INDUCTION, PROPERTIES AND APPLICATION OF XYLANASE ACTIVITY FROM SCLEROTINIA SCLEROTIORUM S2 FUNGUS

Extracellular xylanase activity was produced in a submerged culture by Sclerotinia sclerotiorum S2 fungus, on wheat bran as an inducing substrate. The enzyme was partially purified and biochemically characterized. The optimum pH and temperature for the activity were 5.5 and 65C, respectively. The xylanase was stable over a pH range of 4.0–9.0 and retained more than 75% of its activity after incubation for 24 h without substrate. The enzyme was stable at 50C and 60C for 80 min of incubation at pH 5.0, while the half‐life was 20 min at 70C. The novel xylanase activity was useful as an aid in orange juice clarification. The clarification was observed with concomitant production of reducing sugars (0.350 mg/mL from juice) after 24 h of incubation of juice with the xylanase. Xylanase aided juice clarification, which resulted in a 27% decrease in insoluble materials.     

Purification and partial characterization of psychrotrophic Serratia marcescens lipase

Serratia marcescens isolated from raw milk was found to produce extracellular lipase. The growth of this organism could contribute to flavor defects in milk and dairy products. Serratia marcescens was streaked onto spirit blue agar medium, and lipolytic activity was detected after 6 h at 30 degrees C and after 12 h at 6 degrees C. The extracellular crude lipase was collected after inoculation of the organism into nutrient broth and then into skim milk. The crude lipase was purified to homogeneity by ion-exchange chromatography and gel filtration. The purified lipase had a final recovered activity of 45.42%. Its molecular mass was estimated by SDS-PAGE assay to be 52 kDa. The purified lipase was characterized; the optimum pH was likely between 8 and 9 and showed about 70% of its activity at pH 6.6. The enzyme was very stable at pH 8 and lost about 30% of its activity after holding for 24 h at 4 degrees C in buffer of pH 6.6. The optimum temperature was observed at 37 degrees C and exhibited high activity at 5 degrees C. The thermal inactivation of S. marcescens lipase was more obvious at 80 degrees C; it retained about 15% of its original activity at 80 degrees C and was completely inactivated after heating at 90 degrees C for 5 min. Under optimum conditions, activity of the enzyme was maximum after 6 min. The Michaelis-Menten constant was 1.35 mM on tributyrin. The enzyme was inhibited by a concentration more than 6.25mM. Purified lipase was not as heat-stable as other lipases from psychrotrophs, but it retained high activity at 5 degrees C. At pH 6.6, the pH of milk, purified lipase showed some activity and stability. Also, the organism demonstrated lipolytic activity at 6 degrees C after 12 h. Therefore, S. marcescens and its lipase were considered to cause flavor impairment during cold storage of milk and dairy products.     

Production,Characterization and Application of a Xylanase from Streptomyces sp. AOA40 in Fruit Juice and Bakery Industries

Streptomyces sp. AOA40, which produces halotolerant and thermotolerant xylanase, was isolated from Mersin soil. Various carbon sources were tested for xylanase production with selected fermentation medium. The best carbon source was selected as corn stover. The effect of corn stover concentration and particle size, composition of fermentation medium, fermentation condition such as initial pH and agitation rate on xylanase production was determined. After production, xylanase was partially purified with ion-exchange chromatography and gel filtration chromatography for characterization of xylanase and application in fruit juice and dough improvement. The optimum pH for the activity of xylanase occurred at pH 6.0 in phosphate buffer, while the optimum temperature was 60°C. The relative xylanase activity in the pH ranges of 4–9 remained between 59.93 and 54.43% of the activity at pH 6.0 (100.00%). The xylanase activity showed a half-life of 172 min at 70°C, which was reduced to 75 min at 80°C. The enzyme was highly inhibited by 10–100 mM of Hg⁺², EDTA, Mg⁺², SDS and 100 mM Cu⁺². Clarity of fruit juices increased after enzymatic treatment of apple (17.85%), grape (17.19%) and orange juice (18.36%) with partially purified xylanase and also reducing sugar concentrations of these fruit juices were improved by 17.21, 16.79 and 19.57%, respectively. Also, dough volume was raised 17.06% with using partially purified Streptomyces sp. AOA40 xylanase in bread making.     

The effect of fibrolytic enzymes sprayed onto forages and fed in a total mixed ratio to lactating dairy cows

We investigated the effect of spraying different combinations of fibrolytic enzymes onto forages on their nutritive value for lactating cows. Holstein cows were fed a TMR consisting of 30% corn silage, 15% alfalfa hay, and 55% concentrate (dry matter basis). During a 12-wk treatment period, the forages were treated with no enzymes (control), cellulase D and sultanas B, or cellulase D and xylanase C. Enzymes were diluted in water and sprayed onto the forages while mixing. Both combinations of enzymes supplied similar amounts of fibrolytic activity based on classical enzyme assays conducted at 50 degrees C. Cows fed forages treated with cellulase D and xylanase B tended to produce more 3.5% FCM (+2.5 kg/d) than did cows fed the untreated forages. Dry matter intake, milk production, milk fat, and milk protein were unaffected by treatment. In vitro production of gas from forages treated with enzymes was greater than from untreated forage, but 96-h volatile fatty acid production was not different among treatments. With an alternative enzyme assay based on the depolymerization of dyed substrate at 40 degrees C, activity of xylanase C was greatest at a pH of 6.5 but was substantially reduced as the pH of the assay was decreased. In contrast, xylanase B showed highest activity at pH 5 and enzyme activity was twice that of xylanase B at pH 5.5 and 6. Overall, the results of this study provide more evidence that fibrolytic enzymes can be used to improve milk production in lactating cows.     

不同来源木聚糖酶酶学性质研究

目的 研究比较4种不同来源木聚糖酶的酶学性质,为各酶在不同领域应用提供理论依据.方法 通过摇瓶发酵获得源自4种菌株的木聚糖酶粗酶液,用二硝基水杨酸（DNS）法测定各木聚糖酶的酶活性,并研究酶学性质.结果 嗜热棉毛菌、绿色木霉、泡盛曲霉和橄榄绿链霉菌来源的木聚糖酶的最适温度分别为70,60,50和60℃;最适pH分别为6,4,4和6.嗜热棉毛菌来源的木聚糖酶耐热性最强,高温60℃下,酶活性高于85％,70℃仍保持60％以上的酶活性,且在pH4～11的范围内能保持较高稳定性.另外3种来源的木聚糖酶则有较强的耐酸性.结论 嗜热棉毛菌来源的木聚糖酶有饲料添加剂方面的应用优势.另外3种来源的木聚糖酶可根据不同行业对木聚糖酶的要求,应用于食品、医药、能源、环境等领域.     

Characterization of almond alpha-mannosidase and its application for structure analysis of sugar chain

Almond alpha-mannosidase was purified by separation on columns of DEAE-Sephadex A50 and hydroxyapatite, and characterized. Its optimum pH was approximately 3.8. It was also shown to be stable from pH 6 to 8. Its activity was stable up to 60 degrees C. The thermostability of almond alpha-mannosidase at 73 degrees C appeared to be superior to that of jack bean a-mannosidase. We examined the substrate specificity of the former toward high-mannose-type N-glycan Man9GlcNAc2, and showed that the deduced trimming pathway was more diverse than that of the latter. We could use almond alpha-mannosidase as well as jack bean alpha-mannosidase for analysis of sugar chain structures.     

Properties of an Alkaline‐Tolerant,Thermostable Xylanase from Streptomyces chartreusis L1105, Suitable for Xylooligosaccharide Production

Abstract: An extracellular xylanase was purified to homogeneity from a culture of Streptomyces chartreusis L1105 by a 2‐step method of ammonium sulfate precipitation and carboxymethyl sepharose fast‐flow chromatography (CMSFF). The xylanase was purified by 6.86‐fold, with a recovery yield of 31.96%. The purified xylanase appeared as a single protein band on SDS‐PAGE with a molecular mass of about 34.2 kDa. The optimum temperature and pH of the purified xylanase activity were 70 °C and 7.2 respectively. The xylanase was more stable under alkaline conditions and retained more than 80% activity after 30 min incubation at pH 6 to 10. It also showed specific activity towards different xylans. Hydrolysis of oat‐spelt and corn‐cob xylans by the xylanase yielded xylobiose and xylotriose as principle products without the formation of xylose. These properties indicate that the purified xylanase may potentially be useful in biotechnological applications, such as xylooligosaccharide preparation. This is the first report about the purification and characterization of a xylanase from S. chartreusis.     

Expression of recombinant Thermomonospora fusca xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it

The mature peptide of Thermomonospora fusca xylanase A (TfxA) was successfully expressed in Pichia pastoris under the control of AOX1 promoter. The activity of recombinant T. fusca xylanase A (reTfxA) in culture supernatant was 117.3 ± 2.4 U/mg, which is 3 times higher than that of the native TfxA. The optimal temperature and pH for reTfxA were 60 °C and 6.0, respectively. When treated at 70 °C and pH 6.0 for 2 min, the residual activity of the reTfxA was 70%. The reTfxA was very stable over a wide pH range (5.0–9.0). After incubation over pH 5.0–9.0 at 25 °C for 1 h, all the residual activity of reTfxA was over 80%. The K_m and k_cat values for reTfxA were 2.45 mg/ml and 139 s⁻¹, respectively. HPLC analysis revealed that xylobiose (X2) was the main hydrolysis product released from birchwood xylan and wheat bran insoluble xylan by reTfxA. Hydrolysis results of xylooligosaccharides showed that reTfxA was an endo-acting xylanase and xylobiose, xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) could be hydrolysed. This is the first report on the expression of reTfxA in yeast and on the determining and quantifying of the hydrolysis products released from xylans and xylooligosaccharides by reTfxA.     

Survival,growth, and inactivation of acid-stressed Shigella flexneri as affected by pH and temperature

A study was done to determine the survival, growth, and inactivation characteristics of unadapted, acid-adapted, and acid-shocked Shigella flexneri 2a cells as affected by pH and temperature. The pathogen was grown at 37 degrees C for 18 h in tryptic soy broth containing no glucose (TSBNG) (unadapted cells) and TSBNG supplemented with 1% glucose (TSBG) (acid-adapted cells). Cells grown in TSBNG were acid-shocked by adjusting 18-h cultures to pH 4.5+/-0.05 with lactic acid. All three cell types were separately inoculated into tryptic soy broth (6.6-7.0 log(10) cfu/ml) containing 0.25% glucose (TSB) acidified to pH 3.5-5.5 with lactic acid and incubated at 4, 12, 21, 30, and 48 degrees C for up to 144 h. Overall, inactivation of S. flexneri cells at low pH was enhanced with an increase in incubation temperature. All three types of cells survived for 144 h at 4 degrees C in TSB acidified to pH 3.5, compared to < 24 h at 30 degrees C and 2 h at 48 degrees C. The population of all three cell types increased significantly (alpha = 0.05) within 24 h when cells were incubated at 12, 21, or 30 degrees C in TSB at pH 5.0, 5.5, or 7.3. Prior exposure of the S. flexneri to an acidic environment (acid-adapted or acid-shocked cells) resulted in increased resistance to extreme acid and temperature conditions. Acid-adapted cells decreased by approximately 2.5 log(10) cfu/ml when incubated at 4 degrees C for 144 h, compared to a 6-log(10) reduction in control (unadapted) cells. When cells were exposed to low pH (3.5-4.5) and high temperature (48 degrees C), significantly higher (alpha = 0.05) populations were recovered on tryptic soy agar (TSA) than on TSA supplemented with 4% NaCl (TSAS), indicating that a portion of S. flexneri cells were injured. Results show that the ability of S. flexneri to survive and grow at a given pH is influenced by previous exposure to acidic environments and by incubation temperature.     

高产木聚糖酶嗜热子囊菌固体发酵条件与酶学性质

以本实验室从土壤中分离筛选到的一株高产木聚糖酶的嗜热子囊菌QS7-2-4为生产菌种,进行固体发酵产木聚糖酶的发酵条件研究,结果表明最佳产酶条件为:玉米芯:麸皮为7:3(w/w);最佳氮源为酵母膏和胰蛋白胨的混合氮源,添加量为1.5%;吐温-80添加量为0.5%,初始pH为7.2,培养基含水量为80%,250mL三角瓶装料量为8g,发酵温度50℃,发酵时间72h,该条件下木聚糖酶产量达27952U/g干基。该酶最适反应温度为75℃,最适反应pH为4.5,在70℃以下具有良好的稳定性,在室温下储藏150d仍然保留87%的活性。     

真菌木聚糖酶产生菌的筛选及酶学性质研究

本实验通过土壤稀释涂布法,经过初筛、复筛,筛选出了两株酶活力较高真菌菌株C4 和G1,在此基础上对它们生长的最佳碳源选择、酶学性质进行了研究。结果表明：菌株C4 产酶的最佳碳源为玉米芯－麸皮,培养24h 就可以达到产酶高峰,其酶活力达6582.29U/ml。此酶反应最适温度为40℃,在50℃以下比较稳定,最适反应pH 值为5.0,并且pH 稳定性比较好;菌株G1 产酶的最佳碳源为玉米芯,培养96h 就可以达到产酶高峰,其酶活力到达6301.04U/ml。此酶反应的最适温度是40℃,在40℃以下比较稳定,最适反应pH 值为5.0,但pH 值稳定性不如菌株C4。     

毕赤酵母木聚糖酶的活力测定条件研究

为探讨毕赤酵母木聚糖酶的催化特性以及更好地发挥其催化功能,从pH、温度、底物浓度、反应时间、DNS用量、DNS显色时间等几个方面研究了木聚糖酶活性测定的最佳条件。研究结果表明,木聚糖酶酶活测定的适宜条件为:1%的木聚糖用0.05mol/L pH6.0的柠檬酸缓冲液配制;测定温度70℃,且该酶的热稳定性较好.在55～60℃下放置2h能保持68%以上的酶活;酶促反应时间10min;DNS用量3mL;DNS显色时间5min。     

黑曲霉M_1菌株木聚糖酶复合酶的固态发酵条件及其酶学性质研究

以玉米芯和麸皮 ( 7∶3 )为碳源 ,(NH4) 2 SO4( 2 % )为氮源 ,3 0℃培养 72h ,发酵曲用蒸馏水3 0℃浸提 1h ,得到 β 1,4 木聚糖酶活力高 ,β 木糖苷酶活力低的粗酶液。β 1,4 木聚糖酶和 β 木糖苷酶的最适作用温度分别为 5 0℃和 60℃ ,最适作用pH分别为 4 8和 4 0 ,β 1,4 木聚糖酶在pH5 0～ 10 6范围内稳定 ,β 木糖苷酶在 pH 3 0～ 3 0范围内稳定。β 木糖苷酶的热稳定性比 β 1,4 木聚糖酶高     

竹鼠肠道耐碱性木聚糖酶菌株的筛选及酶学性质研究

为获得良好耐酸碱性的木聚糖酶,以解决木聚糖酶在实际工业中的应用问题。本研究以木聚糖为唯一碳源,从宜宾竹鼠肠道及粪便中,采用刚果红褪色法初筛和DNS法测定木聚糖酶活进行复筛,筛选了1株具有耐碱性木聚糖酶活性的菌株JZF,16S rDNA序列分析,鉴定为蕈状芽孢杆菌（Bacillus mycoides）,对蕈状芽孢杆菌JZF的生长曲线、产酶曲线以及产耐碱性木聚糖酶的酶学性质进行初步的探索。结果显示,该菌株于37 ℃,180 r/min培养48 h酶活达到15.17 U/mL;培养28 h后菌体量达到最大值,72 h后木聚糖酶活力达到最大值为29.65 U/mL,所产木聚糖酶最适合反应温度为50 ℃,最适pH9.0;40~60 ℃,pH8.0~9.0条件下相对酶活能保持在60%以上,金属离子Mn2+与Ca2+对该菌株的木聚糖酶有明显的促进作用,本研究所得菌株所产的木聚糖酶在碱性条件下具备良好的活性,为后续耐碱性木聚糖酶的实际应用研究提供了菌种来源与数据基础。     

Production of crude xylanase from Thermoascus aurantiacus CBMAI 756 aiming the baking process

In recent years, the baking industry has focused its attention on substituting several chemical compounds with enzymes. Enzymes that hydrolyze nonstarch polysaccharides, such as xylanase, lead to the improvement of rheological properties of dough, loaf specific volume, and crumb firmness. The purpose of this study was to find a better solid-state fermentation substrate to produce high levels of xylanase and low levels of protease and amylase, which are enzymes involved in bread quality, from Thermoascus aurantiacus CBMAI 756. Wheat bran, corncob, and corn straw were used as energy sources. The enzyme extract of corncob showed high xylanase activity (130 U/mL) and low amylase and protease activity (<1 and 15 U/mL, respectively). This enzyme profile may be more profitable for the baking industry, because it results in a slower degradation of gluten. Our results confirm this finding, because the enzyme obtained by fermentation in corncob resulted in a gluten with a higher specific volume than all the other substrates that were tested. The crude xylanase presented maximum activity at a pH of 5, and the optimum temperature was 75 °C. It was stable up to 70 °C for an hour and at a pH range from 4 to 10.     

Thermostable collagenolytic activity of a novel thermophilic isolate, Bacillus sp. strain NTAP-1

We isolated an acidophilic thermophile belonging to the genus Bacillus, strain NTAP-1, which secreted a thermostable collagenolytic activity into the culture medium. The collagenolytic activity exhibited an optimum pH for Azocoll hydrolysis of pH 3.9 and was not completely inhibited by 10 mM ethylenediaminetetraacetic acid (residual activity, 63%), suggesting that Bacillus NTAP-1 produces a novel acid proteinase with highest activity for collagen. The collagenolytic activity was thermostable; more than 80% of the original activity was retained after incubation of the culture supernatant at pH 4.0 and 60 degrees C for 4 h.     

基因工程菌1020耐热木聚糖酶的纯化与性质

通过热变性处理和Ni-NTA层析分离纯化基因工程菌1020耐热木聚糖酶。对热变性条件优化,70℃处理30min效果,纯化倍数4.9;利用木聚糖酶C端6个His标记对Ni-NTA琼脂糖凝胶的结合性,一步层析得到电泳纯的木聚糖酶。酶的最适反应温度为110℃,具有高度耐热性,在70℃保温8h,酶活基本不变,100℃保温5h后,酶活残余40%;在pH6.0～10.0范围内都有较高的酶活性和稳定性。低浓度的异丙醇对酶反应有促进作用,Hg2+对酶反应有强抑制作用。该酶对燕麦木聚糖的Km为0.55mg/ml,Vmax=14.26μmol/min·mg。     

一株高产木聚糖酶菌株的筛选鉴定及酶学特性研究

以木聚糖为唯一碳源,在富集培养基的基础上,通过初筛和摇瓶复筛,筛选到一株高产木聚糖酶的菌株RX-9,对菌株RX-9进行菌落形态、生理生化实验及16S rRNA序列遗传分析,该菌株的序列与Enterbacter(肠杆菌属)的多株菌具有99%以上的同源性,将该菌株归属为肠杆菌属(Enterbacter),命名为Enterbacter RX-9。对菌株所产木聚糖酶的酶学特性进行研究,发现该菌株所产的木聚糖酶在100 ℃下保温30 min,其酶活力仍达到2310.03 U/mL;当pH在7.5~9.5之间,木聚糖酶的活性基本稳定在3200 U/mL以上,表明该酶具有较好的热稳定性和pH稳定性。     

The processing of high-molecular-weight xylanase (XynE, 110 kDa) from Aeromonas caviae ME-1 to 60-kDa xylanase (XynE60) in Escherichia coli and purification and characterization of XynE60

A xylanase gene (xynE) encoding XynE (110 kDa) was cloned from a lambda phage genomic library of Aeromonas caviae ME-1 which is a multiple-xylanase-producing bacterium. Upon nucleotide sequence analysis, we found that xynE comprises 2823 by and encodes a protein of 941 amino acid residues (104,153 Da), which was similar to endo-beta-1,4-xylanases which are categorized to glycosyl hydrolase family 10. An Escherichia coli transformant that harbored pXED30 carrying xynE produced 110-, 84-, 72-, and 66-kDa xylanases in the cell-free extract, and 72- and 66-kDa xylanases in the culture supernatant. We purified the 66-kDa xylanase to electrophoretic homogeneity from a culture supernatant by a series of column chromatographies. The calculated molecular mass of the purified xylanase determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was 60,154.50 Da, and the xylanase was designated XynE60. Analysis of the N-terminal 10 amino acid residues and the determined molecular mass of XynE60 revealed that XynE60 is a product processed at the Gly26-Gly27, and Thr565-Ala566 sites of XynE by proteolytic cleavage. XynE60 showed optimal activity at 55 degrees C and pH 8.0, and was stable below 45 degrees C and at pH 7.0-8.5. The K(m) and V(max) of XynE60 were calculated to be 8.1 mg/ml and 6897 nkat/mg, respectively.     

Purification and characterization of the Clostridium josui porphobilinogen deaminase encoded by the hemC gene from a recombinant Escherichia coli

The porphobilinogen deaminase encoded by the Clostridium josui hemC gene was purified from a recombinant Escherichia coli strain and its properties were characterized. The optimal temperature and pH of the purified enzyme were 65 degrees C and 7.0, respectively. This enzyme was quite thermostable: it retained 86% of the original activity after incubation at 70 degrees C for 1 h. The Km and Vmax values of the enzyme were 65 microM and 3.3 micromol/h/mg for porphobilinogen, respectively.