Construction and evaluation of a three-dimensional structure of cytochrome P450choP enzyme (CYP105C1)

The purpose of this work was to develop and carefully evaluateimproved strategies for constructing reliable 3-D models ofP450 isozymes. To this end, a unique combination of steps forbuilding and evaluating a model structure was used to builda homology model of the P450choP isozyme, based on knowledgeof the X-ray structures of P450cam, P450terp, P450BM-3 and P450eryF.Specifically, the reliability of this model was examined bysystematic comparisons of its conformational, energetic, environmentaland packing properties and those of the four reference proteinswith corresponding properties from the database of proteinswith known structures. The results showed that the examinedproperties of this model structure are well within the criteriaestablished for reliable structures and are of nearly as goodquality as those of the reference proteins. In addition, theresult from a 120 ps unconstrained MD simulation of the modelwith structural waters provided evidence that the model is stableat room temperature. This 3-D model can now be reliably usedfor explicit characterization of substrate and inhibitor complexes.Most importantly, although it is envisioned that building modelsfor mammalian P450s will be even more challenging, the stepsdescribed here should be very useful in future constructionof 3-D models of mammalian P450 isozymes.     

Stabilized variant of Streptomyces subtilisin inhibitor and its use in stabilizing subtilisin BPN'

Protein protease inhibitors could potentially be used to stabilize proteases in commercial products such as liquid laundry detergents. However, many protein protease inhibitors are susceptible to hydrolysis inflicted by the protease. We have engineered Streptomyces subtilisin inhibitor (SSI) to resist proteolysis by adding an interchain disulfide bond and removing a subtilisin cleavage site at leucine 63. When these stabilizing changes were combined with changes to optimize the affinity for subtilisin, the resulting inhibitor provided complete protease stability for at least 5 months at 31 degrees C in a subtilisin-containing liquid laundry detergent and allowed full recovery of the subtilisin activity upon the dilution that occurs in a North American washing machine.     

空气负离子对阿维链霉菌发酵周期及产量的影响

为了研究低浓度空气离子对微生物的效应,利用低浓度空气离子对固体培养基上Streptomyces avermitilis作用,分析了其次生代谢产物产生时间早晚,各组分含量变化,并且初步研究有效成分含量提高的机理.用浓度为5×103/cm3的空气负离子连续十天处理固体培养基上的阿维链霉菌,经过高效液相色谱(High performance liquid chromatography,HPLC)检测后发现:经过处理,该菌株提早四天到达阿维菌素(Avermectins,AVM)产素平台期,有效成分B1a和B1b提高约20%,而无效成分含量无显著提高.经过分析,低浓度空气负离子可有效缩短该菌种的发酵周期,提高有效成分含量,刺激次生代谢过程.     

Optimization of nitrilase production from a new thermophilic isolate

A new thermostable nitrilase‐producing isolate identified as Streptomyces sp. MTCC 7546 has been studied extensively for the optimization of enzyme production operating in batch mode. The benzonitrile was observed as inducer of nitrilase production. The isolate showed maximum nitrilase production after 24 h of incubation at optimal conditions. The strain grows well on a variety of carbon sources and produces the nitrilase that catalyses the hydrolysis of nitriles to acids without formation of amides. The enzyme is mostly active against mono‐ and di‐aliphatic nitriles (10 mmol L^?1) at pH of 7.4 and at a temperature of 50 °C. Copyright © 2007 Society of Chemical Industry     

大孔树脂D380固定化橄榄绿链霉菌E-86来源木聚糖酶的研究

选择有代表性的 1 4种吸附和离子交换树脂进行了橄榄绿链霉菌E 86来源的木聚糖酶固定化试验 ,筛选出固定化效果较好的 72 4和D3 80两种树脂 ;对含伯氨基的离子交换树脂D3 80采用戊二醛进行交联固定化 ,研究了其固定化条件。结果表明 ,戊二醛浓度为1 % ,处理 3 0min ,加酶量为 0 8～ 1mL ,酶液pH 5 8,2 5℃ ,5～ 1 0h固定化处理效果最好 ,获得的固定化酶活力可达 64U/ g(载体 )。     

微生物谷氨酰胺转胺酶的分批发酵生产

在首先采用我们自行设计的2L小型、便易的生化反应器,对我们研究室保藏的高产酶菌株链霉菌(Streptomyces sp.)WZFF.L-M168发酵生产微生物谷氨酰胺转胺酶(MTG)过程中发酵培养基的碳源、氮源和初始pH值的影响作用进行研究,确定培养条件后,逐级扩大发酵罐规模,在20L和200L发酵罐上以在线监控技术手段直接监测分析环境因素对MTG发酵生产的作用效果,确立各级发酵罐分批发酵的生产工艺条件。结果表明:碳氮源采用葡萄糖、淀粉和多价胨,初始pH值为7.0,接种量5%～10%,发酵过程中在线控制培养温度、pH、通气量和搅拌速度等各项参数指标分别为30±0.5℃、6.6～6.9,0.8～1.1vvm和200～400r/min较为适宜。在这优化技术条件下,200L发酵罐可以稳定生产酶活在2.75U/ml以上的MTG。     

50L自控式发酵罐中ε-聚赖氨酸发酵条件的优化

在50L自控式发酵罐上进行了ε-聚赖氨酸(ε-PL)发酵条件的优化研究,主要考察了分批发酵和补料分批发酵过程中pH、搅拌转速对ε-PL发酵和菌体生长的影响。结果表明,当控制pH在5.0以上时有利于菌体的生长,但ε-PL基本不合成,甚至出现降解现象;当维持pH在4.0左右时,能促进ε-PL的合成。搅拌转速为300 r/min时,有利于溶氧和传质,400r/min时,由于剪切力过大,导致菌丝断裂,ε-PL产量下降。通过控制发酵中后期pH4.0和搅拌转速300r/min的pH反馈控制自动流加补料培养,可获得最大的ε-PL产量7.36g/L,产率0.072g/g,产量较优化前提高近10倍,产率提高2倍。     

Development of an Intergeneric Conjugal Transfer System for Xinaomycins-Producing Streptomyces noursei Xinao-4

To introduce DNA into Streptomyces noursei xinao-4, which produces xinaomycins, we explored an intergeneric conjugal transfer system. High efficiency of conjugation (8 × 10⁻³ exconjugants per recipient) was obtained when spores of S. noursei xinao-4 were heat-shocked at 50 °C for 10 min, mixed with Escherichia coli ET12567 (pUZ8002/pSET152) in the ratio of 1:100, plated on 2CMY medium containing 40 mmol/L MgCl₂, and incubated at 30 °C for 22 h. With this protocol, the plasmids pKC1139 and pSET152 were successfully transferred from E. coli ET12567 (pUZ8002) with different frequencies. Among all parameters, the ratio of donor to recipient cell number had the strongest effect on the transformation efficiency. In order to validate the above intergeneric conjugal transfer system, a glycosyltransferase gene was cloned and efficiently knocked out in S. noursei xinao-4 using pSG5-based plasmid pKC1139.     

色褐链霉菌磷脂酶D基因pld的克隆与表达

利用表达载体pET-22b( ),实现了色褐链霉菌磷脂酶D基因在大肠杆菌BL21(DE3)中的高效表达.利用镍亲和柱对表达产物进行纯化,将纯化后的重组磷脂酶D作用于底物卵磷脂和丝氨酸定向合成磷脂酰丝氨酸,并用HPLC法检测酶活力.结果表明,目的蛋白可在短时间内进行大量表达.转酯反应6 h后卵磷脂的转化率达到31%,重组磷脂酶D活力达到39 U·(mg蛋白)-1.