嗜热菌Anoxybacillus sp．DL3产蛋白酶条件及其酶学性质研究

目的：对Anoxybacillussp．DL3的产蛋白酶条件及其酶学性质进行研究,为下一步进行蛋白酶基因的克隆、表达提供依据。方法：应用常规方法液体培养细菌,研究温度、pH、培养基中碳源、氮源对菌株产蛋白酶的影响,硫酸铵盐析的方法提取酶液,并采用Folin法测酶活性。用紫外分光光度计在OD680hi／1测吸光值。并对提取的蛋白酶液进行酶的最适温度、pH以及酶的热稳定性和pH稳定性研究,向酶液中添加金属离子和EDTA、PMSF,研究其对酶活性的影响。结果：在培养基初始pH是6．5,培养温度为40℃时菌株产酶酶活性最大;培养基中以乳糖为碳源,酵母膏和硫酸铵为氮源,碳源与氮源的比例为1：2时,酶活最大。酶学性质研究结果显示：该酶的最适反应温度是55℃,最适反应pH是7．0;在50℃保温20min-80min内,酶活力下降幅度较小。60℃保温60min后,仍保持约60％的酶活。70℃保温60min后,残余酶活为30％。该酶在pH为6．0～8．0范围内,相对酶活差别不是很大,下降趋势大致相同。在强碱条件下,相对酶活下降很明显。Fe2＋、Cu2＋和Hg2＋对酶活性有明显的抑制作用;Ca2＋、Mg^2＋、Mn2＋等金属离子对酶活性有明显的促进作用;乙二胺四乙酸（EDTA）和苯甲基磺酰氟（PMSF）对酶活性也有一定抑制作用。结论：Anoxybacillussp．DL3所产的蛋白酶为嗜热中性蛋白酶,此酶具有较好的热稳定性和pH耐受性,该菌株具有进一步开发、利用的价值。     

一株产碱性蛋白酶菌株的选育及其发酵条件的研究

从土壤中分离到的553株芽孢杆菌中选出一株产蛋白酶活力较高菌株,经紫外线和亚硝基胍诱变,获得一株产碱性蛋白酶的菌株,酶活力可达10000u／m1,此菌株经鉴定为地衣状芽孢杆菌(Bacillus licheniformis)。适宜的发酵条件：培养基由6％玉米粉(山芋粉或葡萄糖),4％豆饼粉(或其水解液),Na,HP0．·12Hz0 0．4％,KH：P0·0．03％,Na zc0,0．1％组成,pH7．O,37℃旋转摇床振荡培养{2一q6小时。酶作用的最适条件：60℃,pH9．0一10．5,在pH6．0—10．5范围内稳定。酶受DFP抑制。     

均匀设计法对产几丁质酶细菌C4发酵条件的优化

系统研究了碳源,氮源,起始pH值、培养基装量、培养温度和时间等因素对细菌C4产几丁质酶的影响。结果表明,碳、氮源分别以胶体几丁质、KNO和蛋白胨最好;在起始pH值7．6—8．5,培养基装量为三角瓶体积的12％,培养温度28℃,振荡培养(180r／min)5d时最有利于几丁质酶的产生。在此基础上通过均匀设计法优化了发酵培养基配方。优化后的培养基配方为：胶体几丁质1．5％,蛋白胨0．55％,KNO3 0．3％,MgSO4 0．09％,Tween80 0．005％。在该条件下,几丁质酶活力达2．68U／mL,比在原基础培养条件下的酶活力提高90．1％。     

青霉素酰化酶产生菌的筛选和大肠杆菌AS 1.76产酶条件的研究

为筛选较优的产青霉素酰化酶的菌种,我们从9个属的232株纽菌中选出了19株产青霉素酰化酶的大肠杆菌,其中,AS 1．76和E 110为最好。还对大肠杆菌AS 1.76的产酶条件进行了研究。结果表明,最适的培养基成分是(％)：蛋白胨l,苯乙酸0．2,玉米浆0．3,氯化钠0．5;在250毫升的三角瓶中装30毫升培养基时,通气量正合适：培养基的初始pH以7.0为佳;培养时间15小时。在未加玉米浆的培养基中,少量的Fe2+(5馓克／毫升)对酶形成有刺激作用,过量的Fe2+(大于10教克／毫升)是不利的;在培养基中添加0．3％的玉米浆能降低Fe2+对酶形成毒害作用。     

微生物法生产普鲁兰酶的研究

对产普鲁兰酶的出发菌株进行筛选和诱变,使酶活从22．10u／ml提高到了40．77u／ml,酶活提高了84．4％。随后对菌体产酶培养基进行了确定和优化,得到最佳发酵培养基,在此培养基下,菌体产酶达46．76u／ml,为原酶活的114．7％。     

固态发酵生产腺苷酸脱氨酶

对多株曲霉产腺苷酸脱氨酶的性能进行了比较,发现米曲霉3.800(Aspergillus oryzae)产酶水平较高。该菌株固态发酵产酶的适宜培养基为：以麸皮为主原料,蔗糖2％,鱼粉2％,（NH4）2SO4 0．1％,柠檬酸钠0．2％,MgSO4 0.05%,吐温－80 0．1％,含水量50％。最佳的培养条件为：250mL三角瓶装20g培养基,在28－30℃培养60h。在优化条件下,培养物酶活可达到1543．48u/g鲜曲。     

双酶水解紫贻贝制备酵母调味料研究

利用复合蛋白酶、复合风味蛋白酶、中性蛋白酶对紫贻贝进行双酶水解,水解效果比较后选择使用复合蛋白酶和复合风味蛋白酶作为复合水解酶,同时采用产酯酵母发酵技术制备调味料,通过电位滴定法测定单菌株和多菌株发酵对双酶水解贻贝肉产总酯的影响。结果表明：产酯酵母1274在双酶水解后,接种量为5％,发酵温度28℃,发酵时间72h时总酯含量为0．65％,相同条件下,产酯酵母1274和1202多菌株发酵时总酯含量为0．78％,经产酯酵母发酵后的调味料,酯香味浓郁,给予产品以发酵特有的风味。     

一株产蛋白酶南极耐冷细菌的筛选及研究

从南极中山站地区分离到1株产胞外酸性蛋白酶的革兰氏阴性杆菌,该菌能在7度,20度及30度生长并产酶;其最适生长温度在20度左右,不耐盐。碳源物质中,葡萄糖对菌株的生长有利,但对蛋白酶的生成影响不大。氮源物质中,蛋白胨对菌株的生长及蛋白酶的生成效果最好,而（NH4）2SO4则是效果最好的无机氮源。该菌所产胞外蛋白酶占其蛋白酶总量的83．2％,蛋白酶反应的最适温度为40度,最适PH为5;酶活力在35度以下保持稳定,直接以酪蛋白液为培养基,在20度条件下对该菌进行摇瓶培养,6d后菌液浓度及产酶量皆到达高值并基本保持稳定,而以LB培养基（Luria-Bertani培养基）在相同条件下培养该菌,3d后菌液浓度即到达高值并基本保持稳定,酶活力则在2d后到达高值。     

绿色糖单孢菌产木聚糖酶规律及其耐碱耐热性的初步研究

采用绿色糖单孢菌为实验材料,在不同诱导产酶培养基上经过192h的振荡培养,探索其产酶时程规律．结果表明,不同的诱导底物诱导产生的木聚糖酶活性差异不显著,但诱导产纤维素酶活性差异显著．其中松木粉加棉纱培养基诱导产纤维素酶活性为0．08IU／ml,与空白对照(5．40IU／ml)相比显著下降(P≤0．05)．为了适应纸浆漂白实际应用中纤维素酶越少越好的要求,选择该培养基为最佳诱导产酶培养基．绿色糖单孢菌在上述培养基中培养156h后达到木聚糖酶产酶高峰。粗酶液酶活可达到9．03IU／ml．通过对该酶进行高温及碱性处理。实验结果表明绿色糖单孢菌分泌的木聚糖酶在pH7．0下反应表现最高活性,同时在90℃下保温3h后酶活为原来的63．55％,具有较好的耐碱耐热性．     

假丝酵母尿酸酶形成条件

选出了一株产尿酸酶的产朊候丝酵母(Candida utilis)AS2．117。此菌株尿酸酶形成条件的研究表明：尿酸、黄嘌呤和鸟嘌呤对酶形成起诱导作用;玉米浆对菌株生长和酶形成起十分重要的作用;蔗糖、葡萄塘、D-甘露糖和果糖是酶形成的适合碳源;生物素对酶产生有促进作用;在含有玉米浆培养基中加入无机氮源对产酶无作用,添加有机氮略增加产酶量。尿酸酶形成最适培养基组成为(％)：蔗糖;,玉米浆3,尿酸0．1,蛋白胨0．1,生物素0．05,KCI0．1,NaCl 0.1。最适pH为6．2。在250ml三角瓶中装30ml培养基为最适。在200r／min的旋转摇床上25℃振荡培养21h,在此条件下最终酶活力可达0．6u／ml。     

极地适冷菌Pseudoalteromonas sp. QI-1产适冷蛋白酶发酵条件的优化

对极地适冷菌Pseudoalteromonas sp. QI-1产适冷蛋白酶的发酵条件进行优化。结果表明:菌株QI-1的最适生长和产酶温度均为5℃;最佳接种量为1%;发酵培养基的最适初始pH和最佳装样量分别为5和10%;盐度为2%时对菌株的生长和产酶最为有利;麸皮和醋酸钠分别为最佳N源和C源;添加0.75%酪蛋白时菌株QI-1胞外蛋白酶的活性最高;10 mmol/L Mg2+和0.5%Tween-80有利于产酶。正交试验结果表明:菌株Pseudoalteromonassp. QI-1产蛋白酶较佳培养基配方(g/L)为麸皮5,酵母粉2.5,酪蛋白3,MgCl2.6H2O 3,KCl 1.5;发酵液比酶活为166.20 U/mL,较优化前提高了约56%。     

Production of an extracellular alkaline metalloprotease from a newly isolated, moderately halophile, Salinivibrio sp. strain AF-2004

An extracellular protease was produced under stress conditions of high temperature and high salinity by a newly isolated moderate halophile, Salinivibrio sp. strain AF-2004 in a basal medium containing peptone, beef extract, glucose and NaCl. A modification of Kunitz method was used for protease assay. The isolate was capable of producing protease in the presence of sodium chloride, sodium sulfate, sodium nitrate, sodium nitrite, potassium chloride, sodium acetate and sodium citrate. The maximum protease was secreted in the presence of 7.5 to 10% (w/v) sodium sulfate or 3% (w/v) sodium acetate (4.6 U ml⁻¹). Various carbon sources including glucose, lactose, casein and peptone were capable of inducing enzyme production. The optimum pH, temperature and aeration for enzyme production were 9.0, 32 °C and 220 rpm, respectively. The enzyme production corresponded with growth and reached a maximum level during the mid-stationary phase. Maximum protease activity was exhibited in the medium containing 1% (w/v) NaCl at 60 °C, with 18% and 41% activity reductions at temperature 50 and 70 °C, respectively. The optimum pH for enzyme activity was 8.5, with 86% and 75% residual activities at pH 10 and 6, respectively. The activity of enzyme was inhibited by EDTA. These results suggest that the protease secreted by Salinivibrio sp. strain AF-2004 is industrially important from the perspectives of its activity at a broad pH ranges (5.0–10.0), its moderate thermoactivity in addition to its high tolerance to a wide range of salt concentration (0–10% NaCl).     

Optimization of culture conditions for the production of haloalkaliphilic thermostable protease from an extremely halophilic archaeon Halogeometricum sp. TSS101

AIMS: Isolation and screening of extreme halophilic archaeon producing extracellular haloalkaliphilic protease and optimization of culture conditions for its maximum production. METHODS AND RESULTS: Halogeometricum sp. TSS101 was isolated from salt samples and screened for the secretion of protease on gelatin and casein plates containing 20% NaCl. The archaeon was grown aerobically in a 250 ml flask containing 50 ml of (w/v) NaCl 20%; MgCl(2) 1%; KCl 0.5%; trisodium citrate 0.3%; and peptone 1%; pH 7.2 at 40 degrees C on rotary shaker. The production of enzyme was investigated at various pH, temperatures, NaCl concentrations, metal ions and different carbon and nitrogen sources. The partially purified protease had activity in a broad pH range (7.0-10.0) with optimum activity at pH 10.0 and a temperature (60 degrees C). The enzyme was thermostable and retained 70% initial activity at 80 degrees C. Maximum protease production occurred at 40 degrees C in a medium containing 20% NaCl (w/v) and 1% skim milk powder after 84 h in shaking culture. Enzyme secretion was observed at a broad pH range of 7.0-10.0. Addition of CaCl(2) (200 mmol) to the culture medium enhanced the production of protease. Protein rich flours proved to be cheap and good alternative source for enzyme production. Different osmolytes were tested for the growth and production of haloalkaliphilc protease and found that betaine and glycerol enhanced growth without secretion of the protease. Immobilization studies showed that whole cells immobilized in 2% alginate beads were stable up to 10 batches and able to secrete the protease, which attained maximum production within 60 h under shaking conditions. CONCLUSIONS: Halogeometricum sp. TSS101 secreted an extracellular haloalkaliphilic and thermostable protease. The optimum conditions required for maximum production are 20% NaCl, 1% skim milk powder and temperature at 40 degrees C. Addition of CaCl(2) (200 mmol) enhanced the enzyme production. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of haloalkaliphilic protease. SIGNIFICANCE AND IMPACT OF THE STudy: The low cost protein rich flours were used as an alternative carbon and nitrogen sources for enzyme production. Immobilization of halophilic cells in alginate beads can be used in continuous production of halophilic enzyme. The halophilic and thermostable protease from Halogeometricum sp. TSS101 is good source for industrial applications and can be a suitable source for preparation of fish sauce.     

Production of extracellular protease from halotolerant bacterium, Bacillus aquimaris strain VITP4 isolated from Kumta coast

A newly isolated halotolerant Bacillus sp. VITP4 was investigated for the production of extracellular protease. 16S rRNA gene analysis identified it as Bacillus aquimaris. Enzyme secretion corresponded with growth (G_t, 38 min) in the basal Zobell medium, reaching a maximum during stationary phase (630 U/ml, 48 h). Protease production was investigated in different salt concentrations (0–4 M). While growth was optimum in the basal medium, higher levels of protease activity were observed in 0.5 M salt medium (728 U/ml, 48 h) and 1 M salt medium (796 U/ml, 78 h) with 21% and 32% increase in production, respectively. Salt concentrations above 2.5 M did not support bacterial growth. The optimum pH and temperature for production were pH 7.5 and 37 °C, respectively. A combination of peptone and yeast extract yielded optimum protease secretion. Inorganic nitrogen sources proved to be less favourable. Production was reduced in the presence of readily available carbon sources owing to catabolic repression. Effect of various salts (1–6%) indicated favourable bacterial growth in these conditions for producing proteolytic molecules with increased activity. The study assumes significance in the ability of the halotolerant bacterium to survive in a wide range of salinity and yield optimum levels of extracellular protease.     

Physical factors affecting the production of organic solvent-tolerant protease by Pseudomonas aeruginosa strain K

The physical factors affecting the production of an organic solvent-tolerant protease from Pseudomonas aeruginosa strain K was investigated. Growth and protease production were detected from 37 to 45 degrees C with 37 degrees C being the optimum temperature for P. aeruginosa. Maximum enzyme activity was achieved at static conditions with 4.0% (v/v) inoculum. Shifting the culture from stationary to shaking condition decreased the protease production (6.0-10.0% v/v). Extracellular organic solvent-tolerant protease was detected over a broad pH range from 6.0 to 9.0. However, the highest yield of protease was observed at pH 7.0. Neutral media increased the protease production compared to acidic or alkaline media.     

土壤中高产蛋白酶菌株产酶条件及酶学性质

【背景】微生物蛋白酶已经成为工业用蛋白酶的主要来源,筛选具有特殊环境适应性的微生物成为生物酶资源的开发热点。【目的】通过对青藏高原土壤微生物产蛋白酶菌株的筛选、优化及相关特性研究,寻找新的蛋白酶资源,为高原菌种资源利用提供科学依据。【方法】采用形态学和分子生物学对筛选菌株进行菌种鉴定,利用单因素试验和正交试验对菌株进行发酵条件优化及酶学性质的探究。【结果】筛选出一株高产蛋白酶菌株XC2,经鉴定菌株XC2为枯草芽孢杆菌(Bacillus subtilis)。XC2最优产酶条件:可溶性淀粉4.0%,牛肉膏1.0%,K~+0.6%,培养温度34°C、初始pH 7.0、接种量2.0%的条件下200 r/min振荡培养13 h,所产蛋白酶活力最高为638.5 U/mL。XC2所产蛋白酶最适反应温度60°C,最适pH9.0;40-50°C、pH8.0-10.0条件下酶活稳定性较高;Mn~(2+)对酶活力有明显激活作用,而Zn~(2+)、Cu~(2+)、Fe~(2+)、Fe3+对酶活力有明显抑制作用。【结论】枯草芽孢杆菌XC2有较强的产碱性蛋白酶的能力,具有较好的应用前景。     

Optimization of conditions for protease production by Chryseobacterium taeanense TKU001

A protease-producing bacterium was isolated and identified as Chryseobacterium taeanense TKU001. An extracellular metalloprotease with novel properties of solvent- and surfactant-stable was purified from the culture supernatant of C. taeanense TKU001 with shrimp shell wastes as the sole carbon/nitrogen source. The optimized condition for protease production was found when the culture was shaken at 37 degrees C for 3 days in 50 mL of medium containing 0.5% shrimp shell powder (SSP) (w/v), 0.1% K2HPO4, and 0.05% MgSO4.7H2O. Two extracellular proteases (FI and FII) were purified and characterized, and their molecular weights, pH and thermal stabilities were determined. The molecular masses of TKU001 protease FI and FII determined by SDS-PAGE and gel filtration were approximately 41 kDa and 75 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU001 protease FI were 8, 60 degrees C, pH 6-9, and 60 degrees C, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU001 protease FII were 7, 60 degrees C, pH 7-9, and 50 degrees C, respectively. TKU001 protease FI and FII were both inhibited completely by EDTA, indicating that the TKU001 protease FI and FII were metalloproteases. TKU001 protease FI and FII retained more than 75% of its original protease activity after preincubation for 10 days at 4 degrees C in the presence of 25% most tested organic solvents. Additionally, the TKU001 protease FI retained 79%, 80%, and 110% of its original activity in the presence of 2% Tween 20, 2% Tween 40, and 2% Triton X-100, respectively. However, at the same condition, the activity of TKU001 protease FII retained 100%, 100%, and 121% of its original activity, respectively. This is the first report of C. taeanense being able to use shrimp shell wastes as the sole carbon/nitrogen source for proteases production. The novelties of the TKU001 protease include its high stability to the solvents and surfactants. These unique properties make it an ideal choice for application in detergent formulations and enzymatic peptide synthesis.     

Enhanced production and characterization of a highly thermostable alkaline protease from Bacillus sp. P-2

An obligatory alkalophilic Bacillus sp. P-2, which produced a thermostable alkaline protease was isolated by selective screening from water samples. Protease production at 30 °C in static conditions was highest (66 U/ml) when glucose (1% w/v) was used with combination of yeast extract and peptone (0.25% w/v, each), in the basal medium. Protease production by Bacillus sp. P-2 was suppressed up to 90% when inorganic nitrogen sources were supplemented in the production medium. Among the various agro-byproducts used in different growth systems (solid state, submerged fermentation and biphasic system), wheat bran was found to be the best in terms of maximum enhancement of protease yield as compared to rice bran and sunflower seed cake. The protease was optimally active at pH 9.6, retaining more than 80% of its activity in the pH range of 7–10. The optimum temperature for maximum protease activity was 90 °C. The enzyme was stable at 90 °C for more than 1h and retained 95 and 37% of its activity at 99 °C and 121 °C, respectively, after 1 h. The half-life of protease at 121 °C was 47 min.     

The ineffectuality of potato protease inhibitor on the extracellular protease from Erwinia carotovora subsp. carotovora

Growth conditions are described for optimum production of extracellular protease in batch cultures of the soft rot pathogen Erwinia carotovora subsp. carotovora . This protease was inhibited by approximately 96% by 1 mmol/1 EDTA and by 55–6% by 10 mmol/l cysteine thereby classifying it as a metalloprotease. It was not inhibited by a chymotrypsin inhibitor extracted from potato tubers. This evidence suggests that the potato chymotrypsin inhibitor is not associated with resistance of potatoes to E. carotovora subsp. carotovora .     

响应面法优化重组毕赤酵母表达米曲霉碱性蛋白酶诱导条件的研究

为探索重组米曲霉碱性蛋白酶（rAlp）在毕赤酵母中表达的最佳诱导条件,本研究在单因子实验的基础上,运用Box-Behnken设计的响应面试验对诱导条件进行了优化。根据回归分析确定了影响rAlp表达的因子,求得最佳诱导条件为：诱导温度28．53℃,诱导pH6．63,甲醇浓度1．28％。此工艺条件下碱性蛋白酶活力实测值为49．78U／mL,回归模型的预测值与实测值的相对误差〈1％,说明该回归方程与实际情况拟合很好。