利用低温蒸煮工艺进行高浓度酒精发酵

本研究利用低温蒸煮工艺,探讨了玉米淀粉的高浓度酒精发酵。在80℃下对玉米粉进行蒸煮15min,同时加α—淀粉酶进行液化。经冷却后,在55℃下加糖化酶糖化30min。再度冷却到30℃,加酵母菌悬液发酵70h。并利用这一技术研究了产高浓度酒精酵母菌株的最佳发酵条件。在加糖化酶量200～300u/g原料。酵母接种量为3％(v/v),发酵温度为30℃,pH4～4.5,发酵周期为70h,原料水比为1:2条件下,所选用的菌株之一W_4可以产生17.5％(v/v)乙醇。发现该菌株在发酵速度和耐酒精能力方面明显地优于国内酒精厂常用的酵母菌株1300。在发酵结束时,成熟发酵醪的pH值为4.2。外观糖度为0(Bx),残还原糖为0.19％,残总糖为3.6％,淀粉利用率为90％。     

沉淀法去除糖化酶中转苷酶的研究

研究了磷钨酸，十二烷磺酸钠，单宁酸三种试剂去除液体曲糖化酶中转苷酶的方法，利用正交试验了每种方法的最优工艺条件，在最优工艺条件下，磷钨酸法转苷去除率为７５．９％，糖化酶活力损失９．６％，十二烷基磺酸钠法转苷酶去除率为６９．１％，糖化酶活力基本不变；单宁酸法转苷酶去除率为７０．３％，糖化酶活力提高９．８％。     

硅藻土吸附法去除糖化酶中的转苷酶

探讨了利用硅藻土作吸会剂去除液体曲糖化酶中转苷酶的方法。结果表明：硅藻土具有吸附转苷酶的作用并使糖化酶活力有所损失。发酵液ｐＨ值和硅藻土添加量对转苷酶去除率和糖化酶活力影响较大，而处理时间的影响较小。     

高活力糖化酶发酵技术的研究

本文对糖化酶产生菌ＡＮ１４９的诱变育种方法、发酵产酶工艺、摇瓶培养基配方及小型自动发酵罐条件等进行了系统的研究。实验结果表明，菌种自然分离、紫外线诱变以及ＮＴＧ处理等的配合作用可显著提高产酶量和转化率，出发菌株的产酶活力从８５００ｕ／ｍｌ提高到１５０００ｕ／ｍｌ。按最佳的培养基配方和发酵工艺条件，采用ＷＮＣ－１５突变菌株发酵１６０ｈ，酶活力可达３００００ｕ／ｍｌ加以上，对提高我国的糖化酶活力具有重要的现实意义。     

Stepwise optimisation of enzyme production in solid state fermentation of waste bread pieces

When it is not consumed, bread presents a major source of food waste, both in terms of the amount and its economic value. However, bread also possesses the characteristics of an ideal substrate for solid state fermentation. Yet nearly all wasted bread ends up in landfill sites, where it is converted into methane by anaerobic digestion. Governments are finally taking action and, according to the EU Landfill Directive, for example, biodegradable municipal waste disposed into landfills must be decreased to 35% of 1995 levels, by 2020. Solid state fermentation of waste bread for the production of value added products is a novel idea, which could help with the achievement of this target. In this study, glucoamylase and protease production from waste bread pieces, via solid state fermentation, was investigated in detail. The optimum fermentation conditions for enzyme production were evaluated as, 20 mm particle size, 1.8 (w/w, db) initial moisture ratio, and duration of 144 h. Under these conditions, glucoamylase and protease activities reached up to 114.0 and 83.2 U/g bread (db), respectively. This study confirms that waste bread could be successfully utilised as a primary raw material in cereal based biorefineries.     

Enhancement of secreted production of glucoamylase through fed-batch bioreactor culture of recombinant yeast harboring glucose-controllable <Emphasis Type="Italic">SUC2</Emphasis> promoter

Glucoamylase that hydrolyses starch to glucose is one of the important industrial enzymes for ethanol production industry. Therefore, genetic production of recombinant glucoamylase has been widely studied. Previously, we reported secreted production of Saccharomyces diastaticus-originated glucoamylase in Saccharomyces cerevisiase expression system using its own signal sequence and the SUC2 promoter that is regulated by glucose level in culture medium. In the present work, we performed a comparative study between batch and fed-batch bioreactor cultures for secreted production of recombinant glucoamylase. Through maintaining low glucose levels in the culture broth, we obtained about 7-fold higher secreted production levels of glucoamlyase in fed-batch culture. Fed-batch culture strategy also enhanced (∼3.1-fold) secretion efficiency of recombinant glucoamylase in S. cerevisiae.     

Production,Purification, and Characterization of Thermoanaerobacterium thermosaccharolyticum Glucoamylase

Glucoamylase from Thermoanaerobacterium thermosaccharolyticum ATCC 7956 (DSM 571) was produced in extracellular form. It was purified to homogeneity by two separate methods, one with two chromatographic steps and the other with three. This glucoamylase is closely related to glucoamylases from Clostridium sp. G0005 and T. thermosaccharolyticum DSM 572. Activities and K_M values with maltose substrate are less than one‐tenth and about fourfold, respectively, those of Aspergillus niger glucoamylase. T. thermosaccharolyticum glucoamylase is about twenty times as thermostable as A. niger glucoamylase and its optimal pH is somewhat higher at 4.9; however, it is produced in much lower activities. Sorbitol strongly stabilizes A. niger glucoamylase.     

Enzymatic hydrolysis of potato starches containing different amounts of phosphorus

The rapid hydrolysis of potato starches differing in phosphorus content, as well as sweet potato, cassava and yam starches, was accomplished by treatment of gelatinised starches with bacterial liquefying α-amylase at 50 °C for 1 h, followed by Bacillus licheniformis α-amylase at 55 °C up to 24 h, and then by glucoamylase at 40 °C for a further 24 h. Among the potato starches, the high-phosphorus starches showed higher starch resistant capacity than the medium-phosphorus starches, as well as other tuber and root starches. The hydrolysis rate of tuber and root starches was not greatly influenced by their amylose content and median granule size. Only glucose was detected in the almost completely hydrolysed tuber and root starch samples, indicating that the concomitant enzymes treatment could hydrolyse both the α-1,4 and α-1,6 linkages of the starches examined.     

Characterization of glucoamylase immobilized on magnetic nanoparticles

Magnetic support was prepared by precipitation from an alkaline solution of divalent and trivalent iron ions and subsequently was modified with 3‐aminopropyltriethoxysilane. FTIR analysis showed existence of a new Si–O–Fe bond in obtained particles. Scanning electronic microscopy images shows that the nanoparticles of all samples have particle size below 30 nm. Glucoamylase AMG 300L was immobilized onto the modified magnetic support using glutaraldehyde as a coupling agent. Obtained preparations had specific activity of 148 U/g of the support when measured at 55°C using maltose as substrate. The immobilized enzyme exhibited mass transfer limitation as reflected by a higher apparent K_m value and a lower energy of activation. The immobilization was almost completely terminated after 30 min of the reaction at 30°C. The highest immobilization yield of the enzyme was achieved at glutaraldehyde concentration of 10 mM. The immobilization did not influence considerably on optimum pH and temperature of substrate hydrolysis catalyzed by investigated enzyme (55°C, pH 4.5). Moreover, immobilized glucoamylase was easily separated from the reaction medium by an external magnetic field and retained about 60% of initial activity after nine repeated cycles of enzyme reaction followed by magnetic separation.     

Improvement of Aspergillus niger Glucoamylase Thermostability by Directed Evolution

Directed evolution was used to improve the thermostability of Aspergillus niger glucoamylase (GA) expressed in Saccharomyces cerevisiae. A starch‐plate assay developed to screen GA mutants for thermostability gave results consistent with those of irreversible thermoinactivation kinetic analysis. Several thermostable multiply‐mutated GAs were isolated and characterized by DNA sequencing and kinetic analysis. Three new GA mutations, T62A, T290A and H391Y, have been identified that encode GAs that are more thermostable than wild‐type GA, and that improve thermostability cumulatively. These individual mutations were combined with the previously constructed thermostable site‐directed mutations D20C/A27C (forming a disulfide bond), S30P, and G137A to create a multiply‐mutated GA designated THS8. THS8 GA is substantially more thermostable than wild‐type GA at 80°C, with a 5.1 kJ/mol increase in the free energy of thermoinactivation, making it the most thermostable Aspergillus niger GA mutant characterized to date. THS8 GA and the singly‐mutated GAs have specific activities and catalytic efficiencies (k_cat/K_m) similar to those of wild‐type GA.