里氏木霉与米根霉混合固态发酵产纤维素酶

以里氏木霉及米根霉单菌固态发酵为对象,考察不同混合发酵形式对里氏木霉与米根霉混合固态发酵产纤维素酶的影响。结果表明:同时接种里氏木霉与米根霉,试验考察的两菌种接种量比1∶1(以孢子个数计)及5∶1条件下,两菌未产生明显协同产酶作用。米根霉延时(24 h)接种且菌种量比5∶1以及米根霉延时(48 h)接种且菌种量比1∶1,2种发酵形式产酶情况类似,滤纸酶活(FPA)及羧甲基纤维素酶(CMCase)酶活相对米根霉单菌发酵有所提高,而β-葡萄糖苷酶(β-GA)酶活相对里氏木霉单菌固态发酵结束时分别增加4.66及4.40倍,可以发现两菌产生一定协同作用。在米根霉延时(48 h)接种且菌种量比5∶1的发酵形式下,FPA及CMCase在发酵第7天酶活分别达到44.04 IU/g、627.14 U/g(以1 g干曲计),分别是里氏木霉固态单菌发酵产酶达到稳定期时酶活的1.36和1.63倍,两菌产生了有效的协同作用。     

甘蔗渣固态发酵产纤维素酶

以甘蔗渣和麸皮混合作为固态发酵产酶培养基,采用单因素优化实验对里氏木霉固态发酵产纤维素酶进行优化。结果表明,在50 m L体系培养基中,在底物绝干原料5.2 g、甘蔗渣与麸皮质量比7∶3、氮源((NH4)2SO4)7.5 g/L、产酶诱导物1.6 g/L、表面活性剂(聚乙二醇PEG6000)0.1 g、发酵起始p H 4.4、培养基中里氏木霉孢子接入量5×105个的条件下,温度30℃时发酵120 h,里氏木霉固态发酵产纤维素酶的酶活达76.39 IU/g,是起始优化前20.29 IU/g的3.76倍。     

里氏木霉与黑曲霉混合发酵产纤维素酶及其水解特性

研究了利用里氏木霉和黑曲霉混合培养产纤维素酶,以黑曲霉孢子悬浮液的不同活化浓度及不同的活化时间来寻找2个菌种发挥最大协同作用的结合点以及所产纤维素酶的水解特性。以里氏木霉单一培养和黑曲霉单一培养为参照进行对比研究。底物为农林废弃物之一的玉米秸秆,经过蒸气爆破预处理后,用作产酶C源。结果表明:黑曲霉孢子悬浮液活化浓度为10个/mL,活化时间为12 h时,滤纸酶比酶活最高,达3.32 U/mL,高于里氏木霉单一培养的2.25 U/mL,β-葡萄糖苷酶比酶活达1.32 U/mL,高于里氏木霉单一培养的0.57 U/mL。为进一步验证混合菌产纤维素酶的水解效果,利用混合菌产纤维酶的酶液及里氏木霉产纤维素酶的酶液进行酶水解实验,当酶用量为20 U/g绝干纤维素,底物质量浓度为100 g/L条件下水解48 h,混合菌所产酶液酶解得率达70.00%,高于里氏木霉所产酶液的酶解得率63.05%。实验表明里氏木霉与黑曲霉混合培养产酶是可行的,并优于单一菌种培养。     

里氏木霉LW1固态发酵纤维素酶条件的研究

采用里氏木霉LW 1(Trichoderma.Reesei)固体发酵生产纤维素酶,研究了秸杆粉和麦麸用量、料水比、起始pH值、发酵温度和发酵时间对该菌株产纤维素酶活力的影响。试验结果表明,里氏木霉LW 1的适宜发酵条件为:在秸秆∶麦麸=1∶1,料水比为1∶2的前提条件下,培养温度28℃,发酵周期为72h,起始pH5.5时产酶活力最高。浸出液中FPA酶活为119.417u/g干物质,CMC酶活为452.433u/g干物质。     

里氏木霉306产t-PA固态发酵条件的研究

对基因工程菌株里氏木霉(Trichodermareesei)306生物合成组织型纤溶酶原激活剂(t-PA)的固态发酵培养基成分和发酵条件进行了研究;分析了发酵过程中t-PA的生成、总糖的消耗和菌体生长的规律。在优化的固体发酵条件下,t-PA的最大酶活是924.63IU/g干重培养基,较初始条件下提高了5倍。通过浅盘进行了放大实验,最大酶活为983.64IU/g干重培养基,t-PA合成速率为:13.66IU/g干重培养基.h,较三角瓶固态发酵最高产酶提高了6.38%,产酶速率提高了24.07%。     

纤维素酶的固态发酵及酶学性质的研究

对绿色木霉产纤维素酶的固态发酵条件进行研究,显示其最优产酶条件为:稻草:麸皮=3:2,水分250%,最佳氮源为0.5%(NH4)2SO4,最佳培养时间3天,温度30℃,接种量10%,初始pH6.0～7.0,在此条件下,每克干曲酶活分别高达18.4FPAU/g,56.7CMCU/g。同时对酶解作用条件进行了初步研究,发现pH5.2时对滤纸酶活力最高,pH4.8时对脱脂棉活力最高。降解温度以滤纸55℃,脱脂棉50℃为最佳。实验还发现乙醇对酶的降解有抑制作用。     

黑曲霉与里氏木霉固态混合发酵产β-葡萄糖苷酶

对黑曲霉NL02与里氏木霉RUT-C30固态混合发酵产β-葡萄糖苷酶的发酵培养基进行优化,研究培养基含水率、C源、N源、接种量、温度和2种菌种不同延长接种时间与接种比例对β-葡萄糖苷酶活力的影响。研究表明：麸皮17.5 g、玉米芯7.5 g、（NH4）2SO4 0.40 g、尿素0.37 g、黑曲霉孢子接入量为107个接种到250 mL三角瓶中,温度30 ℃、摇床转速100 r/min时,里氏木霉以105个孢子与黑曲霉同时接入,每克干曲所得β-葡萄糖苷酶的活力为132.45 IU,较黑曲霉单独培养时的104.35 IU提高了26.94%。     

高效转化法定向进化里氏木霉的纤维素酶

优化了根癌农杆菌介导的里氏木霉的转化方法,得出转化的最适条件:共培养pH 5.3、共培养温度24℃、乙酰丁香酮浓度200μmol/L、根癌农杆菌OD660值0.8、里氏木霉孢子预萌发时间3 h。此时转化率为13 000个转化子(以107个里氏木霉孢子计),是未优化条件下的27倍以上。利用此高效转化法,结合强化纤维二糖水解酶基因II表达的操作,从324个转化子中筛选到了1株优良的里氏木霉转化子C10,发酵5 d后,纤维二糖水解酶活力达(122.44±5.91)U/m L,是初始菌株的5.4倍;滤纸酶活力达(28.92±2.45)IU/m L,是初始菌株的4.3倍。与初始菌株所产纤维素酶相比,转化子C10所产纤维素酶降解玉米秸秆和稻草的能力有明显的提高。本研究为里氏木霉的基因工程改造及其纤维素酶的定向进化提供了有价值的实验数据。     

高产纤维素酶的木霉菌株筛选及发酵条件优化

纤维素是由β-1,4糖苷键链接而成的葡萄糖单体聚合物,广泛分布于自然界,是一类易获取且廉价的可再生资源。自然界含纤维素材料的利用和处理是目前亟待解决的问题,筛选产纤维素酶的优良菌株并实现纤维素的有效利用具有重要意义。利用纤维素酶降解纤维素是绿色、高效和可持续的利用方式,广泛应用于工业和农业领域(养殖、能源、纺织等)。因此,实现高效低成本的纤维素酶生产成为研究和发展的目标。木霉属真菌是重要的纤维素酶产生菌,具有产酶量大、酶系全和酶活性高等优点,分泌的胞外酶易于工业生产中的分离提纯。本研究以课题组前期积累和保存的25种木霉150个菌株为材料,通过刚果红平板法筛选、滤纸酶活测定和天然纤维素降解等测试,筛选出14株产纤维素酶能力较强的菌株(优于对照菌株里氏木霉QM9414),从中选取3株进行最佳产酶条件探索,如接种量、吐温80添加量、发酵培养基pH值等。初步测试结果表明,贵州木霉菌株8705在20℃条件下、摇瓶发酵9 d、在优化后的发酵培养基中产酶效果最佳,其上清液酶活可达到6.63 IU/mL。通过优化发酵温度、发酵时间和培养基成分配比等发酵条件,为资源利用打下物质基础并提供理论依据。     

液态发酵条件下拟康宁木霉8985产纤维素酶能力初探

液态发酵条件下,以微晶纤维素为唯一碳源,比较了拟康宁木霉Trichoderma koningiopsis 8985和里氏木霉T. reesei QM9414产纤维素酶的能力。8985发酵12 h开始产生纤维素酶,36 h时酶活达到产酶峰值的50%,此时QM9414尚未诱导产酶。测定8985发酵84 h时上清液中滤纸纤维素酶、羧甲基纤维素酶、β-葡萄糖苷酶和木聚糖酶的酶活分别为1.06、3.62、1.80和6.67 IU/mL,分别是QM9414上述酶活的1.72、1.70、6.35和1.12倍。8985滤纸纤维素酶酶活的最适反应条件为pH 4.5,反应温度50 ℃,在Fe³⁺ (≤ 4 mmol/L)和Cu²⁺ (0-10 mmol/L)存在条件下酶活稳定。     

Studies on mixed fungal culture for cellulase and hemi-cellulase production part-1: Optimization of medium for the mixed culture ofTrichoderma reesei D1-6 andAspergillus Pt 2804

Biotechnology Letters - For enhanced production of cellulase and xylanase by the mixed culture ofT. reesei D1-6 andA. wentii Pt 2804, the composition of medium has been optimized.     

Effect of culture phasing and a polysaccharide on production of xylanase by mixed culture of Trichoderma reesei D1-6 and Aspergillus wentii Pt 2804

A significant increase in extracellular xylanase activity was observed in the mixed culture fermentation of Trichoderma reesei D1-6 and Aspergillus wentii Pt 2804 when A. wentii inoculation was phased by 15 h. A. wentii produced a polysaccharide, chiefly consisting of glucose monomeric units, which was required for expression of maximum xylanase activity. Expression of high activity of xylanase in the A. wentii phased mixed culture compared to that in either T. reesei or A. wentii single cultures appeared to be controlled by the combined action of a polysaccharide produced by A. wentii and the relative growth of the two fungi in the mixed culture.     

Optimization of ellagic acid production from ellagitannins by co-culture and correlation between its yield and activities of relevant enzymes

Aspergillus oryzae was co-cultured with Trichoderma reesei using acorn cups extract containing up to 62% ellagitannins as substrate to produce ellagic acid with relatively high levels of ellagitannin acyl hydrolase, cellulase and xylanase. Ellagitannins concentration, initial pH, T. reesei and A. oryzae during the fermentation were identified as important process parameters effecting ellagic acid accumulation and the enzymes syntheses. These parameters were optimized by uniformity design to determine the optimum condition for ellagic acid production. Under optimum operational condition, ellagic acid yield could be arrived at 24%, when the fermentation run lasted 96h with an initial pH of 4.5, an ellagitannins concentration of 4gl(-1), T. reesei of 3ml and A. oryzae of 3ml. Meanwhile, it was found that the three enzymes activities correlated very well with ellagic acid yield, resulting in model with high coefficient of determination (R(2)=0.98). The results indicate that the mixed culture of T. reesei and A. oryzae is an effective approach to produce an enzyme system of degrading ellagitannins for ellagic acid production.     

Cellulase production by a solid state culture system

Production of cellulase using solid culture systems of Trichoderma reesei QM9414 and Sporotrichum cellulophilum on wheat bran was studied. By using moisture-controlled solid culture equipment, the effect of water content of wheat bran on cell growth and cellulase production was investigated. Cellular biomass grown on solid substrate was estimated by measuring oxygen consumption rate and glucosamine content of the cells. These parameters were shown to have a good linear correlation with the specific growth rate. This reliable method of estimating the cell growth rate enabled us to simulate the enzyme production in a solid culture system by means of multiple linear regression analysis which takes into account of the water content, cell mass, and the oxygen consumption rate as variables. The cell growth and cellulase production were maximized at different water content of the medium. A high water content, 57% for T. reesei and 70% for S. cellulophilum, favored mycelial growth, while the maximum cellulase activity was obtained at a lower water content such as 50% for both fungi. It was observed that cellulase production by T. reesei depended on the culture conditions that support the optimal growth rate for the maximum enzyme production.     

Production of cellulase on sugar cane bagasse by fungal mixed culture solid substrate fermentation

Trichoderma reesei LM-UC4 and its mutant LM-UC4E1 were co-cultured with Aspergillus phoenicis QM329 for cellu-lase production on bagasse by mixed culture solid substrate fermentation. A mutual synergism was observed between the parent Trichoderma strain and the Aspergillus, resulting in enhanced combined biomass production and corresponding increased in cellulase, endoglucanase and b-glucosidase activities. Such synergism was absent with the mutant Trichoderma strain suggesting that in the hypermutation the ability for cooperative interaction with other microbes was lost.     

毛头鬼伞菌丝体对培养料中不同养分的利用特点

将毛头鬼伞菌丝体接种到不同栽培培养基上,分析了菌丝体对培养基中综纤维素、木质素以及淀粉利用的动态变化。结果表明:毛头鬼伞菌丝体对培养基中的综纤维素、木质素以及淀粉都有较好的利用,在淀粉含量高的情况下,毛头鬼伞菌丝体对综纤维素、木质素的利用表现出一种延迟。另外,通过分析毛头鬼伞菌丝体对综纤维素、木质素利用的比值,得出其对木质素利用的比值高。因此,在毛头鬼伞的稻草栽培培养基中添加适量的木屑是可行的。     

Extractive fermentation for improved production of endoglucanase by an intergeneric fusant of Trichoderma reesei/Saccharomyces cerevisiae using aqueous two-phase system

Extractive aqueous two-phase fermentation of endoglucanase, a key enzyme for the conversion of cellulosic substances to fermentable sugars, from an intergeneric fusant of Trichoderma reesei/Saccharomyces cerevisiae is a meaningful approach for better production and simple recovery of this enzyme. A phase composition of 6.5% (w/w) dextran and 7.5% (w/w) polyethylene glycol 6000, having a partition coefficient of 2.89 and 1.31 for endoglucanase from an intergeneric fusant of T. reesei/S. cerevisiae and T. reesei (WT) (being a control in this study), respectively, was chosen for extractive fermentation of the enzyme. Endoglucanase production is higher in medium containing polyethylene glycol (PEG) 6000 than in medium without PEG 6000. Comparative analysis of endoglucanase fermentation by fusant and T. reesei was carried out in shake culture and environment-controlled bioreactor conditions. The fusant produced 0.43U of endoglucanase (overall production: 0.34U) in the top phase of an aqueous two-phase system (ATPS), compared to 0.3U in medium without the phase system in shake culture. In a batch reactor, the endoglucanase level for the fusant in the top phase of ATPS was 0.49U (overall production: 0.40U), compared to 0.38U produced in medium without aqueous two-phase components. To corroborate this study, T. reesei produced 8.41U of endoglucanase (overall production: 5.96U) in the top phase of ATPS, compared to 7.18U in the medium without the phase system in shake culture. On the other hand, in a batch bioreactor, T. reesei produced 10.13U of endoglucanase (overall production: 6.90U) in the top phase of ATPS, compared to 8.56U of the enzyme in medium without aqueous two-phase components. The lower overall enzyme production by T. reesei in the two-phase system might be due to limitation in oxygen transfer to the dispersed phase where the enzyme is produced. A higher cell concentration and a reduced lag phase was obtained in ATPS, compared to a similar medium without phase forming polymers for both the intergeneric fusant of T. reesei/S. cerevisiae and T. reesei.     

Effect of culture phasing and mannanase on production of cellulase and hemicellulase by mixed culture of Trichoderma reesei D 1-6 and Aspergillus wentii pt 2804

Significant increase in extracellular cellulase and hemicellulase activities was observed in the biosynthesis of cellulase enzyme in mixed culture fermentation of Trichoderma reesei D 1-6 and Aspergillus wentii Pt 2804 when the A. wentii inoculation was phased by 15 h. The optimal conditions of fermentation by the mixed culture have been established. Presence of mannanase has been found to affect the release as well as activity of cellulase enzyme produced in mixed culture.