produced greenish fluorescent pigment under aerobic conditions was accidentally isolated from municipal sewage sludge. Using 16S-rDNA sequence analysis, we identified the isolate as Pseudomonas aeruginosa R12, with 100% similarity. We achieved the highest pigment production rate (1.36 mg/L/h) in a 1-L bioreactor under aerobic conditions, using the optimal culture parameters determined in this study: 37°C, pH 8.0, 200 rpm, 5 wm aeration, and medium containing succinate and (NH₄)₂SO₄. The pigment was not a secondary metabolite and had no antibacterial activity on its co-isolates. Under anaerobic conditions, the isolate produced mainly N₂ and behaved as a strong denitrifier, displaying synergistic denitrification with co-isolated denitrifiers. To our knowledge, herein we have described the first instance in which P. aeruginosa R12 produces a fluorescent pigment under aerobic conditions. This newly-isolated strain therefore shows potential as a commercial resource for natural pigment.     

Improved purification of 12-lipoxygenase from rat basophilic leukemia cells and conditions for optimal enzyme activity

12-Lipoxygenase from rat basophilic leukemia cells was purified about 300-fold by protein-HPLC in a single run. Maximal 12-lipoxygenase activity was observed at pH 7.5, while the enzyme became almost inactive at pH 6 and 9. Although Ca2+ was not essential for 12-lipoxygenase activity, the partially purified enzyme was stimulated approx. 2-fold in the presence of 0.1-5.0 mM Ca2+. Contrary to 5-lipoxygenase from RBL-1 cells, 12-lipoxygenase was not inactivated by preincubation with Ca2+ for 1-10 min, nor was it stimulated by 0.1-10 mM ATP.     

Taxonomical examination and characterization of a methanol-utilizing yeast

The morphological, cultural, and physiological characteristics are described of a yeast, LI70, which uses methanol as its source of energy and carbon; these characteristics have made it possible to identify the strain as Candida boidinii Ramirez. The identification was confirmed by a DNA-DNA genetic homology of 99.43% with the type strain of C. boidinii. Strain LI70 is not pathogenic.     

Anaerobic degradation of toluene by a denitrifying bacterium.

A denitrifying bacterium, designated strain T1, that grew with toluene as the sole source of carbon under anaerobic conditions was isolated. The type of agar used in solid media and the toxicity of toluene were determinative factors in the successful isolation of strain T1. Greater than 50% of the toluene carbon was oxidized to CO2, and 29% was assimilated into biomass. The oxidation of toluene to CO2 was stoichiometrically coupled to nitrate reduction and denitrification. Strain T1 was tolerant of and grew on 3 mM toluene after a lag phase. The rate of toluene degradation was 1.8 mumol min-1 liter-1 (56 nmol min-1 mg of protein-1) in a cell suspension. Strain T1 was distinct from other bacteria that oxidize toluene anaerobically, but it may utilize a similar biochemical pathway of oxidation. In addition, o-xylene was transformed to a metabolite in the presence of toluene but did not serve as the sole source of carbon for growth of strain T1. This transformation was dependent on the degradation of toluene.