Lipolytic Enzyme Production by Immobilized Rhizopus oryzae

In this paper, a suitable technique as well as an adequate material for the immobilization of the fungus Rhizopus oryzae were investigated. This organism has been shown to have potential in the field of food aroma due to the production of extracellular lipolytic enzymes. However, an efficient production system at bioreactor scale for its application to the flavor compounds production is still needed. Among the supports studied, alginate beads were the best carrier materials, leading to the highest lipolytic activities of up to 400 U/L after 3 days of cultivation. Repeated batch cultures were carried out to improve cell concentration and lipolytic activity. The gel beads produced lipolytic enzyme under optimized conditions for consecutive batch fermentations without marked activity loss and deformation attained a maximum level of 715 U/L after three batches. The production of lipolytic enzyme by immobilized Rhizopus oryzae in a 2‐litre airlift bioreactor with the optimized conditions was evaluated. Lipolytic activities of 487 U/L were attained, operating in successive batches without operational problems and the bioparticles (the fungus grows in alginate beads) maintained their shape throughout fermentation.     

纤维废渣酶水解及L-乳酸发酵的研究

木糖厂的纤维废渣中,纤维素为主要成份, 本文用纤维素酶对木糖厂的纤维废渣进行酶解，继而研究了用米根霉进行乳酸发酵。结果表明，实验条件下，纤维废渣可用纤维素酶直接水解或经碱处理后进行酶水解, 9%的纤维废渣经酶解后还原糖浓度为39.6mg·mL-1，纤维素糖化率为68.2%. 物料经碱处理后酶解液的还原糖浓度为48.2mg·mL-1, 比碱处理前提高了约16.1%。水解液用米根霉进行乳酸发酵，乳酸的浓度达到了29.5mg·mL-1，比用碱处理前物料酶解液乳酸发酵的乳酸浓度(24.0mg·mL-1)增加了约23%，但过程的总体效率有待进一步提高。     

Insecticidal effect of three diatomaceous earth formulations, applied alone or in combination, against three stored-product beetle species on wheat and maize

Laboratory tests were carried out to examine the insecticidal effect of three commercially available diatomaceous earth (DE) formulations on wheat and maize against three major stored-grain beetle species: Rhyzopertha dominica, Sitophilus oryzae, and Tribolium confusum. The three DEs tested were Insecto^®, PyriSec^®, and Protect-It^®. These DEs were applied alone or in all possible combinations (Insecto^®+PyriSec^®, Insecto^®+Protect-It^®, PyriSec^®+Protect-It^®, and all three DEs together), at three (total) dose rates: 0.25, 0.5, and 0.75 g/kg of each commodity. Adults of the above species were exposed to the treated commodities for 7 d at 26 °C, and 65% r.h., and after this interval the mortality was measured. For each species, adult mortality was significantly affected by the type of DE, the commodity, and the dose rate. All DEs were less effective against T. confusum, where mortality did not exceed 67%, in comparison with the other two species, where 100% mortality was achieved in some combinations. For all species tested, all DEs were more effective on wheat than on maize. Generally, the mix of two or three DEs was more effective than the application of one DE, for all species and commodities. The results of the present work clearly indicate that a blending of several DEs together may produce a new DE formulation that is highly effective at low dose rates.     

常压室温等离子体诱变选育高产曲酸米曲霉

以米曲霉KA2308作为出发菌株,首次利用常压室温等离子体诱变技术选育高产曲酸菌株,通过96孔板高通量筛选获得一株曲酸生产菌KA1515,曲酸产量提高到47.28g·L-1,比出发菌株提高了31.48%,经过多次传代,曲酸产量仍比较稳定。表明采用常压室温等离子体诱变能有效提高米曲霉曲酸产量。     

耐酸性木聚糖酶在清酒酿造中的作用

从华根霉 (RhizopuschinensisY92 )发酵液中通过离子交换色谱和凝胶过滤色谱分离纯化获得一种耐酸性木聚糖酶R ,将它和另外 2种耐酸性木聚糖酶 :米曲霉 (AspergillusoryzaeRIB12 8)木聚糖酶B和白曲菌 (AspergilluskawachiiIFO43 0 8)木聚糖酶C分别应用于清酒酿造中 ,结果表明 ,木聚糖酶B可以促进米细胞的溶解 ,对于原料米的利用率有明显的提高 ,而木聚糖酶C和木聚糖酶R的作用则不太明显     

DJS水系对米曲霉酶活力提高的研究

研究了DJS水系在酿造酱油制曲中产酶及其活力的影响。试验中与自来水介质进行比较,结果表明:DJS水系能改变米曲霉的孢子数,对其酶活力的提高有显著的影响;在DJS-A介质中的孢子产量虽少于DJS-B介质,而其中的酶活力却较高。     

米根霉葡萄糖淀粉酶的分离纯化及特性研究

葡萄糖淀粉酶是米根霉在淀粉质培养基中诱导分泌的一类胞外酶，其表达对米根霉转化淀粉生成L-乳酸的效率，以及L-乳酸分批发酵周期的长短有很大影响。本文对米根霉葡萄糖淀粉酶进行分离纯化，并研究其酶学性质，结论如下：经硫酸铵盐析、透析脱盐、Sephadex G-100柱层析等纯化步骤，葡萄糖淀粉酶比活力提高23倍。SDS-PAGE显示纯化后的酶为一条带，相对分子量约75．5kD。该酶以可溶性淀粉为底物时最适催化反应pH值为4．0～6．0，最适催化反应温度为40-50℃，在50℃下保持稳定。钙离了和锰离子对该酶活力有一定的增强作用，而铅离子和亚铁离子对其活力有明显的抑制作用。     

Rapid Reconstitution of Biosynthetic Machinery for Fungal Metabolites in Aspergillus oryzae: Total Biosynthesis of Aflatrem

Reconstitution of the biosynthetic machinery for fungal secondary metabolites in Aspergillus oryzae provides an opportunity both for stepwise determination of the biosynthetic pathways and the total biosynthesis of fungal natural products. However, to maximize the utility of the reconstitution system, a simple and rapid strategy for the introduction of heterologous genes into A. oryzae is required. In this study, we demonstrated an effective method for introducing multiple genes involved in the biosynthesis of fungal metabolites by using the expression vectors pUARA2 and pUSA2, each of which contains two cloning sites. The successful introduction of all the aflatrem biosynthetic genes (seven genes in total) after two rounds of transformation enabled the total biosynthesis of aflatrem. This rapid reconstitution strategy will facilitate the functional analysis of the biosynthetic machinery of fungal metabolites.     

Analysis of the catalyic mechanism of a fungal lipase using computer-aided design and structural mutants

Both an active enzyme conformation and stabilization of tetrahedraltransition states are essential for the catalysis of ester bondhydrolysis by lipases. X-ray structural data and results fromsite-directed mutagenesis experiments with proteases have beenused as a basis for predictions of amino acid residues likelyto have key functions in lipases. The gene encoding a lipasefrom Rhizopus oryzae was cloned and expressed in Escherichiacoli. Site-directed mutagenesis of this gene was used to testthe validity of computer-aided predictions of the functionalroles of specific amino acids in this enzyme. Examination ofthe kinetic constants of the Rhizopus oryzae lipase variantsallowed us to identify amino acid residues which are directlyinvolved in the catalytic reaction or which stabilize the activegeometry of the enzyme. The combination of these results withmolecular mechanics simulations, based on a homology-derivedstructural model, provided new information about structure-functionrelationships. The interpretation of the data is consistentwith results obtained with other hydrolases, such as proteases.