外切-β-葡聚糖酶基因重组里氏木霉的筛选及其产酶性能

外切-β-葡聚糖酶是纤维素酶的重要组分之一,提高该组分的活力是增强纤维素酶协同降解性能、降低纤维素水解成本的关键。分别采用微晶纤维素琼脂平板法和滤纸崩解法,对已有的基因重组转化子进行筛选试验,获得了6个优良转化子,其滤纸崩解速率和微晶纤维素琼脂平板上的生长速率都较大。进一步在摇瓶条件下进行复筛试验,获得了外切-β-葡聚糖酶(C1)高产转化子Trichoderma reesei ZU-101,液体培养48 h,其C1酶活力可达18.24 U·ml-1,是出发菌株的2.16倍;分析结果表明:重组转化子的纤维素酶体系中内切-β-葡聚糖酶和纤维二糖酶的活力与出发菌株相比变化不大,但由于外切-β-葡聚糖酶活力得到了大幅度提高,纤维素酶的总活力(滤纸酶活力FPA)也提高了61.9%。采用纤维素酶对碱预处理玉米秸秆进行酶解试验,当酶用量为20 FPIU·(g底物)-1,水解48 h,重组转化子T.reesei ZU-101纤维素酶的酶解得率高达94.4%。本文的研究结果在可再生纤维素资源的生物转化与利用方面具有广阔的应用前景。     

重组里氏木霉液体深层发酵产中性内切-β-葡聚糖酶

中性内切-β-葡聚糖酶在纺织、食品和造纸等领域中应用广泛。研究采用一株重组里氏木霉(Trichoderma reesei)生产中性内切-β-葡聚糖酶,发酵液经SDS-PAGE检测,显示了来自特异腐质霉(Humicola insolens)内切-β-葡聚糖酶的蛋白条带(约52 kDa)。对重组里氏木霉的发酵性能进行了研究,表明碳源对内切-β-葡聚糖酶的形成有重要影响,当采用乳糖与微晶纤维素的复合碳源时,酶活力和产率都可明显提高。利用玉米浆粉作为氮源,其适宜浓度为12 g·L-1。培养基初始pH值对发酵酶活力及产率有一定影响,适宜的初始pH值为5.0。在2 m3的发酵罐进行产酶试验,发酵96 h酶活可高达8012 U·mL-1。酶学性质研究表明:该酶在50℃以下稳定,在45~55℃有明显的催化作用,其最适催化温度为50℃。在pH 5.0~7.0稳定性较好,并且有明显的催化作用,其最适催化pH值为6.0。生物整理实验结果显示重组里氏木霉所产酶液用于牛仔布水洗时反染较少,水洗效果良好。     

厌氧真菌内切-β-葡聚糖酶基因在里氏木霉中的重组与表达

里氏木霉(Trichoderm reesei)是重要的纤维素酶生产菌,为了显著提高其产酶性能,拟利用分子生物学技术构建高产的基因工程菌株.将前期优化后的瘤胃厌氧真菌内切-β-葡聚糖酶基因af2置于里氏木霉纤维二糖水解酶Ⅰ强启动子Pcbh1(及其信号肽)和终止子Tcbh1之间,并进一步以pCAMBIA1300为载体骨架,...     

里氏木霉LW1产纤维素酶的酶学性质

研究了里氏木霉LW1所产纤维素酶的主要酶学性质。结果表明,该纤维素酶的最适温度为50℃,最适pH值为4.8;在30～50℃,pH 4.0～6.0范围内有较高的稳定性;90℃处理15 min,酶粉的CMC酶活和FPA酶活保存率分别为38.66%和52.68%;Ca2+、K+、Na+、Mg2+离子对酶活有激活作用,Mn2+、Zn2+、Cu2+离子有抑制作用。     

中性内切-β-葡聚糖酶基因在里氏木霉中的重组与表达

中性内切-β-葡聚糖酶在棉织物生物整理方面具有重要的应用价值,研究首先从特异腐质霉(Humicola insolens)菌株中克隆到一个中性内切-β-葡聚糖酶基因,将该基因置于里氏木霉(Trichoderma reesei)纤维二糖水解酶I强启动子Pcbh1(及其信号肽)和终止子Tcbh1之间,并以pCAMBIA1300为载体骨架构建重组质粒pCB-PHT。采用根瘤农杆菌介导转化技术将重组质粒pCB-PHT导入里氏木霉的分生孢子中,进一步筛选得到八个重组里氏木霉转化子。摇瓶发酵培养72 h时,发酵液的中性内切-β-葡聚糖酶活力最高可达到98.8 IU mL 1左右,是出发菌株的5.1倍。研究成功地实现了外源中性内切-β-葡聚糖酶在丝状真菌里氏木霉中的重组与胞外表达,有关研究结果将在牛仔布水洗工业中发挥出重要作用。     

氮源对里氏木酶合成木聚糖酶的影响

分别以（ＮＨ４）２ＳＯ４、酵母浸膏及蛋白胨为产酶氮源制备木聚糖酶。试验结果表明,酵母浸膏的产酶效果最好,其次是（ＮＨ４）２ＳＯ４。当以酵母浸膏为氮源产酶时,酶活力最高达到２５．４７ＩＵ／ｍＬ,酶得率和酶产率分别为３６３８．６ＩＵ／ｇ木聚糖和８４９０．０ＩＵ／Ｌ·ｄ,酶活力分别是以（ＮＨ４）２ＳＯ４及蛋白胨为氮源时的１．５倍和２．０倍     

间歇出酶/出菌丝的培养方式对里氏木霉产纤维素酶的影响

以微晶纤维素和淀粉水解液作为碳源生产纤维素酶,在里氏木霉Rut C30分批补料生产纤维素酶的过程中通过间歇取出部分酶的培养方式保护纤维素酶。实验结果显示：采用间歇出酶培养方式,在分批补料3 d后,每1、2或者3天取出部分酶液。当平均每天取出15%的酶液,酶活显著增加,总纤维素酶酶活较单纯分批补料提高26.5%~32.6%,而总β-葡萄糖苷酶酶活提高超过46%;采用间歇出酶出菌丝培养方式,当每天取出15%的酶液时,纤维素酶产量比分批补料增长35.4%,去除和酶液等量的菌丝,酶活和分批补料相比增长32.5%,而且这两种培养方式所生产的纤维素酶的酶解效率高达82%,远超商品酶Celluclast（62.03%）。     

黑曲霉纤维二糖酶基因的克隆及其在里氏木霉中的表达

里氏木霉(Trichodema reesei)是目前常用的纤维素酶生产菌种,其分泌的纤维素酶是一个多组分的复合体系,主要包括5种内切型-β-葡聚糖酶(endo-J3-glucanase,EG,EC3.2.1.4),两种外切型β-葡聚糖酶(exo-β-glucanase,EC3.2.1.91,也称纤维二糖水解酶cellobiohydrolase,CBH)和两种纤维二糖酶(cellobiase,EC3.2.1.21,也称β-葡萄糖苷酶β-glucosidase)[1-3].在里氏木霉的纤维素酶蛋白中,纤维二糖水解酶I(CBHI)受强启动子控制,其蛋白质比例最高,约占50%～60%[1],而纤维二糖酶(CB)的比例最低,只占1%左右[4].     

里氏木霉QM9414尿嘧啶缺陷型转化体系改进和β-葡萄糖苷酶的过量表达

里氏木霉是广泛应用于纤维素酶生产的工业真菌,但高产突变株遗传操作困难,限制了菌种改良。首先利用定点整合策略敲除里氏木霉突变株QM9414的pyr4基因,成功构建尿嘧啶营养缺陷型菌株QP4,其遗传转化效率显著提高,而且产酶能力不受影响。进一步在QP4中过量表达β-葡萄糖苷酶(BGL)基因bgl1,经大量平板显色筛选获得2株BGL活力明显增强的工程菌QPB4和QPB5,其酶活分别提高10.01倍和8.26倍。利用发酵酶液对2种不同预处理的玉米芯底物进行水解糖化实验,结果显示以酸处理玉米芯为底物时QPB4和QPB5的葡萄糖得率比QP4分别提高60.98%和52.44%,而以脱木素处理玉米芯为底物时其葡萄糖得率分别提高80.01%和86.00%。研究表明改进里氏木霉高产突变株遗传转化体系可以显著促进菌株改良,提高糖化应用效果。     

Effect of carrier ionic properties on cellulase productivity by immobilized filamentous Trichoderma reesei

Filamentous cells of Trichoderma reesei were immobilized by an adhesion method using carriers prepared by radition polymerization and the effects of the ionic properties of the carriers on cellulase productivity have been investigated. The cationic polymers were better than anionic polymers in the adhesion of the cells. Enzyme productivity was affected by the ionic property of the polymer, the concentration of the coating monomer and the hydrophobicity of the pre-coating polymer.     

Cellulase activity of Trichoderma reesei immobilized on gauze covered with hydrophilic and hydrophobic copolymers

The effect of the surface properties of the matrix for immobilized Trichoderma reesei on cellulase activities was studied. The immobilization was based on an adhesion method. The carriers were obtained by coating cellulosic gauze with hydrophilic and hydrophobic monomers using a radiation copolymerization technique. The cellulase activities of the organisms immobilized on the matrices coated with the copolymers were higher than those of the free organisms. Hydrophilic matrices allowed increased growth of the immobilized organisms with increased cellulase activity.     

低聚木糖生产用里氏木霉木聚糖酶选择性合成的研究现状

对低聚木糖生产用里氏木霉木聚糖酶选择性合成的研究现状进行了较全面的总结和评述。系统地介绍了里氏木霉木聚糖酶的的多样性以及碳源、pH和碳氮比等培养条件对合成内切木聚糖酶和木糖苷酶的影响,提出了调控这些培养条件选择性合成低木糖苷酶活的木聚糖酶的方法。     

The influence of pH upon the hydrolysis of carboxymethylcellulose with cellulases from Trichoderma reesei

We have studied the enzymatic hydrolysis of carboxymethylcellulose (CMC) with Celluclast, a commercial preparation of cellulases deriving from Trichoderma reesei, by monitoring the variation in the concentrations of glucose and reducing sugars at a constant temperature of 50°C and different pH values. We determined both glucose and overall yield, concluding that the production of glucose directly by endoglucanase is higher than that coming from the hydrolysis of cellobiose by β‐1,4‐glucosidase and that the rate at which cellobiose is formed can be calculated via kinetic parameters that are due in the end mainly to exoglucanase activity. We observed the effect of pH upon the kinetic parameters and found that the ideal value for the hydrolysis of CMC is one of pH 4.9.     

麸皮对里氏木霉Rut C-30产纤维素酶的促进作用

本研究尝试通过里氏木霉RutC-30添加适量的麸皮以及利用实验设计软件Design-Expert寻找适宜的麸皮与微晶纤维素的配比来研究麸皮对里氏木霉RutC-30产纤维素酶的影响。结果表明,适当的麸皮添加量能够促进纤维素酶的生产：通过二元二次正交旋转组合设计,确定了微晶纤维素添加量和麸皮添加量分别为12．23和23．50g/L的优化产酶条件：此奈件下,在250mL摇瓶中滤纸酶活达到6．383FPIU／mL,得率系数为521．913FPIU／g;在7．5L发酵罐中,滤纸酶活为6．807FPIU／mL,得率系数为556．582FPIU／g。相比于优化前,优化后摇瓶实验和发酵罐实验中滤纸酶活分别提高了14．247％和17．403％,而纤维素酶的得率系数却分别降低了6．584％和4．005％。分别以酸解杨木残渣和蒸汽爆破杨木浆替代微晶纤维素作为碳源,最终获得最高滤纸酶活1．953FPIU／mL和1．745FPIU／mL。     

Effect of Natural and Pretreated Soybean Hulls on Enzyme Production by Trichoderma reesei

The enzyme induction utility of soybean hulls (SBH), consisting in excess of 50 wt% non-starch polysaccharides (NSP) cellulose, hemicellulose, and pectin, was studied through cultivation of the carbohydrase-producing fungus Trichoderma reesei Rut C-30. Shake flask systems of T. reesei were grown in a medium consisting of defatted soybean flour as a nitrogen source and SBH, some of which were untreated and others pretreated by liquid hot water, alkaline, and supercritical carbon dioxide, as carbon source. Cellulase, xylanase, and polygalacturonase activities were measured for the systems, and the natural hull systems were found to yield optimum enzyme production. Controlled batch fermentation experiments were carried out to compare enzyme production resulting from media with Avicel^® (FMC BioPolymer, Philadelphia, PA, USA) versus natural SBH with and without soybean flour as the nitrogen source. Soybean hull fermentations were also performed at several pH levels to observe the effects on enzyme production. Soybean hulls induced comparable levels of cellulase, and higher levels of xylanase and polygalacturonase, than Avicel^®. With SBH, cellulase and xylanase production were enhanced at higher pH levels (6.0), and polygalacturonase was enhanced at lower pH levels (4.0–4.5). Enzyme production was largely unaffected by the presence of soybean flour as the nitrogen source.     

麸皮对里氏木霉Rut C-30产 纤维素酶的促进作用

本研究尝试通过里氏木霉Rut C-30添加适量的麸皮以及利用实验设计软件Design-Expert寻找适宜的麸皮与微晶纤维素的配比来研究麸皮对里氏木霉Rut C-30产纤维素酶的影响.结果表明,适当的麸皮添加量能够促进纤维素酶的生产;通过二元二次正交旋转组合设计,确定了微晶纤维素添加量和麸皮添加量分别为12.23和23.50 g/L的优化产酶条件.此条件下,在250 mL摇瓶中滤纸酶活达到6.383 FPIU/mL,得率系数为521.913 FPIU/g;在.5 L发酵罐中,滤纸酶活为6.80 FPIU/mL,得率系数为556.582 FPIU/g.相比于优化前,优化后摇瓶实验和发酵罐实验中滤纸酶活分别提高了14.24%和1.403%.而纤维素酶的得率系数却分别降低了6.584%和4.005%.分别以酸解杨木残渣和蒸汽爆破杨木浆替代微晶纤维素作为碳源,最终获得最高滤纸酶活1.953 FPIU/mL和1.45 FPIU/mL.     

Enzymatic Hydrolysis of Carboxymethylcellulose and Filter Paper by Immobilized Cellulases on Lignophenols

The cost of cellulase is a major factor limiting the enzymatic hydrolysis of cellulosic biomass. Thus, immobilization of cellulase would be an important advancement. Lignophenol is a lignin-based functional phenolic polymer synthesized from a lignocellulosic material and a phenol derivative at ambient temperature. Cellulase derived from Trichoderma reesei is easily immobilized by softwood and hardwood lignocresol simply by mixing to produce a water-insoluble lignophenol-cellulase complex. Enzymatic hydrolysis performances of cellulases immobilized on lignocresols are approximately 80–90% and 30–50% relative to that of free cellulase in the hydrolysis of carboxymethylcellulose (CMC) and filter paper, respectively. Cellulase was active enough even after adsorption on lignocresols. Limited physical contact between solid substrates and immobilized cellulase due to the presence of lignocresol seems to lead to lower enzymatic activity for solid substrates. Hardwood lignocresol-immobilized cellulase exhibits slightly higher activity than softwood lignocresol-immobilized cellulase when the same amount of cellulase is used per gram lignocresol. Although cellulase activity gradually decreases with recycling, sufficient enzymatic activity, at least for hydrolyzing soluble substrates, remains that it can be reused.