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Roman Jansen Holger Morschett Dennis Hasenklever Matthias Moch Wolfgang Wiechert Marco Oldiges 《Biotechnology progress》2021,37(4):e3144
In recent years, many fungal genomes have become publicly available. In combination with novel gene editing tools, this allows for accelerated strain construction, making filamentous fungi even more interesting for the production of valuable products. However, besides their extraordinary production and secretion capacities, fungi most often exhibit challenging morphologies, which need to be screened for the best operational window. Thereby, combining genetic diversity with various environmental parameters results in a large parameter space, creating a strong demand for time-efficient phenotyping technologies. Microbioreactor systems, which have been well established for bacterial organisms, enable an increased cultivation throughput via parallelization and miniaturization, as well as enhanced process insight via non-invasive online monitoring. Nevertheless, only few reports about microtiter plate cultivation for filamentous fungi in general and even less with online monitoring exist in literature. Moreover, screening under batch conditions in microscale, when a fed-batch process is performed in large-scale might even lead to the wrong identification of optimized parameters. Therefore, in this study a novel workflow for Aspergillus niger was developed, allowing for up to 48 parallel microbioreactor cultivations in batch as well as fed-batch mode. This workflow was validated against lab-scale bioreactor cultivations to proof scalability. With the optimized cultivation protocol, three different micro-scale fed-batch strategies were tested to identify the best protein production conditions for intracellular model product GFP. Subsequently, the best feeding strategy was again validated in a lab-scale bioreactor. 相似文献
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The xynHB gene, encoding alkaline xylanase was cloned from Bacillus pumilus by a shot-gun method. The gene was cloned into vector pHBM905A, and expressed in Pichia pastoris GS115. Xylanase-secreting transformants were selected on plates containing RBB-xylan. Enzymatic activity in the culture supernatants was up to 644?U?mL?1 and the optimal secretion time was 4 days at 25°C. SDS-PAGE showed two bands, of 32.2?kDa and 29.6?kDa, both larger than the predicted mass of 22.4?kDa based on its amino acid sequence. Zymogram analysis demonstrated that the enzyme in both bands could hydrolyze xylan. Deglycosylation by endoglycosidase H revealed that both were derived from the same protein but contain different extents of glycosylation (30 and 25%). The optimal pH and temperature of the enzyme was pH6–9 and 50°C, respectively. 相似文献
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N-乙酰鸟氨酸脱酰基酶为一种新型手性拆分酶制剂,酶活性依赖于Mg2+、Mn2+、Zn2+及Co2+ 中的某种金属离子。以高表达NAOase的重组菌DH10B/argE-pHsh为研究对象,考察不同培养条件下Mg2+、Mn2+、Zn2+及Co2+ 4种离子对重组菌的生长、酶的表达活性及表达量的影响。结果发现:同种离子在不同条件下对重组菌的生长影响不大,但对酶活性影响显著。4种离子在合适浓度时皆能提高酶活性,促进强度从高到低依次为Mn2+、Mg2+、Co2+和Zn2+。在TB培养基中,Mn2+为15 mmol/L时:NAOase比酶活达到1 272.7 U/mL,是未添加时的4.67倍,激活作用显著高于其他离子。SDS-PAGE电泳实验表明4种离子的蛋白表达量基本相同。 相似文献
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Lactic acid bacteria (LAB) are Gram positive bacteria, widely distributed in nature, and industrially important as they are used in a variety of industrial food fermentations. The use of genetic engineering techniques is an effective means of enhancing the industrial applicability of LAB. However, when using genetic engineering technology, safety becomes an essential factor for the application of improved LAB to the food industry. Cloning and expression systems should be derived preferably from LAB cryptic plasmids that generally encode genes for which functions can be proposed, but no phenotypes can be observed. However, some plasmid-encoded functions have been discovered in cryptic plasmids originating from Lactobacillus, Streptococcus thermophilus, and Pediococcus spp. and can be used as selective marker systems in vector construction. This article presents information concerning LAB cryptic plasmids, and their structures, functions, and applications. A total of 134 cryptic plasmids collated are discussed. 相似文献
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Hisanao Takeuchi Megumi Maeda Yukio Yamaguchi Keiichiro Muramatsu 《Bioscience, biotechnology, and biochemistry》2013,77(4):931-935
Three chitinases (EC 3.2.1.14) were purified from yam, Dioscorea opposita THUMB, by fractionation with ammonium sulfate, chromatographies on DEAE-Cellulose and DEAE-Sephadex A-50, chromatofocusing and gel filtration on Bio-Gel P-60. The purified enzymes (E-l, E-2 and E-3) showed single bands on sodium dodecylsulfate polyacrylamide gel electrophoresis, and the molecular weights were estimated to be 33,500. The pIs were 4.05 (E-l), 4.0 (E-2) and 3.8 (E-3). All enzymes were glycoproteins and the neutral sugar contents were 3.6% (E-l), 3.6 (E-2) and 0.9% (E-3). The N-terminal amino acids of E-l and E-3 were the same and determined to be histidine. All enzymes hydrolyzed glycolchitin, but not p-nitrophenyl-2-acetamido-2-deoxy-β-d-glucopyranoside or Micrococcus lysodeikticus cell walls. E-l and E-3 were stable in the pH range of 5 ~ 11, and below 60°C. These enzymes showed two optimum pHs around 3.5 and 8.0 or 8.5 with glycolchitin as substrate. 相似文献