新分离菌Streptomyces产小分子CMC液化酶的研究

新分离纤维素分解菌经鉴定为链霉菌属中的新种,暂定名为ＳｔｒｅｐｔｏｍｙｃｅｓｓｐＬＸ,革兰氏阳性,产气生孢子,好氧生长,最适生长温度和ＰＨ为３０℃和７．２。该菌能够完全降解纤维素且不产生还原糖,并分泌一种分子量为９．２ｋｕ的液化性质的内切纤维素液化酶,亦即ＣＭＣ液化酶,该酶在裂解滤纸为短纤维的过程中既没有还原糖生成,也没有失重现象发生,而且纤维素的聚合度也几乎没有明显变化,推测可能是一种能打开氢键的小分子解链酶。     

芽孢杆菌E2菌株纤维素酶性质的研究

芽孢杆菌Ｅ２菌在５５℃下生长良好,在培养液中能大量积累胞外纤维素酶（１９０ｍｕ／ｍＬ培养液）,所产生的纤维素普单一的羧甲基纤维素酶（ＣＭＣａｓｅ）。羧甲基纤维素钠（ＣＭＣＮａ）为其专一性底物。该酶作用的ｐＨ为４．５－８．０;最适ｐＨ为６．５－７．０;在ｐＨ４．０－８．０范围内较稳定,酶作用的最适温度为５５℃;在６０℃处理１０,３０,６０,９０以及１２０分钟后,残余酶活分别为９５％,８０．３％,４０     

环状芽孢杆菌C-2几丁酶基因的克隆

环状芽孢杆菌（Ｂａｃｉｌｕｓｃｉｒｃｕｌａｎｓ）Ｃ２总ＤＮＡ经ＰｓｔＩ部分酶切后分离２～１０ｋｂ的片段,插入质粒ｐＵＣ１９的ＰｓｔＩ位点,转化大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ）,利用几丁质平板从约８０００个重组子中筛选到一个几丁酶基因阳性克隆（命名为ｐＣＨＴ１）。用１２种限制酶对重组质粒进行的酶切分析表明,重组质粒中的插入片段长３０ｋｂ,其中各有一个ＫｐｎＩ,ＳａｃＩ和ＳｓｐＩ位点。把该克隆片段反向插入ｐＵＣ１９的ＰｓｔＩ位点所得到的重组子同样具有几丁酶基因表达活性,说明此片段含有一个完整的几丁酶基因,其自身的启动子能被大肠杆菌转录系统所识别。Ｓｏｕｔｈｅｒｎ杂交证实了该片段来自于Ｂ．ｃｉｒｃｕｌａｎｓＣ２基因组,且以单拷贝形式存在,它不能与来自于其它７株几丁酶产生菌的总ＤＮＡ杂交。     

直接加热膨化蔗渣酶法水解的研究

采用加热时间为２０ｍｉｎ,压力为２．０ＭＰａ,温度为１２０℃的直接加热膨化甘蔗渣为水解底物、日本ｙｓｋｕｌｔ生物化学试剂公司生产的ｏｎｏｚｕｋＲＳ型纤维纯洁酶粉进行蔗渣的酶法水解实验,考察了蔗渣中纤维素的酶解还原糖得率与反应时间、酶浓度、ｐＨ值、缓冲液种类、离子强度以及固液比的关系,结果表明：当固液比为５％（ｗ／ｖ）,酶浓度＞１．２０ｍｇ／ｍｌ时,还原糖得率随酶浓度的增加变化不显著;本实验条件下,缓冲液种类和离子强度对还原糖得率几乎没有影响;水解最适宜ｐＨ值为４．２～４．９;最佳反应温度为５０℃。     

纤维二糖脱氢酶生成羟自由基和还原各种自由基的研究

利用电子顺磁共振（ＥＳＲ）技术和硫代巴比妥酸（ＴＢＡ）反应研究了纤维二糖脱氢酶（ＣＤＨ）生成·ＯＨ和还原各种自由基的能力．以纤维二糖为电子供体时,ＣＤＨ可以生成·ＯＨ．·ＯＨ生成量与ＣＤＨ、Ｆｅ３＋和Ｏ２的浓度有关．加入过氧化氢酶可使·ＯＨ的生成明显减少．ＣＤＨ可以还原自旋加合物［ＰＢＮ－ＯＨ］·、氮氧自由基和天然木素分子中的自由基．结果表明,ＣＤＨ具有生成·ＯＨ和还原各种自由基的能力．对该酶在木质纤维素降解中的作用进行了探讨     

嗜热脂肪芽孢杆菌DNA解链蛋白BstH2的纯化及性质研究

以嗜热脂肪芽孢杆菌为材料,通过ＰｏｌｙｍｉｎＰ沉淀、硫酸铵分级及ＤＥＡＥ纤维素、磷酸纤维素、Ｂｌｕｅ－Ｓｅｐｈａｒｏｓｅ、ＦＰＬＣＭｏｎｏＱ、ＦＰＬＣ Ｓｕｐｅｒｏｓｅ１２等柱层析,得到了部分纯化ＤＮＡ解链蛋白ＢｓｔＨ２。ＢｓｔＨ２具有受ＤＮＡ促进的ＡＴＰ酶活力,没类型的核酸对ＢｓｔＨ２的ＡＴＰ酶活力的促进作用没。ＢｓｔＨ２在５５℃有最高ＡＴＰ酶活力。这种活力受大肠杆菌单链ＤＮＡ结合蛋白的抑制及随     

纤维二糖脱氢酶在纤维素降解中的作用研究*

裂褶菌纤维二糖脱氢酶（ｃｅｌｌｏｂｉｏｓｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ, ＣＤＨ）可以提高纤维素酶对纤维素的降解。以纤维二糖为电子供体, ＣＤＨ作用于羧甲基纤维素可降低其溶液的粘度,作用于纤维素 ＣＦ１１和磷酸膨胀纤维素,分别使其悬浊液的浊度提高７％和１４．４％。ＣＤＨ与纤维二糖水解酶或内切纤维素酶在降解棉花纤维素时没有表现出协同作用。但若棉花事先在纤维二糖存在下用ＣＤＨ预处理,则变得易于被水解。     

地衣芽孢杆菌碱性蛋白酶的研究：I.碱性蛋白酶高产菌的筛选及产酶条件初探

从成都佳丰食品厂等处采集的样品中平板分离初筛到１２４株碱性蛋白酶产生菌,进一步复筛出一株高产,且稳定的碱性蛋白酶产生菌株Ｂ．Ｌ．ＪＦ－ｌｄ初步鉴定为地衣芽孢杆菌（Ｂａｃｉｌｌｕｓｌｉｃｈｅｎｉｆｏｒｍｉｓ）。该菌的最适产酶条件为：培养基（％）为麦芽糖７．５,酵母膏３,ＮａＣｌ０．５,Ｋ２ＨＰＯ４·３Ｈ２Ｏ０．５３,ＮａＨ２ＰＯ４·２３Ｈ２Ｏ０．０３,Ｎａ２ＣＯ３０．０５６,ＭｎＳＯ４ｌ×１０＾－４     

中国锈革孔菌科新种及值得注意的种

本文报道了产于我国云南西双版纳热带雨林的多孔菌一新种,版纳嗜蓝孢层孔菌 Ｆｏｍｉｔｉｐｏｒｉａ ｂａｎｎａｅｎｓｉｓ Ｙ．Ｃ． Ｄａｉ,该菌具有平伏的子实体较小的担孢子及大量的子实层刚毛这些特征很容易使该新种区别于同属的其它种,对与其它近似种的不同也进行了讨论锈革孔菌科的另外两种悦目小集毛菌Ｃｏｌｔｒｉｃｉｅｌｌａ　 ａｂｌｅｃｔａｂｉｌｉｓ（Ｌｌｏｙｄ） Ｋｏｔｌ, Ｐｏｕｚａｒ＆ Ｒｙｖａｒｄｅｎ和杜氏齿革菌 Ｈｙｄｎｏｃｈａｅｔｅ ｄｕｐｏｒｔｉｉ Ｐａｔ．被报道为中国新记录种,并根据我国的材料对这两种进行了详细描述。三个种被重新进行了组合它们是 Ｆｏｍｉｔｉｐｏｒｉａ ｓｏｎｏｒａ（Ｇｌｉｂ．）Ｙ．Ｃ． Ｄａｉ, Ｆｏｍｉｔｉｐｏｒｉａ　 ｓｕｂｌａｅｖｉｇａｔａ（Ｃｌｅｌａｎｄ　＆ 　Ｒｏｄｗａｙ） Ｙ．Ｃ． Ｄａｉ和 Ｏｎｎｉａ　ｆｌａｏｖｉｄａ（Ｂｅｒｋ．） Ｙ．Ｃ． Ｄａｉ．     

新菌——吉林链梭菌的分类学研究

从７例阴道炎患者阴道分泌物分离出７株细菌,均具有相同的生物学特性,Ｇ－梭状菌,单、成对或链状。无荚膜,无芽抱,无鞭毛。兼性厌氧菌,在大气中培养不生长,在５％～１０％ＣＯ２中或烛缸培养１８～２４ｈ才能形成菌落,ＭａｃＣｏｎｋｅｙ培养基不生长,最适生长温度３５℃－３７℃。氧化酶阴性,接触酶阴性,不还原硝酸盐,发酵糖类（指示剂用溴甲酚紫）,克氏双糖铁高层和斜面均发生产酸反应,七叶苷水解试验阳性,马尿酸盐水解试验阴性。经ＢｉｏｌｏｇＭｉｃｒｏｓｔａｔｉｏｎＳｙｓｔｅｍ自动化细菌鉴定系统检测无确定结果,ＤＮＡＧ＋Ｃ含量为４２．３ｍｏｌ％、１６ｓｒＲＮＡ序列测定结果经计算机检索国际基因菌库ＥＭＢＬ和ＧｅｎＢａｎｋ所有序列进行比较,表明该白与已知科、属亲缘关系较远,结合其表型特征,建议建立新属,命名为吉林链梭菌（Ｓｔｒｅｐｔｏｆｕｓｉａｇｅｎ．ｎｏｖ．Ｊｉｌｉｎａｓｐ．ｎｏｖ．）该菌已收藏在中国微生物菌种保藏中心ＣＧＭＣＣＮＯ．０２１５Ｔ（Ｔ＝ｔｙｐｅｓｔｒａｉｎ）．该菌１６ＳｒＲＮＡ片断已被国际基因库收录,接收号为Ｕ３４３６５。     

Isolation and partial properties of cellulose-decomposing strain of Cytophaga sp. LX-7 from soil

A new facultatively anaerobic, Gram-negative bacterium, Cytophaga sp. LX-7, degrading crystalline cellulose completely, was isolated from soil by dilution plating on cellodextrin agarose plates. This strain could excrete extracellularly all three types of cellulase and cellulosic substrates were the strongest inducer of endocellulase with CMC-liquefying activity production. No reducing sugar was found in cultures of cellulose during incubation. An enzyme which degrades crystalline cellulose was detected in cultures of cellulose by measuring the formation of soluble carbohydrate but was not detected by determining the reducing sugar released. This strain also synthesized cell-bound cellobiose oxidizing enzyme which was previously noted only in fungi. Both cellulose and soluble sugars could promote the production of cellobiose oxidizing enzyme.     

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.     

CMC-liquefying enzyme, a low molecular mass initial cellulose-decomposing cellulase responsible for fragmentation from Streptomyces sp. LX

Streptomyces sp. LX, newly isolated from soil, was shown to secrete a carboxylmethylcellulose (CMC)-liquefying enzyme that cleaves the CMC chains, releasing negligible reducing terminals. The new enzyme, named component C₂, was purified to homogeneity by dialysation. It has a molecular mass of 9·8 kDa. The pH optimum of the enzyme activity is 6·4 and its temperature optimum is 50°C. It retains full activity at pH 4–6·4 upon incubation at 50°C for 30 min. The enzyme has significant fragmentation activity on filter paper despite the absence of weight loss, release of reducing sugars and depolymerization during incubation with filter paper. The one-electron oxidative reaction is shown not to participate in the fragmentation of filter paper by enzyme C₂.     

Endoglucanase S, a novel endocellulase exhibiting exoglucanase activity from a newly isolated Streptomyces sp. LX

A novel endocellulase, designated as endoglucanase S, was purified to homogeneity from culture supernatant fluids of the newly isolated Streptomyces sp. LX, and shown not to be identical with previously described endo-β-1,4-glucanases. Both endo- and exo-cellulase activities were found to reside on a monomeric protein of 48 kDa. Its temperature optimum is 50 °C, and it was stable at 60 °C. The optimum pH is 5·5, and it has a broad pH stability from pH 3·0–7·0. The action of the enzyme on carboxymethylcellulose (CMC) suggested that the enzyme behaved as endoglucanase, whereas it was also active on crystalline cellulose with cellodextrin as end-product. Fragmentation of filter paper revealed that the degree of polymerization of residual cellulose decreased with time but only 5·2% of filter paper was converted into soluble carbohydrate.     

Isolation and some properties of cellulose-degrading Vibrio sp. LX-3 with agar-liquefying ability from soil

A new bacterium, Vibrio sp. LX-3 was isolated from soil, which was a facultatively anaerobic and polarly flagellated Gram-negative rod. Sodium ions stimulated its growth but were not an absolute requirement. The isolate could digest both crystalline cellulose and agar. Carboxymethylcellulase was produced extracellularly when various celluloses were used as carbon sources, but no reducing sugars were found in the culture on cellulose over the incubation period. Only low agarase activity could be detected in the broth of agar although the strain LX-3 strongly liquefied agar.     

Biodegradation of xanthan by newly isolated Cellulomonas sp. LX, releasing elicitor-active xantho-oligosaccharides-induced phytoalexin synthesis in soybean cotyledons

A Cellulomonas sp. LX newly isolated from soil samples could degrade the extracellular polysaccharide (xanthan) of Xanthomonas campestris. Such degradation was inhibited by glucose addition. Xanthan-degrading enzyme activity was found in the culture supernatant when Cellulomonas sp. LX was grown in the medium with xanthan as carbon source. The optimal pH and temperature for the xanthan-degrading reaction was 6.0 and 40 °C, respectively. The bioactivity of the xantho-oligosaccharide was examined with the soybean cotyledon bioassay and found it was an active elicitor.     

Exo-mode of action of cellobiohydrolase Cel48C from Paenibacillus sp. BP-23. A unique type of cellulase among Bacillales.

Sequence analysis of a Paenibacillus sp. BP-23 recombinant clone coding for a previously described endoglucanase revealed the presence of an additional truncated ORF with homology to family 48 glycosyl hydrolases. The corresponding 3509-bp DNA fragment was isolated after gene walking and cloned in Escherichia coli Xl1-Blue for expression and purification. The encoded enzyme, a cellulase of 1091 amino acids with a deduced molecular mass of 118 kDa and a pI of 4.85, displayed a multidomain organization bearing a canonical family 48 catalytic domain, a bacterial type 3a cellulose-binding module, and a putative fibronectin-III domain. The cloned cellulase, unique among Bacillales and designated Cel48C, was purified through affinity chromatography using its ability to bind Avicel. Maximum activity was achieved at 45 degrees C and pH 6.0 on acid-swollen cellulose, bacterial microcrystalline cellulose, Avicel and cellodextrins, whereas no activity was found on carboxy methyl cellulose, cellobiose, cellotriose, pNP-glycosides or 4-methylumbeliferyl alpha-d-glucoside. Cellobiose was the major product of cellulose hydrolysis, identifying Cel48C as a processive cellobiohydrolase. Although no chromogenic activity was detected from pNP-glycosides, TLC analysis revealed the release of p-nitrophenyl-glycosides and cellodextrins from these substrates, suggesting that Cel48C acts from the reducing ends of the sugar chain. Presence of such a cellobiohydrolase in Paenibacillus sp. BP-23 would contribute to widen up its range of action on natural cellulosic substrates.     

Characterization of an iron- and manganese-containing superoxide dismutase from Methylobacillus sp. strain SK1 DSM 8269

A superoxide dismutase was purified 62-fold in seven steps to homogeneity from Methylobacillus sp. strain SK1, an obligate methanol-oxidizing bacterium, with a yield of 9.6%. The final specific activity was 4,831 units per milligram protein as determined by an assay based on a 50% decrease in the rate of cytochrome c reduction. The molecular weight of the native enzyme was estimated to be 44,000. Sodium dodecyl sulfate gel electrophoresis revealed two identical subunits of molecular weight 23,100. The isoelectric point of the purified enzyme was found to be 4.4. Maximum activity of the enzyme was measured at pH 8. The enzyme was stable at pH range from 6 to 8 and at high temperature. The enzyme showed an absorption peak at 280 nm with a shoulder at 292 nm. Hydrogen peroxide and sodium azide, but not sodium cyanide, was found to inhibit the purified enzyme. The enzyme activity in cell-free extracts prepared from cells grown in manganese-rich medium, however, was not inhibited by hydrogen peroxide but inhibited by sodium azide. The activity in cell extracts from cells grown in iron-rich medium was found to be highly sensitive to hydrogen peroxide and sodium azide. One mol of native enzyme was found to contain 1.1 g-atom of iron and 0.7 g-atom of manganese. The N-terminal amino acid sequence of the purified enzyme was Ala-Tyr-Thr-Leu-Pro-Pro-Leu-Asn-Tyr-Ala-Tyr. The superoxide dismutase of Methylobacillus sp. strain SK1 was found to have antigenic sites identical to those of Methylobacillus glycogenes enzyme. The enzyme, however, shared no antigenic sites with Mycobacterium sp. strain JC1, Methylovorus sp. strain SS1, Methylobacterium sp. strain SY1, and Methylosinus trichosproium enzymes.     

Saccharification of native and degraded cotton cellulose and commercial microcrystalline cellulose by Trichoderma viride cellobiohydrolase I

The degree of polymerization of samples of acid degraded cotton cellulose has no appreciable influence on the saccharification by cellobiohydrolase I from Trichoderma viride. The increase in the number of cellulose molecule ends, achieved by a 30-fold decrease in molecular weight, does not produce the effect which could be expected for a pure end-wise mode of action of this exoglucanase. Microcrystalline celluloses saccharified by the same enzyme yield considerably more reducing sugars than cotton cellulose, either with a similar degree of polymerization or one of about 7000. It appears, therefore, that the difference in the susceptibility of the commercial substrates is not a consequence of their low degree of polymerization.     

A Thermostable Xylanase from a Thermophilic Acidophilic Bacillus sp.

Bacillus sp. 11-IS, a strain of thermophilic acidophilic bacteria, produced an extracellular xylanase during growth on xylan. The enzyme purified from the culture supernatant solution was homogeneous on disc-gel electrophoresis. The molecular weight was calculated to be 56,000 by SDS-gel electrophoresis. The enzyme had a pH optimum for activity at 4.0, and its stability range was pH 2.0 ~ 6.0. The temperature optimum was 80°C (10-min assay); however, the enzyme retained full activity after incubation at 70°C for 15 min. The enzyme acted on carboxymethyl cellulose (CMC) and cellulose, as well as on xylan. The Michaelis constants for larchwood xylan and CMC were calculated to be 1.68 mg xylose eq/ml and 0.465 mg glucose eq/ml, respectively. The predominant hydrolysis products from larchwood xylan were xylobiose, xylotriose, and xylose; the release of arabinose from rice-straw arabinoxylan was not detected. CMC was cleaved to cellobiose and larger oligosaccharides. Thus, the enzyme is considered to be an endoenzyme which degrades the β-1,4-glycosyl linkages in xylan and cellulose.