一株纤维素降解真菌的筛选及鉴定

[目的]分离筛选高效降解纤维素的真菌菌株,并研究其产酶能力.[方法]利用刚果红染色法从甘蔗地土壤中分离纤维素降解真菌,再通过测定滤纸的降解率及发酵酶活复筛.[结果]综合考虑水解圈,水解圈和菌株直径的比值(HC值),滤纸的降解率和复筛酶活,对试验真菌降解纤维素的能力进行综合评价,筛选到具有较强纤维素降解能力的真菌菌株SJ1,经形态学观察及分子生物学鉴定,该菌属于草酸青霉.其滤纸酶活、内切葡聚糖酶酶活(CMC酶活)、β-葡聚糖苷酶酶活和外切葡聚糖酶酶活(CBH酶活)分别为25.15、740.42、58.03和2.442 U/mL.[结论]菌株SJ1是一株十分具有研究开发潜力的纤维素酶生产菌株.     

秸秆纤维素分解菌的酶活力测定

目的:测定秸秆纤维素分解菌的酶活力。方法:从土壤中分离出具有分解纤维素能力的菌株,采用刚果红染色法进行粗选,得到7株透明圈较大的菌株。将这7株菌株液体发酵培养6d,再分别用滤纸分解度观察、羧甲基纤维素酶活法(CMC)、滤纸酶活法(FPA)和天然纤维素酶活法测定其酶活力。结果:在7株菌株中,F-1、F-2、F-3、F-5的酶活力测定结果与其溶解圈的测定结果、滤纸分解结果基本相同。且天然纤维素酶活力高的菌株,其CMC酶活、FPA酶活也高,滤纸分解效果也比较明显。结论:CMC法、FPA法和天然纤维素酶活法适于测定秸秆纤维素分解菌的酶活力。     

产纤维素酶菌株的筛选及离子束诱变

从含腐败秸秆的土壤中分离出刚果红水解圈与菌落比值(D/H)较大的7株纤维素酶产生菌,其中菌株S-1分解羧甲基纤维素钠的酶活(CMC酶活)最高,经鉴定为解淀粉芽孢杆菌。以之为出发菌株进行离子束诱变,采用酶标仪高通量酶活测定法筛选出CMC酶活较高的突变菌株进行传代培养,测定其传5代后菌株的CMC酶活,得到15株CMC酶活较高的突变菌株,其中突变菌株308的菌落形态变化较大,其CMC酶活最高,接种24 h后比出发菌S-1提高了84.4%,且突变菌株308的CMC酶活的提高具有遗传稳定性。表明离子束诱变对于提高菌株产纤维素酶能力具有潜在的应用价值。     

1株产纤维素酶细菌的筛选、鉴定及生长特性

分离筛选高效降解纤维素的菌株,并研究其生物学特性。利用刚果红染色法从腐烂的玉米秸秆中分离纤维素降解菌,再通过测定滤纸的降解率及多种酶活复筛。综合考虑水解圈和菌落直径(HC值),滤纸的降解率和酶活,对所筛选的菌株进行纤维素降解能力综合评价,最终获得1株具有纤维素降解能力的菌株DX4,其滤纸酶活(FPA酶活)、内切葡聚糖酶活力(CMC酶活)和外切葡聚糖酶活力(Cex酶活)分别为256.051、358.276和5.536 U/m L。结合形态学、生理生化特性和分子生物学鉴定,将该菌株鉴定为枯草芽胞杆菌(Bacillus subtilis),命名为Bacillus subtilis DX4,简称BS-DX4。研究表明,BS-DX4的最适生长温度为40℃,最适生长pH为7.0,低盐浓度下生长旺盛,是具有开发潜力的纤维素酶高产菌株。     

纤维素降解菌Aspergillus sp. YN1的产酶条件及酶学特性

从牛羊粪堆肥中筛选出一株纤维素降解菌Aspergillus sp.YN1,主要研究了液体发酵培养基中碳源、氮源、培养温度、起始pH、通气量以及接种菌龄对菌株YN1的羧甲基纤维素酶活(CMC酶活)及滤纸酶活的影响。研究结果表明,在优化条件下,该菌的CMC酶活、滤纸酶活在培养第3天分别达到0.53U/mL和0.15U/mL。在酶学特性研究中,菌株YN1的CMC酶的最适反应温度为70°C,最适反应pH4.0(酶促反应为30min)。用不同温度处理1h或不同pH处理2h,YN1的CMC酶在30°C?50°C或pH3.0?4.0之间仍可保持80%以上的酶活性,对热及酸表现出较高的稳定性。     

发酵床中纤维素降解菌的分离与鉴定

从发酵床垫料中初步分离出43株纤维素降解菌。采用刚果红鉴别培养基及滤纸条培养基初筛,得到5株透明圈较大且使滤纸条产生崩解的菌株,通过进一步液体发酵,测定其CMC酶活、FPA酶活和天然纤维素酶活,获得2株具有较高纤维素降解活性菌株,并分别命名为F7和F21。经16S rRNA基因序列分子生物学鉴定和系统发育分析表明,这2株纤维素降解菌分别归属为枯草芽孢杆菌(Bacillus subtilis)和链霉菌(Streptomyces sp.)。     

真空微波诱变结合化学诱变选育酸性纤维素酶高产菌株

以酸性纤维素酶产生菌绿色木霉（Trichoderma viride）WL0512作为原始出发菌株,首先经自然分离筛选出一株产酶较稳定的菌株TVN-18,其羧甲基纤维素酶活（CMC酶活）达2765．8U／g,滤纸酶活（FPA酶活）达48．5U／g。再经真空微波和甲基磺酸乙酯（EMS）逐级诱变处理,获得了一株高产、稳产酸性纤维素酶的E6—1菌株,其CMC酶活达4396．6U／g,FPA酶活达126．0U／g,分别是菌株TVN-18的1．59倍和2．60倍。通过对固态发酵培养基麸皮和稻草比例、料水比以及初始pH值的优化,突变株的产酶能力进一步得到提高,其产的CIVIC酶活和FPA酶活分别提高了22．3％和22．4％。     

一组降解纤维素细菌的分离筛选及产酶特性研究

用稀释法从腐殖泥中分离菌株,根据它们在羧甲基纤维素钠(CMC-Na)-刚果红培养基上的透明圈直径及对滤纸的崩解能力,获得7株纤维素分解细菌(编号:B-37、B-35、B-31、B-25、B-17、Z-a和Z-b),其中菌株Z-b的纤维素崩解能力最强,分子鉴定结果表明它与Stenotrophomonas maltophilia的16S rDNA序列有99.8%的同源性,初步确定为嗜麦芽窄食单胞菌。适合7个菌株生长的C源为马铃薯浸出液,无机盐组分为:CaCl_2 0.20、MgSO_4 1.25、NaCl 5.00、(NH_4)_2SO_4 1.30、KH_2PO_4 1.35、FeSO_4·7H_2O 0.015、Na-EDTA 0.02g/L。在滤纸为唯一碳源的培养基中,菌株Z-b的滤纸酶(FPase)和CMC酶(CMCase)活性最大,为0.099 U/mL和0.075 U/mL,而在固体PSA上,菌株B-31和B-37的FPase和CMCase活性最高,为0.131 U/mL和0.175 U/mL。7个细菌单独发酵,Z-b对滤纸的崩解能力最强,滤纸块完全崩解成粉未状;与真菌34混合发酵,菌株组合Z-a+34、Z-b+34、B-31+34、B-25+34将滤纸完全水解为水溶性物质。可见,各菌株的纤维素酶活与培养条件密切相关,某些真菌、细菌间存在协同作用,它们混合发酵可大大提高纤维素的水解效率。     

青霉的纤维素酶抗降解物阻遏突变株的选育

从土样中筛出一株生长快,产纤维素酶较高的斜卧青霉(Penicillium decumbens114-2)。能在全纤维素(hlocellulose)双层平板上形成清晰的透明圈。摇瓶培养的滤纸酶活可达8.8mg 葡萄糖/ml.h。 114-2菌株(其纤维素酶合成可为葡萄糖所阻遏)经UV和NG诱变处理后,在含葡萄糖的全纤维素平板上筛选到多株仍能形成明显透明圈的突变株。其中JN15和JU1在含葡萄糖的全纤维素液体培养基中,在残余葡萄糖浓度为 1％左右时,滤纸酶活可分别达到7.3 和13.9mg葡萄糖ml·h。这是出发株114-2和纤维素酶高产菌株木霉EA_3-867和QM9414所不能的。在不加阻遏剂的对比试验中,两个突变株的纤维素酶产量都比较高。其中,JU1的滤纸、CMC和棉花酶活分别达到33mg葡萄糖/ml·h,234mg葡萄糖/ml·h和86mg葡萄糖/ml·24h。     

采用混合策略提高葡萄糖氧化酶在毕赤酵母中的表达水平

为了提高葡萄糖氧化酶 (GOD) 在毕赤酵母中的表达水平,提出了甲醇/山梨醇混合碳源诱导和共表达分子伴侣二硫键异构酶 (PDI) 和透明颤菌血红蛋白 (VHb) 两种策略。利用对照菌株X33/pPIC9k–GOD 在5 L发酵罐放大培养时,采用甲醇/山梨醇混合碳源诱导,GOD最终酶活为456 U/mL,比只采用甲醇作为单一碳源诱导时GOD最终酶活提高了20%。利用整合伴侣蛋白菌株X33/pPIC9k-GOD/pPICZ-PDI-VHb在5 L发酵罐进行高密度发酵,采用甲醇/山梨醇混合碳源诱导,GOD最终酶活达到716 U/mL,蛋白浓度为7.4 g/L。研究结果对提高外源蛋白在毕赤酵母中的表达有重要参考价值。     

Identification of Mycelium-Associated Cellulase from Streptomyces reticuli

Among 180 Streptomyces strains tested, 25 were capable of hydrolyzing microcrystalline cellulose (Avicel) at 30°C. Streptomyces reticuli was selected for further studies because of its ability to grow at between 30 and 50°C on Avicel. Enzymatic activities degrading Avicel, carboxymethyl cellulose, and cellobiose were found both in the culture supernatant and in association with the mycelium and crystalline substrate. The bound enzymes were efficiently solubilized by repeated washes with buffer of low ionic strength (50 mM Tris hydrochloride [pH 7.5]) and further purified by fast protein liquid chromatography. A high-molecular-weight Avicelase of >300 kilodaltons could be separated from carboxymethyl cellulase (CMCase) and β-glucosidase activities (molecular mass, 40 to 50 kilodaltons) by gel filtration on Superose 12. The CMCase fraction was resolved by Mono Q anion-exchange chromatography into two enzymes designated CMCase 1 and CMCase 2. The β-glucosidase activity was found to copurify with CMCase 2. The purified cellulase components showed optimal activity at around pH 7.0 and temperatures of between 45 and 50°C. Avicelase (but not CMCase) activity was stimulated significantly by the addition of CaCl₂.     

Enhanced cellulase production of the Trichoderma viride mutated by microwave and ultraviolet

Cellulase-producing fungi Trichoderma viride were cultured and fermented on the solid-state wheat bran fermentation medium. The characteristics of its carboxymethyl cellulase (CMCase) in the condition of this solid-state fermentation were evaluated, and the optimum culture time, optimum pH and optimum temperature for CMCase activity of T. viride fermented in this solid state were 60 h, 5.0 and 50 °C, respectively. Carboxymethyl cellulose sodium (CMC-Na) and Congo red were used to screen the strains that had stronger ability to produce enzymes. After the compound mutagenesis by microwave and ultraviolet, seven mutant strains (M-B1–M-B7) were selected and their CMCase activities were assayed. Five of them (M-B1, M-B2, M-B3, M-B5 and M-B7) had significantly stronger ability to produce enzymes than the normal wild type, and they were also very stable for a long period up to 9 generations to produce cellulase. Molecular studies showed that there were some base mutations in endoglucanase I (EG I) genes of mutants M-B1, M-B2, M-B3 and M-B5, but no change in M-B7, suggesting that some amino mutations in EG I proteins caused by base mutations could lead to enhanced cellulase production.     

Optimization of cellulase production in batch fermentation byTrichoderma reesei

Maximum cellulase production was sought by comparing the activities of the cellulases produced by differentTrichoderma reesei strains andAspergillus niger. Trichoderma reesei Rut-C30 showed higher cellulase activity than otherTrichoderma reesei strains andAspergillus niger that was isolated from soil. By optimizing the cultivation condition during shake flask culture, higher cellulase production could be achieved. The FP (filter paper) activity of 3.7 U/ml and CMCase (Carboxymethylcellulase) activity of 60 U/ml were obtained from shake flask culture. When it was grown in 2.5L fermentor, where pH and DO levels are controlled, the Enzyme activities were 133.35 U/ml (CMCase) and 11.67 U./ml (FP), respectively. Ammonium sulfate precipitation method was used to recover enzymes from fermentation broth. The dried cellulase powder showed 3074.9 U/g of CMCase activity and 166.7 U/g of FP activity with 83.5% CMCase recovery.     

Characterization and Phylogenetic Diversity of Carboxymethyl Cellulase Producing <Emphasis Type="Italic">Bacillus</Emphasis> Species from a Landfill Ecosystem

Total population of cellulose degrading bacteria was studied in a landfill ecosystem as a part of microbial diversity study. Samples were obtained from 3 and 5 feet depth of a local landfill being operated for past 10 years. Among many isolates, 22 bacterial strains were selected based on their capability to decompose carboxymethyl cellulose (CMC). These isolates were cultivated on agar medium with CMC as the carbon source. All isolates were Gram positive, endospore forming and alkalophilic bacteria with optimum growth pH 9–10. They were grouped based on the phenotypic and chemotaxonomic characters and representative strains of different groups along with high carboxymethyl cellulase (CMCase) producing strains were included for further characterization. Analysis of 16S rRNA gene indicated that these strains belong to different species of the genus Bacillus. Maximum CMCase activity of 4.8 U/ml at 50°C was obtained by strain LFC15. Results in the present study indicated the potential of waste land ecosystems such as landfill are potential source for isolation of industrially important microorganisms.     

Isolation and mutation of cellulolytic fungi

Nineteen fungi were isolated from different soil samples on the basis of clear zones formed on Rose Bengal Cellulose agar medium. In shake flasks th isolate K1 gave 12.1 units/ml of CMCase activity. A mutant of the isolate K1, KM7, was selected after N-methyl-N'-nitro-N-nitrosoguanidine treatment of the wild-type. This mutant differed morphologically from the parent strain on RBCA medium and gave 36.2 units/ml of CMCase activity which represented about 50% of the enzyme yield from the standard organism, Trichoderma viride QM 9414 (80 units/ml of CMCase activity). The isolate K1, which was identified as a Phoma species, produced 48 units of beta-glucosidase. The yield of beta-glucosidase was increased about 8-fold in the mutant KM7 and was about 68% higher than the level found in T. viride QM 9414.     

The cellular location of Prevotella ruminicola beta-1,4-D-endoglucanase and its occurrence in other strains of ruminal bacteria.

Prevotella ruminicola B(1)4, TC1-1, TF1-3, and TS1-5 all produced immunologically cross-reacting 88- and 82-kDa carboxymethyl cellulases (CMCases). P. ruminicola 23, 118B, 20-63, and 20-78 had much lower CMCase activities, and Western blots (immunoblots) showed no cross-reaction with the B(1)4 CMCase antiserum. Fibrobacter succinogenes S85 and Selenomonas ruminantium HD4 and D produced CMCase, but these enzymes were smaller and did not cross-react with the B(1)4 CMCase antiserum. The B(1)4 CMCase antiserum inhibited the B(1)4, TC1-1, TF1-3, and TS1-5 CMCase activities and agglutinated these cells, but it had no effect on the other strains or species. On the basis of these results, the B(1)4 CMCase is a strain-specific enzyme that is located on the outside surface of the cells. P. ruminicola B(1)4 cultures, grown on sucrose, did not have significant CMCase activity, but these cells could bind purified 88- and 82-kDa CMCase but not 40.5-kDa CMCase. Because the 40.5-kDa CMCase is a fully active, truncated form of the CMCase, it appears that the N-terminal domain of the 88-kDa B(1)4 CMCase anchors the CMCase to the cells. Cells grown on cellobiose produced at least 10-fold more CMCase than the sucrose-grown cells, and the cellobiose-grown cells could only bind 15% as much CMCase as sucrose-grown cells. Virtually all of the CMCase activity of exponentially growing cultures was cell associated, but CMCase activity was eventually detected in the culture supernatant. On the basis of the observation that the 88-kDa CMCase was gradually converted to the 82-kDa CMCase when cultures reached the stationary phase without a change in specific activity, it appears that the 82-kDa protein is probably a proteolytic degradation product of the 88-kDa CMCase.     

木质纤维素酶高产菌株的筛选及其对玉米秸秆降解效果的影响

为了提高玉米秸秆中木质素的降解率,从腐烂的树枝和土壤中筛选木质纤维素酶高产菌株,以秸秆为唯一C源富集培养后,采用PDA-愈创木酚法进行初筛,筛选出产木质素酶真菌5株,然后以玉米秸秆为主要C源进行固态发酵和复筛。结果表明:第5号菌株在发酵玉米秸秆5 d后,使木质素的降解率达到最高(34.95%),粗纤维的降解率达到20.00%,显著高于其他4种菌株(P<0.05),其羧甲基纤维素酶比酶活达到116.35 U/g;10 d后,其木质素酶比酶活达到最高(45.64 U/g)。     

Molecular cloning,expression, and characterization of a new endoglucanase gene fromFibrobacter succinogenes S85

A DNA fragment coding for a carboxymethylcellulase (CMCase) ofFibrobacter succinogenes S85 was isolated from a pUC18 gene library inEscherichia coli JM109. The CMCase gene was present as a single copy in theF. succinogenes S85 genome and was found in all the otherF. succinogenes strains tested. The gene was expressed from an endogenous promoter inE. coli and was not subject to glucose repression. Most of the CMCase activity was located in the membrane ofE. coli. Zymogram analysis and³⁵S labeling of the proteins encoded by the CMCase gene-containing plasmid indicated that the enzyme has a molecular mass of 58,000. The optimal pH and temperature of activity on CMC were respectively 6.4 and 30°C. The enzyme was active on CMC, barley -glucan, and lichenan but would not hydrolyze laminarin and exhibited no exoglucanase-type activity, suggesting that it is an endo-(1,4)--d-glucanase.     

Molecular cloning of an endoglucanase gene from an alkalophilic Bacillus sp. and its expression in Escherichia coli

One of the cellulase genes from alkalophilic Bacillus sp. strain N-4 was cloned in pBR322. A recombinant plasmid, pYBC107, expressing carboxymethyl cellulase (CMCase) was isolated, and the size of the cloned HindIII fragment was found to be 5.5 kilobases. The restriction map of pYBC107 showed a different pattern from those of pNKI and pNKII (N. Sashihara, T. Kudo, and K. Horikoshi, J. Bacteriol. 158:503-506, 1984). When the HindIII fragment from pYBC107 was subcloned into pYEJ001, there was a 3.8-fold increase in CMCase activity over that observed with pYBC107. Plasmid pYBC108 constructed by treatment of pYBC107 with HindIII and EcoRI expressed the CMCase activity, although to a limited extent. To verify the originality of cloned pYBC107 from Bacillus sp., we analyzed the restriction digest by Southern blotting.     

从万古霉素抗性突变体中筛选碱性纤维素酶高产菌株

以芽胞杆菌 Ｘ—６ 为出发菌株,经甲基磺酸乙酯（ Ｅ Ｍ Ｓ） 和紫外线（ Ｕ Ｖ） 复合诱变,选育万古霉素抗性突变体。研究结果表明,抗药性突变株碱性羧甲基纤维素酶（ Ｃ Ｍ Ｃａｓｅ） 产量提高的正变率和正变幅度明显高于非抗药性菌株。从抗性突变株中获得 Ｅ Ｖ２３ 菌株,其产酶活力比出发株 Ｘ—６ 提高３２０ ％ ,酶活力达３５３ｕ／ ｍｌ。