南昌霉素高产菌株的链霉素抗性基因突变诱变筛选研究

通过对链霉素对南昌霉素（Nanchangmycin)产生菌NS－41－80菌株孢子的致死浓度测定基础上,采用诱变剂甲基磺酸乙酯（EMS）的不同诱发剂量对菌株孢子进行诱变处理,诱变处理的孢子涂布在含链霉素（10ug/mL)致死浓度的高氏平板上,获得了大量的链霉素抗性基因（str)突变株。然后从3,000株链霉素抗性基因（str)突变株中通过初筛获得比诱变出发菌株产素能力提高20％以上的菌株202株,再进一步通过摇瓶复筛,获得比出发菌株产素能力分别提高100％,200％,300％高产菌株为48株,7株和1株,分别为复筛菌和初筛菌株的23．76％和1．60％,3．46％和0．23％,0．5％和0．03％,将产素能力提高240％以上5个菌株连同出发菌株连续3批次进行摇瓶发酵结果,5个突变株的产素能力均比出发菌株的产素能力提高57％－96．4％,其中突变株80－5．3－165菌株摇瓶发酵单位达6,000ug/mL以上,3批次摇瓶平均发酵单位达5,855ug/mL,建立了南昌霉素高产菌株的链霉素抗性基因突变诱变快速高效的筛选方法。     

米曲霉木聚糖酶生产菌的理性选育及发酵优化

目的：米曲霉木聚糖酶生产菌的理性选育及发酵优化。方法：以米曲霉FS018为出发菌株,采用紫外和激光诱变先后处理3代,以抗高浓度葡萄糖阻遏效应为筛选模型获得一株高产木聚糖酶菌株FST036,并对该菌株的发酵培养基进行了初步优化。结果：突变株FST036产酶水平可达4200．57U／mL,产酶水平比出发菌株提高了24％。对该菌株的发酵培养基初步优化结果表明：培养基的最适氮源为牛肉膏;最适碳源为麸皮与玉米芯组合,总浓度为4％。二者最适比例为7：3;对该菌株的发酵条件初步优化结果表明：最适接种量1％,初始pH6．78,250mL三角瓶装液量为45mL,培养72h酶活可达5404．24U／mL。结论：反复多次诱变处理,并结合抗高浓度葡萄糖阻遏效应作为一种理性筛选模型,为通过诱变育种来获得曲霉木聚糖酶高产菌株提供了一条有效的途径。     

链霉素抗性突变--纳他霉素高产菌株的选育研究

应用链霉素抗性筛选法,将经过紫外线诱变处理的纳他霉素生产菌——褐黄孢链霉菌(Streptomyces gilvosporeus)ATC13326的孢子涂布在含有链霉素最小抑制浓度(0．6μg／mL)的培养基平板上,获得了122株链霉素抗性突变株。其中纳他霉素产量高于出发菌株的有13株,产量阳性效率达到10．6％,同时获得了产抗生素能力为出发菌株1．46倍的突变株SG-56。     

α-乙酰乳酸脱羧酶产生菌的微波诱变

为获得α-乙酰乳酸脱羧酶的高产突变株,以产α-ALDC的枯草芽孢杆菌3226—5为出发菌株进行了诱变处理。经过微波（小火）物理诱变得到3株高产正突变株W181、W184、W195,经过多次传代实验,表明W181、W195是稳定的突变株。突变株W195的α-ALDC相对酶活（OD522）由出发菌株的0．35提高到0．617,提高了76％,突变株W181提高了66．9％。     

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。     

β-葡聚糖酶和木聚糖酶高产菌株的选育及在小麦加工中的应用

以Aspergillus nigerJ5为出发菌株,经Co60γ-射线诱变,筛选到一株β-葡聚糖酶和木聚糖酶活力都较出发菌株高的突变株A-25,其产β-葡聚糖酶和木聚糖酶的合适发酵条件为:大麦粉4%、玉米浆2.5%、NaNO30.4%、Na2HPO40.1%、MgSO4.7H2O0.03%、FeSO4.7H2O0.01%、CaCO30.5%、吐温-800.25%,初始pH6.7,300mL三角瓶的装液量为50mL,在此条件下培养84h,β-葡聚糖酶活力达到1203.9I U/mL,较出发菌株提高35.9%,木聚糖酶活力达到395.2I U/mL,较出发菌株提高27.8%。突变株粗酶液降解工业面粉非淀粉多糖的能力明显高于出发菌株。     

新一代糖化酶高产菌株的选育及其工业应用

【目的】建立对糖化酶生产菌种黑曲霉随机突变文库进行筛选的方法,以获得糖化酶酶活提高的突变菌株。【方法】以一株可产糖化酶的黑曲霉菌株Aspergillus niger X1为出发菌株,经硫酸二乙酯诱变获得突变文库,采用葡萄糖的结构类似物——2-脱氧葡萄糖进行筛选,并在筛选过程中逐渐提高2-脱氧葡萄糖浓度,定向选育具有2-脱氧葡萄糖抗性、高产糖化酶的突变株。【结果】获得的高产突变菌株DG36摇瓶发酵糖化酶产量比出发菌株A.niger X1提高22.2%–33.8%,经工业水平50 m~3罐发酵测试,突变株DG36发酵128 h糖化酶活可达49094 U/m L,在相同发酵时间内,其酶活较出发菌株A.niger X1提高32.8%,发酵时间缩短16.9%。【结论】本研究开发了一种以2-脱氧葡萄糖为抗性标记选育高产糖化酶突变株的方法,所得突变株DG36遗传性状稳定,与出发菌相比具有菌丝粗壮、产酶期提前、糖化酶活高、发酵时间短、有利于发酵后处理的优点。     

E.aero高产1.3-丙二醇菌株选育及发酵培养基研究(Ⅰ)

以淡水湖泊泥土中分离出的300多株肠杆菌(Enterobacter)为出发菌株,利用常规筛选方法选出2株1．3-丙二醇产生菌(Enterobacter)。经UV、DES、NTG、EMS、LiCl单独及复合诱变,选育出一株(E．aero-N-56)1．3-PD高产突变株。通过单因素实验,确定了E．aero—N-56菌株1．3-PD发酵培养基为：甘油90g/L,NH4CL1．50g/L,Fe^2 0．005％,Co^2 0．004％。微量元素液12mL／L。该突变株1．3-PD产量为36．8g/L。     

产适冷木聚糖酶的海洋产青霉的筛选和诱变

从黄海深层海底泥样中分离到1株产低温木聚糖酶的青霉,经EMS诱变得到11株酶活性提高的菌株,对其中1株产低温木聚糖酶活力最高的菌株产酶性质进行了初步研究。所产木聚糖酶在pH4．6,45℃时酶活可达25．8u／ml,比出发菌株提高126％。诱变后菌株所产木聚糖酶在0℃仍有显著酶活性,达8．2u／ml。     

枯草芽孢杆菌B-903菌株的诱变选育

枯草芽孢杆菌B-903菌株是由河南省农业科学院植物保护研究所从郑州果园中分离得到,其代谢产生的抗菌物质对多种植物病原真菌具有较强抑制作用。以此菌株为出发菌株,进行亚硝基胍（NTG）和微波诱变处理,确定了,二者诱变处理的最佳处理剂量：NTG最佳处理浓度为200μg／mL,微波诱变为HI微波挡（850W、脉冲频率2450MHz）处理100s,筛选出13个高效突变株。经传代实验,2株高效突变株N1和W2抑菌圈直径分别稳定在26mm和24mm以上,比出发菌株提高21．8％和14．8％．     

Xylanase production by Aspergillus awamori. Development of a medium and optimization of the fermentation parameters for the production of extracellular xylanase and beta-xylosidase while maintaining low protease production

A growth medium was developed for maximal production in batch culture of extracellular xylanase and beta-xylosidase by Aspergillus awamori CMI 142717 and a mutant (AANTG 43) derived from the wild-type strain. The optimum pH for the production of xylanase and beta-xylosidase was 4.0. The best temperature of xylanase production was 30 degrees C; 35 degrees C was optimal for beta-xylosidase. Protease production was never completely suppressed under any of the conditions tested. However, protease titre was 3.5-fold less than the control in medium in which proteose peptone and yeast extract were omitted: the level of xylanase was not affected (8.6 U mL(-1)) but beta-xylosidase titre was increased 4.7-fold to 1.5 U mL(-1). When corn steep liquor was used as the sole nitrogen source, xylanse and beta-xylosidase titres were further increased by 1.5- and 1.9-fold, respectively. Of the carbon sources investigated, ball-milled oat straw or oat spelt xylan produced the highest titres of xylanse and beta-xylosidase. None of the soluble carbon sources investigated produced the high titres of xylanase or beta-xylosidase induced by either oat straw for xylanse and beta-xylosidase was 2% and the optimum spore inoculum was between 10(6) and 10(7) spores/mL(-1) final concentration. The level of xylanse activity obtained in the culture filtrates of the mutant was a remarkable 820 U mL(-1) when the reducing sugar released was measured by the dinitrosalicylic acid method. This enzyme titre would appear to be the highest reported so far. The xylanases system contained the correct balance of enzymes to effect extensive hydrolysis of oat spelt xylan. The protease titre was very low.     

Cloning, disruption, and expression of two endo-beta 1, 4-xylanase genes, XYL2 and XYL3, from Cochliobolus carbonum.

In culture, the filamentous fungus Cochliobolus carbonum, a pathogen of maize, makes three cationic xylanases, XYL1, which encodes the major endoxylanase (Xyl1), was earlier cloned and shown by gene disruption to encode the first and second peaks of xylanase activity (P. C. Apel, D. G. Panaccione, F. R. Holden, and J. D. Walton, Mol. Plant-Microbe Interact. 6:467-473, 1993). Two additional xylanase genes, XYL2 and XYL3, have now been cloned from C. carbonum. XYL2 and XYL3 are predicted to encode 22-kDa family G xylanases similar to Xyl1. Xyl2 and Xyl3 are 60% and 42% identical, respectively, to Xyl1, and Xyl2 and Xyl3 are 39% identical. XYL1 and XYL2 but not XYL3 mRNAs are present in C. carbonum grown in culture, and XYL1 and XYL3 but not XYL2 mRNAs are present in infected plants. Transformation-mediated gene disruption was used to construct strains mutated in XYL1, XYL2, and XYL3. Xyl1 accounts for most of the total xylanase activity in culture, and disruption of XYL2 or XYL3 does not result in the further loss of any xylanase activity. In particular, the third peak of cationic xylanase activity is still present in a xyl1 xyl2 xyl3 triple mutant, and therefore this xylanase must be encoded by yet a fourth xylanase gene. A minor protein of 22 kDa that can be detected immunologically in the xyl1 mutant disappears in the xyl2 mutant and is therefore proposed to be the product of XYL2. The single xylanase mutants were crossed with each other to obtain multiple xylanase disruptions within the same strain. Strains disrupted in combinations of two and in all three xylanases were obtained. The triple mutant grows at the same rate as the wild type on xylan and on maize cell walls. The triple mutant is still fully pathogenic on maize with regard to lesion size, morphology, and rate of lesion development.     

The interaction of xylanases with commercial pulps

When purified xylanases from Trichoderma harzianum E58 or from a clone of Bacillus circulans were incubated with various low-yield wood pulps, little of the original enzyme activity could be detected in the filtrate at the end of the reaction. Partial bleaching of the pulps prior to enzymatic treatment generally resulted in an increased recovery of the xylanase activity. It appears that both nonspecific adsorption and soluble inhibitors may be responsible for the loss of much of the xylanase activity. However, xylanases from Aureobasidium pullulans and Schizophyllum commune were not as inhibited by the pulps, and the activity of the latter enzyme actually increased after incubation with several high-yield pulps. Although a lignin preparation from spent sulfite liquor at a concentration of 0.06 mg/mL could inhibit the xylanase activity of T. harzianum and B. circulans by 65% and 50%, respectively, xylanases from Thermoascus aurantiacus, S. commune, and A. pullulans were activated at similar lignin concentrations. At higher concentrations these latter xylanases were also inhibited. Water-soluble lignins extracted from a variety of pulps and used at a lignin concentration of 2.5 mug/mL resulted in inhibition of more than 65% of the original activity of the xylanase from T. harzianum. Kinetic studies showed that lignin from spent sulfite liquor resulted in noncompetitive inhibition of this enzyme.     

斜卧青霉纤维素酶和木聚糖酶高产菌株的选育

以纤维素酶高产菌株斜卧青霉A50为出发菌株,通过紫外诱变原生质体获得1株木聚糖酶活力提高80%而纤维素酶活力没有改变的6号菌。蛋白质电泳和酶谱检测结果显示,纤维素酶谱基本无差别,而木聚糖酶谱显示6号菌比A50多了一条带。6号菌优化后的产酶培养基组成为:麸皮7%、葡萄糖0.1%,该条件下,纤维素酶活为19.7IU/mL,木聚糖酶活力为215.4IU/mL。     

In vitro mutagenesis of a xylanase from the extreme thermophile Caldocellum saccharolyticum

Summary Six mutant xylanases were obtained by in vitro mutagenesis of a xylanase gene from the extremely thermophilic bacterium Caldocellum saccharolyticum. The temperature stability of all enzymes was affected by mutation to various degrees and one of the xylanases had an altered temperature optimum. The mutations had no effect on the pH optimum. The C. saccharolyticum xylanase showed strong homology to several thermophilic and mesophilic xylanases, and comparison of primary sequences allowed the localization of probable active sites and residues involved in thermostability. Offprint requests to: P. L. Bergquist     

毛壳霉CQ31的鉴定及固体发酵产木聚糖酶条件的优化

从土壤中筛选出一株产木聚糖酶的真菌CQ31, 经鉴定后命名为毛壳霉CQ31。该菌能够利用几种农业废弃物固体发酵高产木聚糖酶, 玉米杆为最佳碳源。单因素优化试验表明: 以玉米杆为碳源, 胰蛋白胨为氮源, 初始水分含量80%, 初始pH值9.0为最佳产酶条件。在优化后的条件下培养7 d产木聚糖酶水平高达4897 U/g干基碳源, 此时甘露聚糖酶酶活达803 U/g干基碳源。因此, 毛壳霉CQ31固体发酵产木聚糖酶和甘露聚糖酶具有一定的工业化应用前景。     

短小芽胞杆菌产碱性木聚糖酶发酵条件的研究

碱性木聚糖酶在碱性条件下催化水解木聚糖,广泛应用于造纸、纺织等领域.着重对短小芽胞杆菌M-11产碱性木聚糖酶的发酵条件进行初步的探索.研究了菌株的生长曲线、确定最佳接种龄为16 h、最佳接种量为1％;确定最适碳源浓度为7％、最适单一氮源为氯化铵、其浓度为1.0％、最适无机盐为氯化铁、其浓度为3 mmol/L;在此基础之上进行6因素3水平的正交试验,确定最适产酶培养基组成:麸皮5％,接种量3％,氯化铵1.2％,氯化铁3.5 mmol/L,硫酸镁0.03％,氯化钠5 mmol/L,磷酸氢二钾0.4％;最适培养条件:接种龄16 h,初始pH 8.0,温度37℃,300 mL摇瓶装液量50 mL,摇床转速220 r/min,发酵周期48 h.通过对发酵条件的优化使发酵液酶活达613 IU/mL.无机氮源为其最适氮源,因此短小芽胞杆菌M-11在碱性木聚糖酶的产品开发上优于短小芽胞杆菌M -26.     

Xylanase production in solid state fermentation by Aspergillus niger mutant using statistical experimental designs

The initial moisture content, cultivation time, inoculum size and concentration of basal medium were optimized in solid state fermentation (SSF) for the production of xylanase by an Aspergillus niger mutant using statistical experimental designs. The cultivation time and concentration of basal medium were the most important factors affecting xylanase activity. An inoculum size of 5 x 10(5) spores/g, initial moisture content of 65%, cultivation time of 5 days and 10 times concentration of basal medium containing 50 times concentration of corn steep liquor were optimum for xylanase production in SSF. Under the optimized conditions, the activity and productivity of xylanase obtained after 5 days of fermentation were 5,071 IU/g of rice straw and 14,790 IU l(-1) h(-1), respectively. The xylanase activity predicted by a polynomial model was 5,484 IU/g of rice straw.     

Cloning, characterization, and expression of xylanase gene from Bacillus lyticus in Escherichia coli and Bacillus subtilis.

A genomic library of Bacillus lyticus was constructed in lambda GEM 11 vector and screened for the xylanase gene using Congo red plate assay. A 16-kb fragment containing the xylanase gene was obtained which was further subcloned using Mbo I partial digestion in an E. coli pUC 19 vector. A 1.3-kb sub-fragment was obtained which coded for a xylanase gene of Mr 23,650 Da. This fragment was sequenced and the homology was checked with known xylanases. The maximum homology was 97%, which was obtained with an endo xylanase gene from Bacillus species at the DNA level, while the translated sequence showed only one amino acid change from alanine to serine at position number 102. Expression was checked in E. coli, using the native promoter, and an extracellular activity of 5.25 U/mL was obtained. Cloning of the gene was done in Bacillus subtilis using a shuttle vector pHB 201, which resulted in increasing the basal level xylanase activity from 14.02 to 22.01 U/mL.     

离子注入选育高产木聚糖酶黑曲霉及其发酵条件研究

以黑曲霉A3为出发菌,利用离子注入技术选育出一株遗传性状稳定的木聚糖酶高产突变株AN497,其产酶水平较出发菌从野生型A3菌株的405.6IU/ml提高到586.2IU/ml,即酶产量增加了44.5%;对高产菌进行发酵条件优化,发现以玉米芯粉为主要碳源、用蔗糖代替葡萄糖作为附加碳源,对木聚糖酶的发酵具有明显的促进作用;采用复合的无机氮源 (NH4)2SO4和NaNO3,(1: 2)浓度以10g/L为宜;菌株对发酵通氧量具有较高的要求,摇瓶转速在230r/min时的产酶水平较200r/min要高;通过发酵条件的优化,高产菌株的产酶活力最高可达671.1IU/mL,比出发菌株的产酶量提高了65.5%。