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。     

斜卧青霉A10产纤维素酶的酶学性质研究

以斜卧青霉（Penieillium decumbens）A10为出发菌株,经450Gy ^60Coγ-射线诱变处理,选育出一株具有较高纤维素酶活力且传代稳定的正突变株A50,发酵60h后,其CMC酶活和滤纸酶活分别为27．28IU／mL和1．98IU／mL,较出发菌株A10分别提高了33．2％和45．59％。对突变株A50的产酶组分进行研究,通过SDS—PAGE电泳分析,从蛋白质水平上证明突变株A50确实是A10在遗传物质上发生改变的菌株,其CMC酶活最适作用pH值为4．0,最适作用温度为60℃;而滤纸酶活的最适作用pH值为5．2,最适作用温度45℃,二者在一定范围内具有较高的稳定性。     

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.)。     

里氏木霉液体发酵产纤维素酶的研究

在摇瓶试验基础上,采用里氏木霉(Trichoderma reesei)HC-415菌株进行5L自控罐产纤维素酶深层发酵试验。在通气量为 0.2—0.6vvm、搅拌速度为 400r/min、发酵液pH控制在5.8—6.1的条件下,发酵液的羧甲基纤维素(CMC)酶酶活最高为325.0mg糖/ml,滤纸糖酶(FPA)酶活最高达17.9mg糖/ml。发酵周期为108h。所得冻干纤维素酶粉CMC酶活最高3111IU/g,FPA最高135IU/g ,对发酵液得率平均6.7g/L。酶活总收率CMC酶活平均78.2％,FPA酶活平均73.5％。     

产黄曲霉毒素B1降解酶菌株的筛选鉴定及发酵条件优化

以香豆素为唯一碳源筛选到27株能高效降解黄曲霉毒素B1(AFB1)的微生物菌株.用高效液相色谱检测AFB1含量的方法进行AFB1降解酶活力测定.以不同菌株发酵上清液中AFB1降解酶活力高低为复筛条件,筛选到AFB1降解酶活力最高的一株菌并命名为HSD8.该菌株经形态学、生理生化及系统发育学方法鉴定为Sinomonas sp..筛选所得最优菌株的发酵上清液中酶活达443 U/mL,通过单因素试验对其产酶发酵条件进行优化,以提高酶活.优化所得最佳发酵条件为:装液量50 mL/250 mL,发酵周期48 h,初始pH 5.0,接种量8％,发酵温度37℃,摇床转速160r/min.在最佳发酵条件下,该菌株发酵上清液中酶活可达548 U/mL,比优化前提高23.7％.优选菌株HSD8在生物降解黄曲霉毒素B1方面具有应用潜力,值得进一步研究开发.     

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％。     

紫外诱变选育木聚糖酶高产菌株及产酶条件初步研究

以短小芽孢杆菌(Bacillus pumilus)HJ-04为出发菌株,通过紫外诱变的方法筛选得到一株木聚糖酶高产菌株B6,其酶活提高约30%.通过因素轮换试验和L9(34)正交试验结果得出B-6菌株产酶的最佳营养条件及发酵条件.结论:4%啤酒糟、2%玉米芯、2%牛肉膏、0.15%FeSO4、1.5%Na2HPO4,pH 8～9,35℃,170r/min培养60h,产酶活力达到536.72IU/mL.     

绿色木霉原生质体诱变筛选纤维素酶高产菌株

以绿色木霉F264为出发菌株制备其原生质体,对其进行紫外线诱变处理,筛选出一株纤维素酶高产突变株绿色木霉F-UV264,其滤纸酶活由出发菌株的5.2IU/g干料提高到15.78IU/g干料,产酶能力提高了3倍。经过10代PDA斜面继代培养及发酵试验,表明该菌株是比原菌株更优秀的稳定高产株。     

拟康氏木霉（Trichoderma pseudokoningii）UV Ⅲ产纤维素酶液体发酵研究

以拟康氏木霉（Trichoderma pseudokoningii)TH为出发菌株,经紫外诱变获得一抗高浓度葡萄糖阻遏突变株UV Ⅲ,其液体发酵最适产酶培养基为（W／V）：豆皮粉3％,硝酸铵0．6％,磷酸二氢钠0．65％,硫酸镁0．25％,氯化钙0．15％,pH5.0;最佳发酵条件为：30℃,125r/min。发酵7d CMCase活力可达103．55IU／mL,滤纸酶活可达5．51IU／mL,β-葡萄糖苷酶活可达0．96IU／mL,分别比出发菌株TH提高了1．40、2．34、0．60倍。     

白腐真菌AH28-2菌株发酵合成漆酶初步研究

从224个野外采集的真菌样品中筛选分离到一株产漆酶活性较高菌株AH28－2,经初步鉴定为白腐真菌,采用单因子相互比较法,研究了该菌株最适发酵产酶条件。应用添加有1g／LKraft木素的液体发酵培养基,接种量5％（V／V）,初始pH8．5,装液量为50％,28℃、150r/min摇瓶振荡培养4－5d,漆酶酶活水平达20184IU／L。     

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。     

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。     

Glucose oxidase activity of actinomycetes.

The ability of 311 actiomycete, belonging to 12 species to produce glucose oxidase was studied. It was found that 174 of them formed exoenzymes on solid medium and 133 in liquid medium. The composition of the nutrient medium has an essential effect on the amount of enzyme formed. Strains with considerably higher activity form a greater amount of exoenzymes on soya meal medium and on synthetic medium with KNO2. The highest activity of the culture liquid of some strains was observed between the 6th and 7th day of cultivation. During this phase of growth the highest productivity of the biomas was established.     

白腐菌液体和固体培养产生木质纤维素降解酶的比较研究

侧耳sp2(Pleurotus sp．2)和粗毛栓菌(Trametes gallica)是产木质纤维素降解酶能力强,且产酶较快的菌株。对其在液体培养基、固体培养基中产生木质纤维素降解酶能力和行为进行了比较分析和研究。结果表明,Pleurotus sp．2在低氮高碳高无机盐培养基中的锰过氧化物酶(Manganese peroxidases, MnPs)、木质素过氧化物酶(Lignin peroxidases．LiPs)、漆酶(laccases,Lacs)和半纤维素酶(Hemicellulases, Hcels)的活性最高。当该菌株培养在含有低氮无碳高无机盐液体培养基的麦草粉中时,MnPs和Lacs的活性峰值均出现在10d,而Hcels的活性在40d时达到峰值。Trametes gallica在高氮低碳高无机盐培养基中的Lacs和LiPs的活性最高,在低氮高碳高无机盐培养基中的MnPs和Hcels的活性最高。当该菌株培养在含有高氮无碳高无机盐和低氮无碳高无机盐液体培养基的麦草粉中时,MnPs存10d、Lacs和Hcels在40d、LiPs存50d,分别达到峰值。Pleurotus sp．2和Trametes gallica在液体培养基中具有很强的木质纤维素降解酶产生能力且产酶速度较快,在固体培养基中具有很强的降解麦秸生物质能力,但这两株菌在液体和固体培养基中,产木质纤维素降解酶的能力和行为都有较大的差异,相关性小。     

Ethanol Production by Thermophilic Bacteria: Relationship Between Fermentation Product Yields of and Catabolic Enzyme Activities in Clostridium thermocellum and Thermoanaerobium brockii

Significant quantitative differences in end-product yields by two strains of Clostridium thermocellum and one strain of Thermoanaerobium brockii were observed during cellobiose fermentation. Most notably, the ethanol/H₂ and lactate/acetate ratios were drastically higher for T. brockii as compared with C. thermocellum strains LQRI and AS39. Exogenous H₂ addition (0.4 to 1.0 atm) during culture growth increased the ethanol/acetate ratio of both T. brockii and AS39 but had no effect on LQRI. All strains had an operative Embden-Meyerhof glycolytic pathway and displayed catabolic activities of fructose-1,6-diphosphate–activated lactate dehydrogenase, coenzyme A acetylating pyruvate and acetaldehyde dehydrogenase, hydrogenase, ethanol dehydrogenase, and acetate kinase. Enzyme kinetic properties (apparent K_m, V_max, and Q₁₀ values) and the specificity of electron donors/acceptors for different oxidoreductases involved in pyruvate conversion to fermentation products were compared in the three strains. Both species contained ferredoxin-linked pyruvate dehydrogenase and pyridine nucleotide oxidoreductases. Ferredoxin-nicotinamide adenine dinucleotide (NAD) reductase activity was significantly higher in T. brockii than in AS39 and was not detectable in LQRI. H₂ production and hydrogenase activity were inversely related to ferredoxin-NAD reductase activity in the three strains. Ferredoxin-NAD phosphate reductase activity was present in cell extracts of both species. Alcohol dehydrogenase activity in C. thermocellum was NAD dependent, unidirectional, and inhibited by low concentrations of NAD and ethanol. Ethanol dehydrogenase activity of T. brockii was both NAD and NADP linked, reversible, and not inhibited by low levels of reaction products. The high lactate yield of T. brockii correlated with increased fructose-1,6-diphosphate. The relation of catabolic enzyme activity and quantitative differences in intracellular electron flow and fermentation product yields of these thermophilic bacteria is discussed.     

纳豆激酶的发酵工艺研究

以实验室选育的纳豆菌— 6号 (Bacillusnatto 6)为出发菌株 ,研究其固液两种发酵条件对纳豆激酶溶栓活力的影响。研究表明 ,纳豆菌适于固体发酵 ,37℃下 2 4h ,其活力可达 1 60 0IU/g以上。     

黑曲霉纤维素酶突变子T-DNA插入位点的遗传分析及性质鉴定

采用羧甲基纤维素钠筛选培养基,对黑曲霉(Aspergillus niger)T-DNA突变子文库进行筛选,分离到一株纤维素酶分泌水平较低的菌株AN-108,为野生型菌株的83.3%。进一步测定该突变子固体发酵的纤维素酶活力,与野生型菌株相比没有明显差别,推测与固体发酵培养基中含有的天然糖类有关。在添加不同糖类的CMC-Na平板上培养该突变子,菌落周围均出现较明显的水解圈,结果显示糖类可能作为诱导物克服突变带来的影响。为了确定突变子AN-108中何种基因被阻断,采用反向PCR方法分析了T-DNA插入位点的序列,获得序列经过比对分析发现,该序列与黑曲霉An14g03730同源程度达90%,编码富含脯氨酸蛋白(proline-rich protein,PRP)。     

两株高产纤维素酶细菌的筛选、鉴定及酶学特性

从腐烂枯叶及附近土壤筛选分离得到2株产纤维素酶的菌株。经细菌形态观察、生理生化实验并结合16S rRNA序列分析,将其初步鉴定为地衣芽孢杆菌CT1(Bacillus licheniformis CT1)和枯草芽孢杆菌CM2(Bacillus subtilis CM2)。经摇瓶发酵,测定其CMCase、FPA酶活力,结果表明CT1和CM2在液体摇瓶培养4 d后的CMC酶活最大,分别可达163.3 U/mL和167.17 U/mL;CT1摇瓶培养2 d后,FPA酶活达到了211.17 U/mL,CM2摇瓶培养3 d后,FPA酶活为207.83 U/mL。进行不同碳源对菌株产酶能力影响的试验,并通过SDS-聚丙烯酰胺凝胶电泳、银染后初步分析纤维素酶谱条带,发现菌株对不同来源纤维素的降解能力及产纤维素酶的种类均有所不同。     

Production of novel oligosaccharide oxidase by wheat bran solid-state fermentation

A search for oxidases that catalyze the oxidation of oligosaccharides has resulted in the isolation of several soil-derived fungus strains which produced novel oligosaccharide oxidases with different substrate specificity on wheat bran solid culture. One of these oxidases produced by Acremonium strictum T1 strain has been characterized. This enzyme showed high reactivity toward maltose, lactose, cellobiose and maltooligosaccharides composed of up to seven glucose units, and was named as glucooligosaccharide oxidase based on its substrate specificity. Strain T1 was subjected to a strain improvement program, and an enzyme hyper-producing mutant strain T1-38 was selected. This mutant strain produced glucooligosaccharide oxidase 75 times higher than the wild type strain T1. When cultivated in a solid medium comprised of 1 part of wheat bran and 1 part of water (w/w), enzyme activity reached a maximum level of 6 units per g of culture medium after 4 days cultivation. Characteristics of the enzyme including the substrate specificity were compared with two other novel oligosaccharide oxidases isolated in this laboratory. Batch type conversion of lactose to lactobionic acid using crude enzyme was also discussed.