交联法固定化米曲霉菌体光学拆分DL-色氨酸的研究

采用戊二醛作交联剂,对米曲霉3042产氨基酰化酶液态发酵菌体进行固定化,固定化酶活保留率最高达到30.4%。固定化米曲霉菌体扫描电镜图可看出,通过固定化后菌体之间结合紧密程度提高,有利于米曲酰化酶附着在菌体上。对固定化米曲霉菌体分批拆分制备L-色氨酸进行了研究,固定化菌体酶活的半衰期约为27d。     

米曲霉1228产α-淀粉酶特性的研究

对米曲霉1228产α-淀粉酶的特性及部分酶学性质进行了研究.结果表明,在发酵培养基中豆粕含量20%、NaCl浓度2%、初始pH6.6的条件下,采取3%的接种量,31 ℃培养108 h,此时的酶活最大.该酶的最适作用温度为55 ℃,最适pH为5.0,Ca2+对酶有激活作用,Fe2+、Fe3+、Mn2+、Ba2+和Cu2+对其有不同程度的抑制.     

米曲霉LZF-5制曲工艺的研究

为了提供米曲霉LZF-5在酿造方面的科学理论依据,以米曲霉LZF-5为研究对象,研究了制曲过程中原料配比、加水量、接种温度、接种量和翻曲温度等因素对曲的蛋白酶活力的影响,通过实验确定了最佳制曲条件,即采取2∶1的豆粕和二级粉为原料,加水量控制在50%,接种温度为35℃,接种量控制在0.5%,翻曲温度为38℃,其蛋白酶活力达到3 968 μ/g.     

米曲霉GX0011β-果糖基转移酶的性质研究

β-果糖基转移酶是酶法由蔗糖生产低聚果糖所必需的酶,本文对从米曲霉GX0011分离纯化得到的β-果糖基转移酶性质进行了研究。结果表明,该酶催化蔗糖转化成蔗果三糖的Km和Vmax分别为0.319mol/L和0.713mol/min/L,最适pH和最适温度分别为5.0～6.0和45℃。葡萄糖是该酶的竞争性抑制剂,其抑制常数Ki=0.608mol/L。经苯甲磺酸氟(PMSF)、N-溴代二酰亚胺(NBS)、1-乙基-3-(3-二甲氨基丙基)碳二亚胺盐酸盐(EDC)和间硝基苯磺酸钠(TNBS)对酶进行化学修饰及底物保护实验,推测该酶活性中心可能包括丝氨酸/苏氨酸、色氨酸、天冬氨酸(谷氨酸)残基,而赖氨酸残基可能与维系酶活性中心构象有关,但不是酶活性必需基团。实验还表明,超声波能在一定程度上提高酶活力,在300W功率下作用5min可提高酶活12%左右。低浓度乙醇对酶活影响并不十分显著,乙醇浓度达20%时,酶活仅损失19%。     

米曲霉GX0011β-果糖基转移酶的性质研究

β-果糖基转移酶是酶法由蔗糖生产低聚果糖所必需的酶,本文对从米曲霉GX0011分离纯化得到的β-果糖基转移酶性质进行了研究。结果表明,该酶催化蔗糖转化成蔗果三糖的Km和Vmax分别为0.319mol/L和0.713mol/min/L,最适pH和最适温度分别为5.0～6.0和45℃。葡萄糖是该酶的竞争性抑制剂,其抑制常数Ki=0.608mol/L。经苯甲磺酸氟（PMSF）、N-溴代二酰亚胺（NBS）、1-乙基-3-（3-二甲氨基丙基）碳二亚胺盐酸盐（EDC）和间硝基苯磺酸钠（TNBS）对酶进行化学修饰及底物保护实验,推测该酶活性中心可能包括丝氨酸/苏氨酸、色氨酸、天冬氨酸（谷氨酸）残基,而赖氨酸残基可能与维系酶活性中心构象有关,但不是酶活性必需基团。实验还表明,超声波能在一定程度上提高酶活力,在300W功率下作用5min可提高酶活12%左右。低浓度乙醇对酶活影响并不十分显著,乙醇浓度达20%时,酶活仅损失19%。      

复合诱变对米曲霉2336产酸量的影响

以米曲霉(Aspergillus oryzae)2336为出发菌株,经紫外线、60Co复合诱变处理,采用快速筛选法获得不产黄曲霉毒素的曲酸高产菌株UC690,以玉米淀粉为碳源,发酵7d,曲酸产量由原来的12.34g/L提高到50.47g/L。经遗传稳定性试验,这一变异菌株可应用于工业化生产。     

固定化米曲霉发酵甘蔗糖蜜生产曲酸的研究

以甘蔗糖蜜为原料,采用聚氨酯泡沫固定米曲霉进行发酵试验,探究聚氨酯泡沫固定化米曲霉的条件,及固定化米曲霉发酵糖蜜的最优条件。试验得出,固定化最优条件为:聚氨酯泡沫载体最佳尺寸为2 mm×2 mm×2 mm,最佳填充量为1.0 g/60 m L发酵液,曲酸最高浓度可达15.43 g/L,用此固定化酶发酵酸处理的糖蜜,当糖蜜培养基中糖浓度80 g/L时,获得的曲酸最高浓度为9.02 g/L。     

乙醇对米曲霉发酵产曲酸的影响

以从湖南冰糖橙皮上筛选到的优良产曲酸菌种米曲霉为研究对象,研究乙醇对生成曲酸的影响。结果表明,在添加质量分数10%的乙醇条件下,当葡萄糖含量为6%时,发酵5 d,曲酸产量提高到43.80 g/L,比未添加乙醇的曲酸得率(15.68 g/L)提高179.34%。其作用机理是乙醇增加了菌株与发酵液的比接触面积,降低了发酵液的粘稠度,使传质、传氧系数增大,葡萄糖转化为曲酸的转化率提高。在米曲霉发酵产曲酸的培养基中添加乙醇,能有效提高曲酸产量和生产效率。     

米曲霉40188产中性蛋白酶、α-淀粉酶特性的研究

通过对米曲霉40188产酶特征及中性蛋白酶及α-淀粉酶酶学特征的研究,得出米曲霉40188制曲最佳时间为50～72 h,中性蛋白酶和α-淀粉酶酶活在40℃稳定,随着温度升高酶活降低,但α-淀粉酶的热稳定性较中性蛋白酶强,中性蛋白酶的最适pH为7～8,α-淀粉酶的最适pH为6～8,α-淀粉酶的耐盐性较中性蛋白酶强,中性蛋白酶酶活随着盐浓度的增加呈下降趋势。     

米曲霉形态与曲酸生产的关系

以米曲霉为出发菌株生产曲酸,通过改变培养基浓度和孢子接种量来研究曲酸生产关键因素对A.oryzae菌体形态的影响,进一步通过不同形态菌体发酵分析菌体形态与曲酸生产的关系。培养基中的碳氮比对A.oryzae菌球的直径的大小具有较大影响。当孢子浓度为109个/m L时,A.oryzae主要呈菌丝形态;但当孢子浓度为107~108个/m L时,菌体形态呈现为球状,并随着孢子浓度的增大菌球直径减少。A.oryzae发酵生产曲酸的最佳形态为菌球,并且菌球直径与曲酸合成能力关系密切,菌球直径为0.25~0.35 mm时,单位细胞曲酸积累量最高。     

Molecular cloning of the isoamyl alcohol oxidase-encoding gene (mreA) from Aspergillus oryzae

Isoamyl alcohol oxidase (IAAOD) is a novel enzyme that catalyzes the formation of isovaleraldehyde, which is the main component of mureka that gives sake an off-flavor (Yamashita et al. Biosci. Biotechnol. Biochem., 63, 1216–1222, 1999). We cloned the genomic DNA sequence encoding IAAOD from a koji mold, Aspergillus oryzae, using a PCR-amplified DNA fragment corresponding to the partial amino acid sequences of the purified protein as a probe. The cloned gene comprises 1903 bp of an open reading frame with three putative introns and encodes 567 amino acids with a presumed signal peptide consisting of 24 amino acids at the N-terminus. Moreover, nine potential N-glycosylation sites were present. Homology search on amino acid sequence showed that IAAOD has a region significantly similar to those conserved in FAD-dependent oxidoreductases. Southern hybridization analysis revealed that the cloned gene exists as a single copy in the A. oryzae RIB 40 chromosome. The cloned gene was overexpressed under the control of the amyB promoter in A. oryzae. The isovaleraldehyde-producing activity in the culture supernatant of one transformant was over 800 times as high as that of transformant with the control vector. This result demonstrates that the cloned gene encodes IAAOD. We named this novel alcohol oxidase gene “mreA”.     

Purification and identification of proteolytic enzymes from Aspergillus oryzae capable of producing the antihypertensive peptide Ile-Pro-Pro

Two proteolytic enzymes capable of releasing the angiotensin I-converting enzyme (ACE) inhibitor Ile-Pro-Pro from casein were identified by purification of an Aspergillus oryzae extract by three-step column chromatography. First, proteins capable of producing Ile-Pro-Pro from β-casein were eluted using a DEAE-sepharose FF column with a linear sodium chloride gradient. An endopeptidase capable of releasing Pro-Ile-Pro-Gln-Ser-Leu-Pro-Gln-Asn-Ile-Pro-Pro from Pro-Ile-Pro-Gln-Ser-Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu-Thr-Gln and an aminopeptidase producing Ile-Pro-Pro from Gln-Asn-Ile-Pro-Pro were separated from the resultant fraction using a hydroxyapatite column. Each active enzyme was then loaded onto a Develosil 300Diol gel filtration column for high performance liquid chromatography (HPLC) and purified to homogeneity.The endopeptidase had a molecular mass of approximately 46,000 Da and exhibited an N-terminal amino acid sequence identical to that of neutral protease I (NP I) of A. oryzae. Meanwhile, the aminopeptidase had a molecular mass of 36,000 Da and an N-terminal amino acid sequence similar to that of Leucine aminopeptidase (LAP), as reported in Aspergillus sojae and A. oryzae. The eluted endopeptidase and aminopeptidase were thus identified as NP I and LAP, respectively.Analysis of peptide production using synthetic proteins containing an Ile-Pro-Pro sequence showed that NP I processed the C-terminal end and LAP processed the N terminus to produce Ile-Pro-Pro. While Ile-Pro-Pro was successfully produced from casein by the addition of these two purified enzymes, it was not generated with the addition of only a single enzyme. Based on our experimental findings, we suggest that NP I and LAP are key proteolytic enzymes in the release of Ile-Pro-Pro from casein in A. oryzae.     

Efficient production of a heterologous peroxidase, DyP from Geotrichum candidum Dec 1, on solid-state culture of Aspergillus oryzae RD005

The productivity of a peroxidase (DyP) originating from Geotrichum candidum Dec 1 was enhanced in the solid-state culture using Aspergillus oryzae RD005. When the humidity, water content, and temperature were adjusted to 60%, 50% and 27°C, respectively, the productivity of DyP reached 5.3 g per kilogram wheat bran, which was used as the solid medium. The yield of 5.3 g per kg wheat bran corresponded to the yield of a 56 kg submerged culture. The productivity per gram carbon of the medium in the solid-state culture was 4.1-fold that in the submerged culture.     

农杆菌介导转化黑曲霉条件优化及脂肪酶表达

优化了农杆菌介导转化黑曲霉的方法,优化条件包括共培养材料、农杆菌种类、诱导剂浓度、共培养时间、共培养温度以及共培养时农杆菌的菌体浓度。结果表明,农杆菌介导转化黑曲霉的最适条件为107个/mL不萌发的新鲜孢子与OD_(600)培养至0.9～1.0的农杆菌以1∶1的比例混合后,在乙酰丁香酮浓度为200μmol/L的IM平板上,23℃避光培养48 h后进行转膜,转化子个数可达到(60±5)个转化子/10~6个孢子,且阳性率达到90%以上。并且构建了同源黑曲霉脂肪酶的组成型和诱导型启动子表达载体,通过优化后的农杆菌介导转化方法转化至黑曲霉中,利用罗丹明橄榄油平板对产酶转化子进行筛选鉴定,并获得了阳性克隆。     

Environmental factors modify carbon nutritional patterns and niche overlap between Aspergillus flavus and Fusarium verticillioides strains from maize

This study examined the utilization patterns of key carbon sources (CS, 24: including key sugars, amino acids and fatty acids) in maize by strains of Aspergillus flavus and Fusarium verticillioides under different water activity (a_w, 0.87–0.98 a_w) and temperature (20–35 °C) values and compared the niche overlap indices (NOI) that estimate the in vitro CS utilization profiles [Wilson, M., Lindow, S.E., 1994. Coexistence among epiphytic bacterial populations mediated through nutritional resource partitioning. Applied and Environmental Microbiology 60, 4468–4477.]. The ability to grow in these key CS in minimal media was studied for 120 h in 12 h steps. The NOI was calculated for inter-species (F. verticillioides–A. flavus) and for intra-species (A. flavus–A. flavus) using CS utilization patterns over the range of interacting environmental conditions. 30 °C, over the whole a_w range examined, was found to be optimal for utilization of the maximum number of CS by A. flavus. In contrast, for F. verticillioides this was more so at 20 °C; 25 °C allowed a suboptimal usage of CS for both species. NOIs confirmed the nutritional dominance of A. flavus at 30 °C, especially at lower a_w levels and that of F. verticillioides at 20 °C, mainly at 0.95 a_w. In other conditions of a_w, based on CS utilization patterns, the data indicated that A. flavus and F. verticillioides occupied different ecological niches. The variability in nutritional sources utilization between A. flavus strains was not related to their ability to produce aflatoxins (AFs). This type of data helps to explain the nutritional dominance of fungal species and strains under different environmental conditions. This could be useful in trying to find appropriate natural biocontrol microorganisms to compete with these mycotoxigenic species.     

Influence of environmental conditions on thermal stability of recombinant Aspergillus aculeatus pectinmethylesterase

The thermal stability of purified recombinant Aspergillus aculeatus pectinmethylesterase (PME) in different media was studied. The influence of pH, ionic strength and additives (salts and polyols) was evaluated. At pH 5.0 and a high ionic strength (0.50 M), the enzyme showed a high thermostability (inactivation at temperatures 60 °C). Interestingly, an enhancement of its heat stability was observed at pH 7.0 and temperatures above 55 °C, this behaviour was reflected in an atypical evolution of structural changes in the overall conformation of the enzyme, according to FTIR spectroscopy results. Recombinant A. aculeatus PME thermal inactivation at pH 7.0 could be described by a fractional-conversion model. Addition of NaCl increased the thermal stability at pHs 5.0 and 7.0, while addition of CaCl₂ had no influence. With regard to sugars (sucrose, trehalose, glucose and maltose) and polyols (sorbitol, lactitol and glycerol) addition, at the same concentration and pH, the polyols showed a higher protective effect than sugars. Also, the thermostability of recombinant A. aculeatus PME increased with the additive concentration, although the source of OH groups was the main parameter involved.