新分离Microbacterium sp. XT11菌产黄原胶降解酶生产条件的优化研究

新分离Microbacteriumsp.XT11菌能够合成黄原胶降解酶,将植物病原菌野油菜黄单孢菌分泌的毒素因子黄原胶分解,生成具有激发子和抗微生物活性的黄原胶寡糖。实验确认,黄原胶和酵母浸粉分别是XT11菌生产黄原胶降解酶的最适碳源和氮源,获得最高酶活力的最低碳源和氮源浓度均为0.3%。XT11菌生产黄原胶降解酶的最适条件为:培养温度28℃,培养基起始pH7.0,转速150r/min。     

微杆菌属ZZJ4-1菌株的耐热尿酸氧化酶基因的克隆及重组酶性质

为了研究微杆菌Microbacterium sp.ZZJ4-1菌株的耐热尿酸氧化酶(Uox)的性质,克隆其基因(uox),得到1个894 bp的开放阅读框。该基因与多数已报道的uox无明显同源性,仅与球形节杆菌Arthrobacterglobiformis的uox有72%的同源性。将基因插入质粒pET-15b构成pET-15b-uox表达载体,转化至Escherichiacoli BL21(DE3)中诱导表达。对重组Uox的主要理化性质研究表明:该酶由大小约为35 kDa的亚基组成;其最佳反应温度和pH分别为30℃和7.5;在65℃以下和pH 8.5～11.0范围内稳定;以尿酸为底物的Km值为0.22 mmol/L;Ag+、Zn2+、Cu2+和SDS均能完全抑制酶活,Tween 20、Tween 80和Triton X-100对酶活有一定的促进作用。该重组酶的耐热性是目前报道的重组Uox中最好的,这一特性有利于其在诊断治疗中的开发应用。     

Thermostable phenylalanine dehydrogenase from a mesophilic Microbacterium sp. strain DM 86-1

Bacteria that produced NAD⁺-dependent phenylalanine dehydrogenase (EC 1.4.1.20) were selected among l-methionine utilizers isolated from soil. A bacterial strain showing phenylalanine dehydrogenase activity was chosen and classified in the genus Microbacterium. Phenylalanine dehydrogenase was purified from the crude extract of Microbacterium sp. strain DM 86-1 (TPU 3592) to homogeneity as judged by SDS-polyacrylamide disc gel electrophoresis. The enzyme has an isoelectric point of 5.8 and a relative molecular weight (M _r) of approximately 330,000. The enzyme is composed of eight identical subunits with an M _r of approximately 41,000. The apparent K _m values for l-phenylalanine and NAD⁺ were calculated to be 0.10 mM and 0.20 mM, respectively. No loss of the enzyme activity was observed upon incubation at 55° C for 10 min. Received: 30 July 1997 / Accepted: 4 November 1997     

新分离Microbacterium sp．XT11菌产黄原胶降解酶生产条件的优化研究

新分离Microbacterium sp．XT11菌能够合成黄原胶降解酶,将植物病原菌野油菜黄单孢菌分泌的毒素因子黄原胶分解,生成具有激发子和抗微生物活性的黄原胶寡糖。实验确认,黄原胶和酵母浸粉分别是XT11菌生产黄原胶降解酶的最适碳源和氮源,获得最高酶活力的最低碳源和氮源浓度均为0.3％。XT11菌生产黄原胶降解酶的最适条件为：培养温度28℃,培养基起始pH7.0,转速150r／min。     

一株抑制黄曲霉毒素的深海微杆菌的分离与鉴定

从南大西洋深海海水中分离到一株放线菌菌株R104,该菌株的发酵无细胞上清液对黄曲霉毒素合成的抑制率高达96.2%,经16S rDNA序列分析,初步将该菌株鉴定为微杆菌（Microbacterium）,这是首次报道深海来源的微杆菌具有抑制黄曲霉毒素合成的能力。     

Purification and Characterization of Two Types of Chitosanase from a Microbacterium sp.

Two extracellular chitosanases (ChiX and ChiN) were extracted from Microbacterium sp. OU01 with Mr values of 81 kDa (ChiX) and 30 kDa (ChiN). ChiN was optimally active at pH 6.2 and 50°C and ChiX at pH 6.6 and 60°C (assayed over 15 min). Both the activities increased with the degree of deacetylation (DDA) of chitosan. ChiN hydrolyzed oligomers of glucosamine (GlcN) larger than chitopentaose, and chitosan with 62–100% DDA; but ChiX acted on chitosan and released GlcN. Hydrolysis of chitosan with 99% DDA by ChiN released chitobiose, chitotriose and chitotetraose as the major products.     

Detoxification process of tolaasins,lipodepsipeptides, by Microbacterium sp. K3-5

Tolaasins are antimicrobial lipodepsipeptides. Here, we report the tolaasins-detoxifying properties of Microbacterium sp. K3-5 (K3-5). The detoxification of tolaasins by K3-5 was performed by hydrolyzation of cyclic structure of tolaasins depending on the tolaasin-K3-5 cell interaction. Our data suggest that the cyclic structure of tolaasins is critical for its interaction to target cells.     

Isolation and characterization of heterocysts from Anabaena sp. strain CA

A comparative study has been made on the pigment composition and nitrogenase activity of whole filaments and isolated beterocysts from a mutant strain of Anabaena CA. The whole cell absorption spectra of intact filaments and isolated heterocysts showed close resemblance especially between 550–700 nm region. On a quantitative basis the chlorophyll a content was found almost equal between the vegetative cell and heterocyst but the c-phycocyanin content in the heterocyst was about 1/2 that of the vegetative cell. The purification of the phycobiliprotein on DEAE-cellulose showed the presence of c-phycocyanin (_max 615 nm) and allophycocyanin (_max 645 nm, shoulder 620 nm). Isolated heterocysts under H₂ showed acetylene reduction rates of 57 nmol C₂H₄/mg dry wt·min (342 mol C₂H₄/mg chl a·h), whereas intact filaments reduced at the rate of 18 nmol C₂H₄/mg dry wt·min (108 mol C₂H₄/mg chl a·h). This rate accounts for 30% recovery of nitrogenase activity in isolated heterocysts compared to whole filaments. The activity was strictly light dependent and was linear under H₂ for more than 3 h. Addition of as little as 5% H₂ under argon stimulated the C₂H₂ reductionseveral fold. The acetylene reduction (nitrogenase activity) also showed tolerance to 5% added O₂ either under H₂ or argon. The results suggest that the heterocyst of Anabaena CA-V is different in some characteristics (viz., higher endogenous C₂H₂ reduction rate, prolonged activity and higher levels of phycobiliproteins) than those reported in other Anabaena species.     

一株对硝基苯胺降解菌Microbacterium sp. PNA8的分离鉴定及其降解条件研究

从污泥中分离得到一株能以对硝基苯胺为唯一碳源、氮源和能源生长的细菌菌株PNA8。经过对其形态特征、生理生化特性、以及16S rRNA序列分析, 该菌株初步鉴定为Microbacterium sp.。进一步研究表明, 菌株PNA8利用对硝基苯胺生长和降解的最适温度和pH分别是30°C和7.0。培养基中添加定量酵母膏有利于菌株的生长及其对对硝基苯胺的降解。最适条件下, 在培养液中添加0.4 g/L酵母膏, 4 d内0.3 mmol/L对硝基苯胺降解率可达100%。     

Purification and characterization of a thermostable uricase from Microbacterium sp. strain ZZJ4-1

In order to study the properties of a thermostable uricase produced by Microbacterium sp. strain ZZJ4-1, the enzyme was purified by ammonium sulfate precipitation and DEAE-cellulose ion exchange, hydrophobic and molecular sieve chromatography. The molecular mass of the purified enzyme was estimated to be 34 kDa by SDS-PAGE. The enzyme was stable between pH 7.0 and 10.00. The optimal reaction temperature of the enzyme was 30 °C at pH 8.5. The K _m and K _cat of the enzyme were 0.31 mM and 3.01 s⁻¹, respectively. Fe³⁺ could enhance the enzyme activity, whereas Ag⁺, Hg²⁺, o-phenanthroline and SDS inhibited the activity of the enzyme considerably. After purification, the enzyme was purified 19.7-fold with 31% yield. As compared with uricases from other microbial sources, the purified enzyme showed excellent thermostability and other unique characteristics. The results of this work showed that strains of Microbacterium could be candidates for the production of a thermostable uricase, which has the potential clinical application in measurement of uric acid.     

Isolation and partial characterization of cellulose-degrading strain of Streptomyces sp. LX from soil

X. LI AND P. GAO. 1996. A new bacterium, Streptomyces sp. LX, was isolated from soil, which was aerobic Gram-positive and could decompose crystalline cellulose completely. Endo-cellulase with CMC-liquefying activity was detected when α-cellulose, Avicel, Whatman CF₁₁ or CMC was used as carbon source, and its production varied with nature of the carbon source. Only traces of reducing sugar were found in cultures during incubation. This strain could produce FPase, β-glucanase and short fibre generating activity. Exo- and endo-cellulase were detected in cultures by measuring formation of total sugar but were not detected by determining release of reducing sugar.     

Microbacterium marinum sp. nov., isolated from deep-sea water

Two Gram-positive, rod-shaped bacterial strains, H101(T) and H207, were isolated from deep sea water collected from South-West Indian Ocean. Phylogenetic analysis of 16S rRNA gene sequences showed that the two strains were closely related to one another (100% similarity), and had the closest relationship with Microbacterium hominis NBRC 15708(T) and Microbacterium insulae KCTC 19247(T) (98.2-98.3% similarities). DNA-DNA hybridization value between strains H101(T) and H207 was 87.2 ± 3.7%, and the values between the two strains and the closely related type strains were well below 70%. The two strains also shared a number of physiological and biochemical characteristics that were distinct from the closely related species, and grew at 2-37 ° C, pH 5-11 and 0-8% (w/v) NaCl. Both strains contained MK-12, MK-13 and MK-11 as the detected menaquinones. The peptidoglycan was of type B1γ with an interpeptide bridge D-Glu(Hyg)→ Gly(2)→ l-Lys. The major cellular fatty acids were anteiso-C(15:0), anteiso-C(17:0), and iso-C(16:0). Based on the genetic and phenotypic properties, it is proposed that strains H101(T) and H207 be classified as representatives of a novel species of the genus Microbacterium, with the name Microbacterium marinum sp. nov. The type strain is H101(T) (= CGMCC 4.6941(T) = DSM 24947(T)).     

Chlorobenzene Degradation via Ortho-Cleavage Pathway by Newly Isolated Microbacterium sp. Strain TAS1CB from a Petrochemical-Contaminated Site

Chlorobenzene (CB), a dense nonaqeuous phase liquid (DNAPL), is categorized as a priority pollutant by the US EPA. It enters into ecosystems via solid and liquid waste discharge. Bioremediation is a key technique to remediate such contaminated sites. The present study aimed to isolate a chlorobenzene-degrading bacterium, determine the metabolic pathway for chlorobenzene degradation, and characterize biosurfactant production. Microbacterium sp. strain TAS1CB was isolated from contaminated sites and identified by 16S rRNA gene sequencing. Cells possessing positive chemotaxis for CB indicated their ability to degrade CB. Cells degraded CB via production of chlorobenzene dioxygenase, which converted CB to chlorocatechol. Chlorobenzene dioxygenase production was higher at 7 pH and 30°C. Intermediate metabolite analysis by UV scanning, HPLC, and GC-MS analysis revealed production of chlorocatechol and cis-cis muconate. Thus, Microbacterium was able to degrade CB via an ortho-cleavage pathway. In addition to chlorobenzene dioxygenase production, cells also produced biosurfactant which pseudosolubilized CB and increased degradation rate. Chemical characterization showed it to be a glycolipid-type biosurfactant. A phytotoxity study showed 60% of toxicity decreased after 72 hrs of degradation by isolate.     

Isolation and characterization of an atrazine-degrading Rhodococcus sp. strain MB-P1 from contaminated soil

Aims:  The aim of this study is to isolate and characterize organisms capable of utilizing high concentration atrazine from the contaminated sites.
Methods and Results:  A selective enrichment was used for isolating atrazine-degrading organisms from the contaminated sites resulting in isolation of an efficient atrazine-degrading organism designated as strain MB-P1. On the basis of 16S rRNA gene sequencing, total cellular fatty acid analysis and physiological and biochemical tests, strain MB-P1 was identified as a member of genus Rhodococcus . High performance liquid chromatography was performed to identify the atrazine degradation intermediates demonstrating that the degradation proceeds via formation of 'de-ethylatrazine' and 'de-isopropylatrazine'. Further, plasmid curing by SDS method showed atrazine-degrading gene(s) to be plasmid-encoded.
Conclusions:  We have successfully isolated a Rhodococcus sp. strain MB-P1 which is capable of utilizing atrazine as sole source of carbon and energy at very high concentrations of 1000 ppm. The pathway for degradation of atrazine has also been determined. The metabolic gene(s) responsible for atrazine degradation was found to be plasmid-encoded.
Significance and Impact of the Study:  Rhodococcus sp. strain MB-P1 could be used as an ideal model system for in-situ degradation and restoration of ecological niches which are heavily contaminated with atrazine.     

白蚁栖息环境中蜡状芽孢杆菌的分离及其纤维素酶活性分析

【目的】了解白蚁栖息环境中有无降解纤维素的微生物。【方法】以羧甲基纤维素钠为唯一碳源,利用刚果红染色,根据透明圈大小进行筛选。通过显微形态、革兰氏染色及16S rRNA基因序列分析对菌株进行鉴定。DNS法测定菌株产纤维素酶与生长周期的关系,并进一步分析纤维素酶性质。【结果】从台湾乳白蚁(Coptotermes formosanus Shiraki)栖息环境中筛选到一株具有较高纤维素酶活性,革兰氏阳性菌株TT15,16S rDNA序列分析鉴定为蜡状芽孢杆菌(Bacillus cereus Gd2T)。菌株培养前12 h没有纤维素酶活性,随着培养时间的增加,纤维素酶活性逐渐增大;当生长达到稳定期(48 h),酶活性达到最大并保持稳定。菌株TT15纤维素酶活性的最适pH和最适反应温度分别为5.0和50°C。【结论】从白蚁栖息环境中分离到一株具有较高纤维素酶活的蜡状芽孢杆菌TT15,可作为产细菌纤维素酶的优良菌株。     

Reduction of Cr(VI) by immobilized cells of Desulfovibrio vulgaris NCIMB 8303 and Microbacterium sp. NCIMB 13776

Hexavalent chromium, a carcinogen and mutagen, can be reduced to Cr(III) by Desulfovibrio vulgaris NCIMB 8303 and Microbacterium sp. NCIMB 13776. This study examined Cr(VI) reduction by immobilized cells of the two strains in a common solution matrix using various entrapment matrices. Chitosan and PVA-borate beads did not retain integrity and supported low or no reduction of Cr(VI) by the cells. A commercial preparation (Lentikats) was stable but also did not support Cr(VI) reduction. K-carrageenan beads were stable in batch suspensions but gel integrity was lost after only 5 h in a flow-through system in the presence of 100 microM Cr(VI). The best immobilization matrices were agar and agarose, where the initial rates of reduction of Cr(VI) (from 500 microM solution) for D. vulgaris NCIMB 8303 and Microbacterium sp. NCIMB 13776 were 127 (agar) and 130 (agarose), and 15 (agar) and 12 (agarose) nmol h(-1) mg dry cell wt(-1), respectively. The higher removal of Cr(VI) by D. vulgaris was also seen in 14-mL packed-bed flow-through columns, where, at a flow rate of 2.4 mL h(-1), the percentage removal of Cr(VI) was approximately 95% and 60% for D. vulgaris and Microbacterium sp., respectively (agar-immobilized cells). The Cr(VI) reducing activities of D. vulgaris and Microbacterium sp. were lost after 159 and 140 h, respectively. Examination of the beads for structural integrity within the columns in situ using magnetic resonance imaging after 24 and 100 h of continuous operation against Cr(VI) (with negligible Cr retained within the columns) showed that agar beads were more stable with time. The most appropriate system for development of a continuous bioprocess is thus the use of D. vulgaris NCIMB 8303 immobilized in an agar gel matrix.     

来自红海榄根际土壤的曲霉属真菌F7菌株及其生物活性分析

基于菌落和菌体的形态特征以及ITS序列分析结果,将分离自中国海南省海口市东寨港红树林保护区红海榄根际土壤的菌株F7鉴定为曲霉属黄绿组的一种真菌。在确定和优化菌株F7培养基的基础上,初步确定了适合其分泌活性代谢产物的改良查氏培养基组成:3%乳糖,1%蛋白胨,0.3%NaNO3,0.05%KCl,0.05%MgSO4·7H2O,0.001%FeSO4,0.1%K2HPO4,pH7.0。将菌株F7接种于该培养基中,28℃下160r/min振荡培养7d后收获发酵液,经乙酸乙酯和正丁醇萃取后获得了乙酸乙酯萃取物(EAE)、正丁醇萃取物(BE)以及水萃取物(WSE),其中EAE和BE对金黄色葡萄球菌、藤黄八叠球菌和枯草芽孢杆菌的生长显示出明显的抑制活性,最低抑菌浓度(MIC)介于0.625-5.0mg/mL之间。同时,上述3种萃取物对人肝癌细胞株HepG2的增殖也表现出一定的抑制活性,IC50约为120μg/mL左右。     

Molecular cloning, expression and characterization of a chitosanase from Microbacterium sp.

A gene encoding a chitosanase (mschito) was cloned from Microbacterium sp. OU01. The ORF consists of 801 bp which encoded a polypeptide of 266 amino acid residues. The deduced amino acid sequence shows 98% identity to that of the chitosanase reported in Pseudomonas sp. A-01. In addition, the fusion protein containing MSCHITO was expressed in E. coli and purified using Ni-NTA affinity chromatography. The purified rMSCHITO protein degraded the chitosan (the degree of deacetylation of 99%) and produced a mixture of chitooligosaccharides. The MSCHITO is thus an endo-chitosanase.     

Isolation and characterization of Bacillus sp. strain BC01 from soil displaying potent antagonistic activity against plant and fish pathogenic fungi and bacteria

Fungal and bacterial pathogens infect a diverse range of hosts including various plant and animal species. Fungal and bacterial diseases, especially of plants and aquatic animals, such as fish, lead to significant damage to crops and aquaculture, respectively, worldwide. The present study was conducted to isolate and characterize potent Bacillus strains with significant antagonistic activity against the major plant and fish pathogenic fungi and bacteria. We randomly collected 22 isolates of Bacillus from the soil, rhizosphere, and sediment from different parts of Bangladesh. Initial characterization, based on in vitro antagonistic activity on the culture plate, resulted in the selection of four gram-positive Bacillus sp. isolates. Among these, the isolate BC01, obtained from soil demonstrated the highest broad-spectrum anti-bacterial and anti-fungal activities. We confirmed the genus of BC01 to be Bacillus by morphological and biochemical tests as well as using molecular data analysis tools, including the study of 16s rDNA, phylogenetic relationship, and evolutionary divergence scores. The isolate significantly inhibited the mycelial growth of the plant pathogen, Penicillium digitatum and fish pathogen, Aphanomyces invadans in vitro. The anti-bacterial effect of the isolate was also evaluated against Pseudomonas spp. and Xanthomonas spp., the two deadliest plant pathogens, and Aeromonas veronii, Pseudomonas fluorescens, and Streptococcus iniae, three major fish pathogens that are primarily responsible for global aquaculture loss. The results of the present study could pave the way for developing potent drugs to combat microbial infection of plants and fish.