用快中子法选育细菌脂肪酶高产菌株

研究了从土壤中筛选产脂肪酶的菌株,利用紫外线,快中子,快中子和磁场复合,γ射线,γ射线和磁场复合诱变,以酶活为筛子进行诱变育种,结果,出发菌酶活较低的一株得到了一株酶活为396．22U/mL的诱变株,此酶活比出发菌株高92倍,并发现比菌对紫外线和快中子比较敏感,而出发菌酶活较高的一株得到了酶活为424．60U/mL发酵液的诱变株,此酶活为出发菌株3．0倍,在此基础上,初步探讨了快中子,γ射线及磁场复合处理在产脂肪酶菌种诱变中的作用,并认为,在产脂肪酶菌株的诱变中快中子诱变更为有效。     

一种胆固醇氧化酶高产菌株筛选方法的建立

用亚硝基胍(1 mg/mL)与超声波(200 W, 50 kHz)复合诱变的方法, 对产胆固醇氧化酶短杆菌Brevibacterium sp. DGCDC-82进行诱变处理, 得到一株桔红色突变株其产胆固醇氧化酶能力提高140%, 酶活达到1.24 U/mL, 又用同样的方法对桔红色突变株进行回复突变处理, 得到一株白色回复突变株和一株淡粉色回复突变株, 两株回复突变株产胆固醇氧化酶的能力又明显下降, 酶活分别为0.17 U/mL和0.69 U/mL。说明短杆菌Brevibacterium sp.产胆固醇氧化酶能力与其产红色素成正相关偶联关系, 这种相关性模型的建立可以作为以后诱变或定向进化研究的筛子。     

通过紫外与伽马射线的复合诱变筛选高糖化酶活力的菌株（英文）

糖化酶,也称淀粉转葡萄糖酶,是淀粉水解过程中一种极其重要的酶类。通过紫外线和伽马射线的复合诱变得到一株酶活性极高的黑曲霉菌株,命名为AnUVγ‐147。该菌株的酶活性(1 182.2U/mL)是原菌株酶活性(86.5U/mL)的13.7倍,并且具有良好的稳定性,经过8代传代培养后酶活性为932.5U/mL。     

一种胆固醇氧化酶高产菌株筛选方法的建立

用亚硝基胍(1 mg.mL)与超声波(200 W,50 kHz)复合诱变的方法,对产胆固醇氧化酶短杆菌 Brevibacterium sp.DGCDC-82 进行诱变处理.得到一株桔红色突变株其产胆固醇氧化酶能力提高140%,酶活达到1.24 U/mL,又用同样的方法对桔红色突变株进行回复突变处理,得到一株白色回复突变株和一株淡粉色回复突变株,两株回复突变株产胆固醇氧化酶的能力又明显下降,酶活分别为0.17 U/mL和0.69 U/mL.说明短杆茵 Brevibacterium sp.产胆固醇氧化酶能力与其产红色素成正相关偶联关系,这种相关性模型的建立可以作为以后诱变或定向进化研究的筛子.     

利用根霉和酿酒酵母转化生玉米粉为高浓度酒精

利用从自然界中新分离得到的生淀粉酶产生菌-根霉W-08,并首次采用固态和液态相结合的培养方法,使生淀粉糖化酶活量达到72IU/mL,然后以生玉米粉为底物,利用根酶W-09和酿酒酵母Z-06采用同步糖化与发酵工艺,30℃,48h,发酵醪酒精浓度达到21%(V/V),转化率为理论转化率的94.5%。     

酯酶产生菌株的分离筛选

利用溴甲酚紫染色法和发酵测酶活的方法,从土壤中筛选出一株酯酶高产菌株ZM1。对ZM1进行形态特征及5.8SrDNA基因两侧的内转录间隙进行序列分析推测该菌株为灰绿犁头霉(Absidia glauca Hagem)。经过紫外诱变处理,得到1株正突变株ZMM1,在适合条件下,酶活达到58.76U/mL,比出发菌株提高了104.3%。传代实验表明ZMM1具有良好的遗传稳定性。     

菊粉酶产生菌的筛选及60Co诱变选育简

目的：从新疆石河子盐碱地菊芋生长根际土壤中分离筛选高产菊粉酶活力菌株。方法：通过稀释平板涂布法分离微生物;利用^60Co诱变选育,96孔板筛选突变菌株;采用3,5-二硝基水杨酸比色法测定菊粉酶酶活。结果：分离到12株具有菊粉酶活力的菌株,复筛得到1株高产菊粉酶活力菌株,将其命名为G-60;以此菌株为出发菌株进行^60Co诱变,利用96孔板对诱变菌株进行筛选,经摇瓶发酵酶活测定,得到1株高产菊粉酶酶活的突变株,酶活达46．62U/mL,是未诱变菌株酶活的2．72倍。结论：经诱变得到1株高产菊粉酶活力的突变菌株。     

菊粉酶产生菌的筛选及~(60)Co诱变选育

目的:从新疆石河子盐碱地菊芋生长根际土壤中分离筛选高产菊粉酶活力菌株。方法:通过稀释平板涂布法分离微生物;利用60Co诱变选育,96孔板筛选突变菌株;采用3,5-二硝基水杨酸比色法测定菊粉酶酶活。结果:分离到12株具有菊粉酶活力的菌株,复筛得到1株高产菊粉酶活力菌株,将其命名为G-60;以此菌株为出发菌株进行60Co诱变,利用96孔板对诱变菌株进行筛选,经摇瓶发酵酶活测定,得到1株高产菊粉酶酶活的突变株,酶活达46.62 U/mL,是未诱变菌株酶活的2.72倍。结论:经诱变得到1株高产菊粉酶活力的突变菌株。     

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。     

紫外、~(60)Co复合诱变选育烟色烟管菌漆酶高产突变株

对烟色烟管菌(Bjerkandera fumosa 5.0172)的孢子悬浮液进行紫外诱变筛选得到一株诱变株ZW-7,其产漆酶的活力是原始菌株的1.33倍,继代培养5代后,未见酶活下降。将诱变株ZW-7的孢子悬浮液用60Co-γ射线进行辐射诱变筛选得到一株诱变株Co-11,其产漆酶的活力是ZW-7的1.18倍,为380.5 U/L,较原始菌株的250.6 U/L提高了约58.1%,继代培养5代后,酶活稳定。     

一株产生淀粉分解酶犁头霉的分离鉴定及其酶学性质

从保宁麸醋醋曲中定向筛选得到一株产生淀粉分解酶的菌株CQB43,其酶活力为105.2 U/mL,RDA值达到27.9%。通过形态观察和分子生物学鉴定确定该菌株为伞枝犁头霉Absidia corymbifera。对该菌株分泌生淀粉酶酶学性质的研究结果表明,该酶在pH为4.0-5.6的范围相对稳定,最适pH是5.0;在60°C以下的范围内具有较好的热稳定性,最适作用温度为40°C。研究金属离子对其活力影响的结果表明,Co2+对该生淀粉糖化酶有激活作用,Fe3+和Ca2+对该酶有抑制作用。     

嗜热菌来源的生淀粉酶分离纯化及其酶学性质

从嗜热菌库中分离到两株能水解生淀粉的菌株173和174,通过扩增和测定两株菌的16S rDNA序列并进行比对结果表明,所分离两株菌属于Geobacillus属的细菌.液体摇瓶发酵菌株173、174,其产生的生淀粉酶(简称RSDE173、RSDE174)活力分别达14.5 U/mL和12.9 U/mL.通过生淀粉吸附-熟淀粉洗脱系统和TOYOPEARL HW-55F系统进行分离纯化,得到纯化的RSDE173和RSDE174,纯化倍数分别为50和29,活力回收率分别为34%和41%.有关RSDE173和RSDE174酶学性质研究显示.对熟淀粉水解的最适作用温度均为70℃,而对生淀粉水解则分别在50℃～60℃和40℃～60℃下表现出高水解活力;对不同底物的最适作用pH值均为5.0～5.5;它们对大多数试验离子的敏感性较低,但个别离子如Co2 、Cu'2 对RSDE173或u'2 对RSDE174的酶活力有一定的抑制作用.纯化的这两种生淀粉酶对不同来源生淀粉的底物专一性并不相同.RSDE173底物专一性顺序为红薯淀粉>小麦淀粉>玉米淀粉>木薯淀粉>糯米淀粉;而RSDE174的糯米淀粉>小麦淀粉>红薯淀粉>玉米淀粉>木薯淀粉.RSDE173对生红薯淀粉有很好的降解,其水解糊化淀粉与生红薯淀粉的比值为1.48;而RSDE174优先降解生糯米淀粉,其相应比值为1.69.     

生淀粉酶生产菌株Paenibacillus sp.的筛选和酶的纯化及酶学性质

分离得到1株产生淀粉酶的菌株,通过扩增和测定16S rDNA序列并进行比对,发现是Paenibacillus属的细菌。液体摇瓶发酵结束后,其产生的生淀粉酶比酶活达108.5U/mL。通过饱和(NH4)2SO4沉淀、Sephacryl S-300层析的方法对其所产的生淀粉酶进行分离纯化,得到纯化的酶蛋白比酶活为5112.04U/mg,纯化倍数为14.1,相对分子质量约为1.0×105。该酶以木薯生淀粉为底物时,最适pH5.6,最适温度50℃。金属离子Ca2+、Mg2+对该酶具有激活作用,Zn2+、Cu2+、Fe2+、Ni2+、Mn2+、Co2+和EDTA2-对该酶均具有抑制作用。     

Alteration of the properties of Aspergillus sp. K-27 glucoamylase on limited proteolysis with subtilisin

An active derivative (mol. wt. 48,000) of Aspergillus sp. K-27 glucoamylase (mol. wt. 76,000) was obtained by limited proteolysis with subtilisin. The amino acid sequences of native and modified enzymes at the N-termini were Ala-Gly-Gly-Thr-Leu-Asp and Ala-Val-Leu, respectively. The proteolysis greatly decreased the affinity of the enzyme for amylopectin and glycogen, but not for oligosaccharides. It also reduced the ability of the enzyme to degrade raw starch, abolished the ability of the enzyme to adsorb onto starch granules, and eliminated the synergistic action of the enzyme in the hydrolysis of starch granules with alpha-amylase. These findings imply that the enzyme has a specific affinity site for polysaccharide substrates besides the catalytic site, i.e., a starch-binding site, and that the former is removed by proteolysis. The extent of the reduction in the activity for raw starches caused by the modification varied with the starch source, as the modified enzyme digested raw potato starch better than either raw corn or sweet potato starches. A new method for evaluation of the raw starch-digesting activity of glucoamylase is described.     

Production and Characteristics of Raw Starch-Digesting Glucoamylase O from a Protease-Negative, Glycosidase-Negative Aspergillus awamori var. kawachi Mutant

Production of a raw starch-digesting glucoamylase O (GA O) by protease-negative, glycosidase-negative mutant strain HF-15 of Aspergillus awamori var. kawachi was undertaken under submerged culture conditions. The purified GA O was electrophoretically homogeneous and similar to the parent glucoamylase I (GA I) in the hydrolysis curves toward gelatinized potato starch, raw starch, and glycogen and in its thermostability and pH stability, but it was different in molecular weight and carbohydrate content (250,000 and 24.3% for GA O, 90,000 and ca. 7% for GA I, respectively). The chitin-bound GA O hydrolyzed raw starch but the chitin-bound GA I failed to digest raw starch because chitin was adsorbed at the raw starch affinity site of the GA I molecule. The removal of the raw starch affinity site of GA O with subtilisin led to the formation of a modified GA O (molecular weight, 170,000), which hydrolyzed glycogen 100%, similar to GA O and GA I, and was adsorbed onto chitin and fungal cell wall but not onto raw starch, Avicel, or chitosan. The modified GA I (molecular weight, 83,000) derived by treatment with substilisin hydrolyzed glycogen up to only 80% and failed to be adsorbed onto any of the above polysaccharides. The N-bromosuccinimide-oxidized GA O lost its activity toward gelatinized and raw starches, but the abilities to be adsorbed onto raw starch and chitin were preserved. It was thus suggested that both the raw starch affinity site essential for raw starch digestion and the chitin-binding site specific for the binding with chitin in the cell wall could be different from the active site, located in the three respective positions in the GA O molecule.     

Isoglucose production from raw starchy materials based on a two-stage enzymatic system

A new low-cost glucoamylase preparation for liquefaction and saccharification of starchy raw materials in a one-stage system was developed and characterized. A non-purified biocatalyst with a glucoamylase activity of 3.11 U/mg, an alpha-amylase activity of 0.12 WU/mg and a protein content of 0.04 mg protein/mg was obtained from a shaken-flask culture of the strain Aspergillus niger C-IV-4. Factors influencing the enzymatic hydrolysis of starchy materials such as reaction time, temperature and enzyme and substrate concentration were standardized to maximize the yield of glucose syrup. Thus, a 90% conversion of 5% starch, a 67.5% conversion of 5% potato flour and a 55% conversion of 5% wheat flour to sweet syrups containing up to 87% glucose was reached in 3 h using 1.24 glucoamylase U/mg hydrolyzed substrate. The application of such glucoamylase preparation and a commercially immobilized glucose isomerase for the production of glucose-fructose syrup in a two-stage system resulted in high production of stable glucose/fructose blends with a fructose content of 50%. A high concentration of fructose in obtained sweet syrups was achieved when isomerization was performed both in a batch and repeated batch process.     

Repeated fermentation from raw starch using Saccharomyces cerevisiae displaying both glucoamylase and α-amylase

A diploid yeast strain displaying both α-amylase and glucoamylase was developed for repeated fermentation from raw starch. First, the construct of α-amylase was optimized for cell surface display, as there have been no reports of α-amylase-displaying yeast. The modified yeast displaying both glucoamylase and α-amylase produced 46.5 g/l of ethanol from 200 g/l of raw corn starch after 120 h of fermentation, and this was 1.5-fold higher when compared to native α-amylase-displaying yeast. Using the glucoamylase and modified α-amylase co-displaying diploid strain, we repeated fermentation from 100g/l of raw starch for 23 cycles without the loss of α-amylase or glucoamylase activity. The average ethanol productivity and yield during repeated fermentation were 1.61 g/l/h and 76.6% of the theoretical yield, respectively. This novel yeast may be useful for reducing the cost of bio-ethanol production and may be suitable for industrial-scale bio-ethanol production.     

嗜热糖化酶基因在重组黑曲霉工程菌中的表达及酶学性质初步研究

为了提高糖化酶的耐热性能,降低淀粉糖化发酵工艺的生产成本,构建了同源整合载体pEasy-glaAdir以及pEasyssg,将黑曲霉(Aspergillus niger)的糖化酶基因(glaA)灭活,并将硫磺矿硫化叶菌(Sulfolobus solfataricus)的嗜热糖化酶基因(ssg)插入到黑曲霉基因组中,筛选得到表达嗜热糖化酶的重组黑曲霉工程菌(A.nigerWW1)。重组菌的发酵结果显示,嗜热糖化酶在黑曲霉中得到了分泌表达,发酵液酶活达到3 030 U/mL。重组嗜热糖化酶的最适反应温度为90℃,最适pH为6.0,该酶具有较高的热稳定性,在80℃时的半衰期在60 min以上,具有良好的工业应用前景。     

General Biochemical Characterization of Thermostable Pullulanase and Glucoamylase from Clostridium thermohydrosulfuricum

Cell extracts of Clostridium thermohydrosulfuricum, an anaerobic bacterium which ferments starch into ethanol at 65°C, contained both pullulanase and glucoamylase activities. The general physiochemical and catalytic properties of these enzyme activities were compared. Pullulanase and glucoamylase activities were stable and optimally active at 85 and 75°C, respectively. The pH optima for activity and pH stability ranges were, respectively, 5.5 to 6 and 4.5 to 5.5 for pullulanase and 4 to 6 and 5 to 6 for glucoamylase. The apparent [S]_0.5v and V_max for pullulanase activity on pullulan were 0.33 mg/ml and 2.6 U/mg of protein. The apparent [S]_0.5v and V_max for glucoamylase activity on starch were of 0.41 mg/ml and 0.31 U/mg of protein. These enzymes were active and stable in the presence of air or 10% (vol/vol) ethanol. These enzyme activities allowed the organism to actively degrade raw starch into glucose in the absence of significant α-amylase activity.