一株降解牛血液蛋白细菌的筛选及酶学性质研究

为了对屠宰血液进行有效处理与利用,以甘南藏族自治州美仁草原秃鹫粪便为分离基质,筛选并鉴定能够降解牦牛血液蛋白的菌株,并探究其生长条件及酶学特性。通过酪蛋白平板和血平板筛选能够分解血液蛋白的菌株,应用液体培养菌株探究碳源、氮源、温度、pH对菌株生长的影响,采用福林酚法测定蛋白酶活性,并提取蛋白酶研究其最适pH、最适温度以及热稳定性和pH稳定性。结果显示,通过选择培养基筛选得到1株高效降解血液蛋白的菌株,编号为NwMCC01910069,经过形态学鉴定、生理生化鉴定和16S rRNA基因鉴定,该菌株属于Providencia属,其生长的最适条件:碳源麦芽糖、氮源酵母粉、温度30℃及pH 8.5,该菌株产蛋白酶最适温度为40℃,当pH值为7～10时,该酶具有较高的酶活性且所产碱性蛋白酶具有较好的温度和pH稳定性。此外,通过菌株对牛血液蛋白降解效果的初步验证,当血液添加量为95%时,发现其降解率达到18.35%。说明该菌株对牦牛血液蛋白有一定的降解能力,有潜力作为废弃血液分解为氨基酸从而生产有机水溶肥的候选功能菌株。     

嗜热菌Anoxybacillu sp.产淀粉酶培养基优化及产酶条件研究

目的：对产淀粉酶嗜热菌Anoxybacillu sp.菌株进行培养基优化及产酶条件研究,以便提高菌株的产酶能力,并为下一步菌 株的诱变育种研究提供基础。方法：常规方法液体培养菌株,用平板初筛和DNS法复筛选择产淀粉酶能力较高的菌株;单因素筛 选培养基最适的碳源、氮源、Ca2+浓度和Mg2+浓度,对单因素筛选的最佳碳源、氮源、Ca2+和Mg2+的三个较佳浓度进行四因素三 水平正交试验优化培养基;对培养基不同pH值及不同培养温度进行培养条件研究。结果：产淀粉酶菌株筛选结果显示：六株菌中 淀粉酶酶活力值最大的是菌株Anoxybacillu sp.DL4,差异有统计学意义（P＜0.05）。培养基单因素筛选结果显示：最适碳源为麦芽糖、最适氮源为 硝酸铵、最适Ca2+、Mg2+浓度均为0.02%,差异有统计学意义（P＜0.05）。培养基优化结果显示：C 源0.1 %,N源0.2 %,Mg2+ 0.04%, Ca2+ 0.04 %为最佳的培养基成分组合。产酶条件筛选结果显示：培养基pH 值为6、培养温度为55 ℃时菌株产酶水平最高,差异有 统计学意义（P＜0.05）。结论：培养基的优化及最适的产酶条件能提高嗜热菌Anoxybacillu sp.DL4 产淀粉酶能力,Ca2+、Mg2+离子 对菌株产淀粉酶有促进作用。     

柑桔全爪螨高致病力菌株芽枝状枝孢霉的生物学特性研究

【目的】明确柑桔全爪螨Panonychus citri(Mc Gregor)高致病力菌株芽枝状枝孢霉Cladosporium cladosporioides(CQBBW3)的生物学特性。【方法】通过单因素试验,测试了不同培养基、碳源、氮源、温度及pH对CQBBW3菌株生长和产孢的影响。【结果】CQBBW3菌株的LC50为4.66×108个孢子/m L,LT50为6.2353 d。最佳生长培养基是淀粉琼脂培养基,最适产孢培养基为察氏培养基;菌丝生长最适碳源和氮源为甘露醇和甘氨酸,产孢最适碳源和氮源为乳糖和赖氨酸;最适生长温度为25℃,最适产孢温度为15℃和30℃;最适生长pH值为6.0~8.0,最适产孢pH值7.0。【结论】CQBBW3菌株对柑桔全爪螨有较好的杀虫活性,喜好25℃、中性、富含有机营养的环境。本研究为菌株快速培养及其真菌杀虫制剂的开发与田间应用奠定基础。     

絮凝剂产生菌的筛选、培养及产物性质研究

从活性污泥中筛选出一株高效产絮凝剂菌株,经鉴定为嗜温鞘氨醇杆(Sphingobacterium thalpophilum).采用单因素实验,结果显示该菌产絮凝荆的最适碳源为可溶性淀粉,最适氮源为硫酸铵,最适初始pH值7.0,最适培养温度为30℃.性质研究分析显示,絮凝剂纯品的热稳定性差,其在pH值2.0～8.0的范围内可以保持较高的絮凝活性.     

高酶活菌株的筛选及漆酶特性

通过Bavendamn氏反应和液体发酵实验筛选出漆酶高产菌株 ,并对其产酶条件和酶活性进行了研究。结果表明 71株实验真菌中有 64株Bavendamn氏反应呈阳性 ,且阳性菌株都具有漆酶活性 ;不同菌株产酶培养基最适碳源、氮源不同 ,采绒革盖菌以淀粉为碳源、干酪素为氮源 ,毛栓菌以麦草粉为碳源、硫酸铵为氮源 ,有利于酶的分泌 ;不同来源漆酶性质不尽相同 ,采绒革盖菌漆酶最适酶解温度为 2 5℃ ,最适酶解pH值为4.6,毛栓菌则分别为 3 0℃和 pH 4.0 ;K+ ,Zn2 + 等对 2种漆酶均有激活作用 ,Ag+ 则能明显抑制漆酶活性。     

耐盐碱菌株的分离筛选及生物学特性和盐碱去除效率的研究

将采集于黑龙江省大庆市盐碱地的土壤样品经适当稀释后,涂布于高盐碱的培养基中,分离纯化获得41株菌。经过不断分别或同时提高培养基的盐、碱浓度进行耐盐碱菌株的筛选,获得1株耐盐碱菌株7,其在pH值13且盐(NaCl)浓度达到200g.L-1的培养基中仍可生长,该菌株既属于极端嗜盐微生物又属于极端嗜碱微生物。分别于不同碳源、氮源、碳氮比、初始pH值和培养温度条件下,对菌株7的生物学特性和盐、碱去除能力进行研究。结果表明,该菌株生长和去除盐碱的最佳碳源为牛肉膏;最佳氮源为蛋白胨;碳氮比为4:1时,最有利于菌株的生长和对盐、碱的去除;菌株生长的最适初始pH值为9.5,最适温度为20℃,而盐、碱去除的最佳温度则为30～35℃。     

极区低温海洋细菌及其产酶情况的初步研究

通过对大量极区低温海洋菌株的分离、筛选及进一步的生理生化特性研究,获得1株最适生长温度为15℃、生长温度上限为35℃、产蛋白酶及多种多糖水解酶的耐冷细菌。该菌过氧化氢酶为阳性,具有弱嗜盐性;蔗糖、可溶性淀粉是有利于菌株生长的碳源物质,而酵母膏则是效果最佳的氮源物质。该菌株所产蛋白酶的最适作用温度为55℃,而淀粉酶、琼脂酶及纤维素酶的最适作用温度皆为35℃。     

极端微生物产碱性蛋白酶菌株的筛选及发酵条件研究

从深海的38份水样和泥样中筛选到一株产蛋白酶的菌株DY－A,其最适生长温度为10℃,能适应较大范围的pH值和盐度,此菌株只在酵母膏存在的情况下产酶,不利用单一氮源,最适产酶条件为,pH10.0,10℃,接种量0．5％,200r/min摇床培养40－72h,粗酶液的最适作用温度为40℃,最适pH值为10,在pH9-12内稳定,是一碱性蛋白酶。     

一株中温淀粉酶菌株的分离鉴定及其产酶条件分析

利用固体淀粉筛选培养基,从安阳市郊区面粉厂附近的土壤里分离筛选出1株产淀粉酶的菌株,编号为MF-3-2.经过菌株形态、革兰氏染色、16S rDNA鉴定及系统进化树分析,初步确定其为枯草芽孢杆菌(Bacillus subtilis).摇瓶培养后对其酶学性质研究发现,该菌株淀粉酶的最适温度为65℃,最适pH值为6.0,在pH值4.8～6.0范围内仍能残余70％以上的酶活力.该菌株的最适生长温度为40℃,最适生长pH值为6.5.产酶条件优化结果表明:最适碳源为马铃薯淀粉,最适氮源为豆粕粉,最适碳氮比为1∶15,发酵温度30℃,发酵pH值6.0,装液量10％,种龄10h,接种量5％,转速200 r/min,48 h达到产酶高峰.通过发酵产酶条件优化,其淀粉酶活性达到86.8 U/mL,是优化前的35倍.另外,在酸性条件下还具有较好的活性.因此,该菌株的淀粉酶具有潜在的工业应用前景.     

链霉菌产木聚糖酶条件和酶学性质以及重离子诱变对产酶的影响研究

以一株由青藏高原牦牛粪中分离出的链霉菌为出发菌株,对其培养特性、产酶条件和酶学性质进行初步研究.通过重离子诱变,筛选出遗传稳定的高产菌株.结果表明,该菌以玉米芯和麸皮(1∶1)为碳源能高效地诱导木聚糖酶的胞外分泌,其最适培养基和培养条件氮源为酵母膏、初始pH7、培养温度为25℃,在此条件下,第4天酶活力达到峰值3480.25 U/mL.说明该酶能够利用农业废弃物高效生产木聚糖酶.该菌株所产木聚糖酶的最适反应温度为15℃、pH4,属低温酸性木聚糖酶.经重离子诱变后,筛选出一株高产菌株SZ10-7,其酶活力可达5 338.42 U/mL.     

嗜热菌Anoxybacillus sp．DL3产蛋白酶条件及其酶学性质研究

目的：对Anoxybacillussp．DL3的产蛋白酶条件及其酶学性质进行研究,为下一步进行蛋白酶基因的克隆、表达提供依据。方法：应用常规方法液体培养细菌,研究温度、pH、培养基中碳源、氮源对菌株产蛋白酶的影响,硫酸铵盐析的方法提取酶液,并采用Folin法测酶活性。用紫外分光光度计在OD680hi／1测吸光值。并对提取的蛋白酶液进行酶的最适温度、pH以及酶的热稳定性和pH稳定性研究,向酶液中添加金属离子和EDTA、PMSF,研究其对酶活性的影响。结果：在培养基初始pH是6．5,培养温度为40℃时菌株产酶酶活性最大;培养基中以乳糖为碳源,酵母膏和硫酸铵为氮源,碳源与氮源的比例为1：2时,酶活最大。酶学性质研究结果显示：该酶的最适反应温度是55℃,最适反应pH是7．0;在50℃保温20min-80min内,酶活力下降幅度较小。60℃保温60min后,仍保持约60％的酶活。70℃保温60min后,残余酶活为30％。该酶在pH为6．0～8．0范围内,相对酶活差别不是很大,下降趋势大致相同。在强碱条件下,相对酶活下降很明显。Fe2＋、Cu2＋和Hg2＋对酶活性有明显的抑制作用;Ca2＋、Mg^2＋、Mn2＋等金属离子对酶活性有明显的促进作用;乙二胺四乙酸（EDTA）和苯甲基磺酰氟（PMSF）对酶活性也有一定抑制作用。结论：Anoxybacillussp．DL3所产的蛋白酶为嗜热中性蛋白酶,此酶具有较好的热稳定性和pH耐受性,该菌株具有进一步开发、利用的价值。     

Production of alkaline protease from a newly isolated Exiguobacterium profundum BK-P23 evaluated using the response surface methodology

Protease producing bacteria were isolated from soil in South Korea. These bacteria were screened in skim milk agar medium using skim milk as the substrate. The highest clear zone producing bacterial strain (BK-P23) was selected for further optimization studies. The strain was identified as Exiguobacterium profundum BK-P23 based on morphological, biochemical and molecular characterizations. The results of the 16S rRNA analysis showed that this strain was highly similar to E. profundum. The strain was able to grow under alkaline conditions at pH 8.5 and a temperature of 30°C. In the preliminary optimization experiments, five different parameters, i.e. carbon source (lactose), nitrogen source (corn steep solid), pH, temperature and incubation period were varied with the goal of optimizing enzyme production in low cost medium using the Box-Behnken design combined with response surface methodology. The optimal conditions were determined to be pH 9.0, a temperature of 30°C, lactose (1.0%) as the carbon source and corn steep solid (1.0%) as the cheap additional nitrogen source. In addition, 24 h of incubation was shown to produce the highest protease yield. Overall, the amount of enzyme produced was significantly higher in the optimized medium when compared with the original medium.     

葡萄糖3-脱氢酶产生菌的筛选和产酶培养

从土壤中筛选分离得到1株葡萄糖3-脱氢酶产生菌,经生理生化初步鉴定为产酸克雷伯菌(Klebsiella oxyto-ca)。将菌株D8进行产酶优化培养,经过单因素法确定优化培养条件:乳糖为C源,酵母膏为N源,250 mL摇瓶装液量为50 mL,培养基的初始pH为7,33℃培养时G3DH酶比活力最高,达到1.45 U。     

Optimization of conditions for protease production by Chryseobacterium taeanense TKU001

A protease-producing bacterium was isolated and identified as Chryseobacterium taeanense TKU001. An extracellular metalloprotease with novel properties of solvent- and surfactant-stable was purified from the culture supernatant of C. taeanense TKU001 with shrimp shell wastes as the sole carbon/nitrogen source. The optimized condition for protease production was found when the culture was shaken at 37 degrees C for 3 days in 50 mL of medium containing 0.5% shrimp shell powder (SSP) (w/v), 0.1% K2HPO4, and 0.05% MgSO4.7H2O. Two extracellular proteases (FI and FII) were purified and characterized, and their molecular weights, pH and thermal stabilities were determined. The molecular masses of TKU001 protease FI and FII determined by SDS-PAGE and gel filtration were approximately 41 kDa and 75 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU001 protease FI were 8, 60 degrees C, pH 6-9, and 60 degrees C, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU001 protease FII were 7, 60 degrees C, pH 7-9, and 50 degrees C, respectively. TKU001 protease FI and FII were both inhibited completely by EDTA, indicating that the TKU001 protease FI and FII were metalloproteases. TKU001 protease FI and FII retained more than 75% of its original protease activity after preincubation for 10 days at 4 degrees C in the presence of 25% most tested organic solvents. Additionally, the TKU001 protease FI retained 79%, 80%, and 110% of its original activity in the presence of 2% Tween 20, 2% Tween 40, and 2% Triton X-100, respectively. However, at the same condition, the activity of TKU001 protease FII retained 100%, 100%, and 121% of its original activity, respectively. This is the first report of C. taeanense being able to use shrimp shell wastes as the sole carbon/nitrogen source for proteases production. The novelties of the TKU001 protease include its high stability to the solvents and surfactants. These unique properties make it an ideal choice for application in detergent formulations and enzymatic peptide synthesis.     

Nutritional factors affecting organic solvent-tolerant alkaline protease production by a newBacillus cereus strain 146

An organic solvent-tolerant bacterium designated as 146 capable of producing an organic solvent-stable alkaline protease was isolated from contaminated soil of a wood factory. The strain was a Gram-positive, spore-forming, nitrate-positive, rod-shaped organism capable of hydrolysing gelatine, starch, skim milk and identified asBacillus cereus. Activity of the protease was drastically increased in the presence of 1–decanol, isooctane, n-dodecane and n-tetradecane, but reduced in the presence of ethyl acetate, benzene, toluene, 1-heptanol, ethylbenzene and hexane. The bacterium was shown to require lactose as a carbon source and peptone as a nitrogen source. The optimum fermentation condition for the production of alkaline protease was in the presence of beef and yeast extract. Optimum pH was determined to be at 10.0 at incubation temperature of 37 °C for 48 h. Results from the studies suggest that 146 is a new strain of Bacillus cereus capable of producing organic solvent-tolerant alkaline protease with potential use in industries.     

丝氨酸生产菌的筛选及静息细胞培养系统中L-丝氨酸的合成

从广西隆安县沼气池里的残渣中筛选到一株能以甲醇为唯一碳源生长的MB200菌株。根据常规形态特征、生理生化性状及16S rDNA基因序列分析将其鉴定为甲基杆菌属(M ethylobacteriumsp.)。其最佳生长条件为:温度32℃、pH值8.0、甲醇体积分数1.25%。建立了MB200生成L-丝氨酸的静息细胞培养系统。确定静息细胞培养的条件为:甘氨酸质量浓度为10 g.L-1,甲醇50 g.L-1,菌体质量浓度为30 g.L-1,pH8.9,于摇床250 r.m in-1,32℃静息培养48 h,L-丝氨酸产量为7.2 g.L-1。     

Optimization of culture conditions for the production of haloalkaliphilic thermostable protease from an extremely halophilic archaeon Halogeometricum sp. TSS101

AIMS: Isolation and screening of extreme halophilic archaeon producing extracellular haloalkaliphilic protease and optimization of culture conditions for its maximum production. METHODS AND RESULTS: Halogeometricum sp. TSS101 was isolated from salt samples and screened for the secretion of protease on gelatin and casein plates containing 20% NaCl. The archaeon was grown aerobically in a 250 ml flask containing 50 ml of (w/v) NaCl 20%; MgCl(2) 1%; KCl 0.5%; trisodium citrate 0.3%; and peptone 1%; pH 7.2 at 40 degrees C on rotary shaker. The production of enzyme was investigated at various pH, temperatures, NaCl concentrations, metal ions and different carbon and nitrogen sources. The partially purified protease had activity in a broad pH range (7.0-10.0) with optimum activity at pH 10.0 and a temperature (60 degrees C). The enzyme was thermostable and retained 70% initial activity at 80 degrees C. Maximum protease production occurred at 40 degrees C in a medium containing 20% NaCl (w/v) and 1% skim milk powder after 84 h in shaking culture. Enzyme secretion was observed at a broad pH range of 7.0-10.0. Addition of CaCl(2) (200 mmol) to the culture medium enhanced the production of protease. Protein rich flours proved to be cheap and good alternative source for enzyme production. Different osmolytes were tested for the growth and production of haloalkaliphilc protease and found that betaine and glycerol enhanced growth without secretion of the protease. Immobilization studies showed that whole cells immobilized in 2% alginate beads were stable up to 10 batches and able to secrete the protease, which attained maximum production within 60 h under shaking conditions. CONCLUSIONS: Halogeometricum sp. TSS101 secreted an extracellular haloalkaliphilic and thermostable protease. The optimum conditions required for maximum production are 20% NaCl, 1% skim milk powder and temperature at 40 degrees C. Addition of CaCl(2) (200 mmol) enhanced the enzyme production. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of haloalkaliphilic protease. SIGNIFICANCE AND IMPACT OF THE STudy: The low cost protein rich flours were used as an alternative carbon and nitrogen sources for enzyme production. Immobilization of halophilic cells in alginate beads can be used in continuous production of halophilic enzyme. The halophilic and thermostable protease from Halogeometricum sp. TSS101 is good source for industrial applications and can be a suitable source for preparation of fish sauce.     

产高温纤维素酶木霉菌株的筛选、鉴定及酶学性质研究

从稻草堆肥中筛选得到一株产高温纤维素酶的霉菌M1,通过形态学观察和分子生物学鉴定,确定其为木霉属(Trichoderma)。在稻草液体发酵培养基中,木霉M1的CMC酶(carboxymethyl cellulase,CMCase)合成模式为同步合成型。酶学性质研究表明,此CMC酶的最适反应pH为4.4,在pH 4.0～6.0保温4h仍可保持95%以上的酶活力;其最适反应温度为75℃,在50℃下保温4h,可保持87%的酶活力;60℃下保温4h,可保持65%的酶活力,具有较好的热稳定性。     

Isolation and Culture Conditions of Thermophilic Hydrogen Bacteria

Isolation of thermophilic hydrogen bacteria was performed at 50°C using enrichment culture method. One of the four strains isolated, strain TH-1 grew most rapidly. Culture conditions of strain TH-1 were investigated. Optimum temperature and pH for growth proved to be 52°C and 7.0, respectively. There existed a positive correlation between the specific growth rate and the partial pressure of carbon dioxide in the gas phase. Ammonium and nitrate are the good nitrogen sources in that order. Effect of concentrations of nitrogen source, magnesium, ferrous and phosphate ions on the cell growth was also investigated. The maximum specific growth rate (μ_max) of strain TH-1 was determined as 0.68 hr^?1 by the cultivation at 52°C in a jar fermentor containing the optimal medium at pH 7.0.