响应面法优化链霉菌A0901产几丁质酶抑制剂的发酵条件

用响应面法优化链霉菌A0901产几丁质酶抑制剂的发酵条件,以提高几丁质酶抑制剂的产率。利用单因素试验筛选出最佳碳源为可溶性淀粉、氮源为KNO3。采用两水平Plackett-Burman法筛选出对产几丁质酶抑制剂有重要影响的3 个因素：可溶性淀粉、ZnCl2和培养温度,通过最陡爬坡试验逼近最佳响应面区域,最后通过中心组合试验设计,利用SAS软件进行回归分析,得到最佳发酵培养条件：可溶性淀粉3.76 g/100 mL、NaCl 0.05 g/100 mL、KNO30.1 g/100 mL、K2HPO4·3H2O 0.05 g/100 mL、MgSO4·7H2O 0.04 g/100 mL、ZnCl2 0.024 g/100 mL、FeSO4·7H2O 0.001 g/100 mL、初始pH 6、温度28.54 ℃、转速250 r/min。在最优培养条件下,发酵液对几丁质酶的抑制率达到67.58%,较原发酵培养基的几丁质酶抑制率提高36.8%。     

Utilization of shrimp shellfish waste as a substrate for solid-state cultivation of Aspergillus sp. S1-13: evaluation of a culture based on chitinase formation which is necessary for chitin-assimilation

The utilization of shrimp shellfish waste as a substrate for solid-state cultivation of a filamentous fungus, Aspergillus sp. S1-13, was investigated. The organism was selected from among 220 isolates based on the productivity of its chitinolytic enzyme (chitinase), which might reflect microbial growth. The enzyme was produced only when the organism was grown on medium containing the shellfish waste. The addition of 58-65% water (w/w) to the medium was effective in enhancing production, and a certain amount of enzyme was observed in media of higher water content (up to about 75%). The initial pH and nitrogen source (ammonium sulfate) of the solid-state medium also affected the amount of enzyme. The amount of enzyme increased 2-fold in an optimum solid-state medium: 5 g of shrimp shellfish waste and 3 ml of basal medium (pH 5) containing 0.1% (NH4)2SO4 was inoculated with 4 ml of spore suspension; static cultivation at room temperature. The amount increased further (1.5-fold) when the cultivation was carried out at 37 degrees C, with 1.85 units of the enzyme formed from 1 g of shrimp shellfish waste. An analysis by ion-exchange column chromatography suggested the presence of at least two colloidal chitin-hydrolyzing enzymes and one p-nitrophenyl beta-D-N-acetylglucosaminide-hydrolyzing enzyme in an extract of the solid-state culture. The elution profile was similar to that obtained with a liquid culture filtrate.     

Preventing protein haze in bottled white wine

Slow denaturation of wine proteins is thought to lead to protein aggregation, flocculation into a hazy suspension and formation of precipitates. The majority of wine proteins responsible for haze are grape‐derived, have low isoelectric points and molecular weight. They are grape pathogenesis‐related (PR) proteins that are expressed throughout the ripening period post véraison, and are highly resistant to low pH and enzymatic or non‐enzymatic proteolysis. Protein levels in un‐fined white wine differ by variety and range up to 300 mg/L. Infection with some common grapevine pathogens or skin contact, such as occurs during transport of mechanically harvested fruit, results in enhanced concentrations of some PR proteins in juice and wine. Oenological control of protein instability is achieved through adsorption of wine proteins onto bentonite. The adsorption of proteins onto bentonite occurs within several minutes, suggesting that a continuous contacting process could be developed. The addition of proteolytic enzyme during short term heat exposure, to induce PR protein denaturation, showed promise as an alternative to bentonite fining. The addition of haze‐protective factors, yeast mannoproteins, to wines results in decreased particle size of haze, probably by competition with wine proteins for other non‐proteinaceous wine components required for the formation of large insoluble aggregations of protein. Other wine components likely to influence haze formation are ethanol concentration, pH, metal ions and phenolic compounds.     

甲壳素水解酶产生菌的分离选育

从沿海滩涂土壤、海鱼消化道、海水和海底污泥等自然环境中采集样品34个，从中分离获得能降解甲壳素的微生物114株，经过初筛和复筛，从中获得产酶能力较高的菌株C43，用比色法测定其酶活，发酵液粗酶活达0．115u。初步鉴定C43菌株为噬纤维菌科、噬纤维菌属。暂命名为噬纤维菌C43（Cytophaga.sp C43)。研究表明：噬纤维菌C43产生的甲壳素水解酶为诱导型。其产酶的最佳条件为：起始pH6．0，温度28℃，摇瓶转速200r/min，发酵周期为60h。     

萝卜CBPs的分离及部分酶学特性研究

通过脱氨再生几丁质凝胶亲和层析和CM-纤维素离子交换层析从萝卜中分离出一组CBPs，并对CBP1和CBP2溶菌酶酶学特性作了研究。活力分析表明：CBP1和CBP2均为溶菌酶／几丁质酶双功能酶；CBP3和CBP4只有几丁质酶活力。SDS—PAGE纯度分析表明：CBP1和CBP2已达到电泳纯，相对分子质量分别为26900和24800；CBP3仍具有两个组分，CBP4则由于含量过低，在凝胶上无蛋白带。CBP1和CBP2部分酶学特性分析表明：CBP1的最适反应条件为PH5．8，55C，0．4g／L溶壁微球菌；CBP2的最适反应条件为pH6．8，45℃，0．4g／L溶壁微球菌；CBP1在pH3．4～10．6，0～55℃较稳定，CBP2在pH2．0～10．6，0～60℃较稳定。     

Preparation of polyclonal antibodies specific for wine proteins

Immunology is an expanding area of research with potentially important applications in the analysis of many biological molecules. Polyclonal antibodies were raised in rabbits against specific proteins as well as against the total protein from a Portuguese wine. FPLC cation exchange chromatography was used to isolate the total protein fraction and, when in combination with denaturing electrophoresis, to purify individual wine polypeptides. To obtain a high titre, an injection of each antigen followed by three boosters were given in the immunisation of each rabbit. The titre of the antisera was measured by the ELISA technique and the specificity of the antibodies detected by immunoblotting. The antibodies produced were shown to be highly specific for the corresponding antigens. However, antibodies obtained specifically against a highly purified wine polypeptide seem to recognize the other major wine polypeptides, raising the possibility of structure similarity between different wine proteins. Neither the anti‐total wine protein antibodies not the anti‐specific wine protein antibodies originated a signal when used to probe thaumatin or chitinase. © 1999 Society of Chemical Industry     

A Chitinase and a β-1,3-Glucanase Isolated from the Seeds of Cowpea (Vigna unguiculataL Walp) Inhibit the Growth of Fungi and Insect Pests of the Seed

A chitinase (22000 Da) and a β-1,3-glucanase (26000 Da) were isolated from cowpea ( Vigna unguiculata ) seeds and shown to deter development, in an in vitro assay, of the phytopathogenic fungi Colletotrichum lindemuthianum (agent of anthracnose, a disease of many plants) and Colletotrichum musae (agent of banana peel anthracnose). The isolated chitinase was also shown to negatively affect the development of the cowpea weevil ( Callosobruchus maculatus ) in an artificial seed system. These results suggest that the cowpea seed contains defence proteins that could be, if their levels are properly managed, utilised to promote increased protection of the plant towards attacking fungi and insects.     

Expression of a gene encoding chitinase (pCA 8 ORF) from Aeromonas sp. no. 10S-24 in Escherichia coli and enzyme characterization

A gene encoding chitinase from Aeromonas sp. no. 10S-24 was expressed using pTrc99A in Escherichia coli JM 105 which yielded a 5-fold higher activity than when pUC19 was used. Three different truncated enzymes (SA-1, SA-2 and SA-3) were obtained after purification. Their isoelectric points were 7.0, 6.9, and 6.7, respectively. The enzymes showed two optimum pHs, 4.0 and 7.0, when incubated with ethylene glycol chitin as the substrate, and were stable over a wide pH range (3.0–9.0). The optimum temperature was 60°C and the enzymes were stable up to 50°C. The chitinases exhibited wide substrate specificities for chitin-related compounds.