Properties of a lipase produced by Beauveria bassiana: purification and biochemical studies

Lipases are some of the crucial enzymes during the fungal penetrating process of the insect integument. Due to the importance and lack of information on the microbial lipases of Beauveria bassiana, investigations were carried out to purify and biochemically characterize these enzymes. The results obtained on growth medium demonstrated the highest activity of lipase 6 days after inoculation while the pH of the medium was 7.1. After three purification steps, the purified enzyme was 9.91-fold with specific activity of 20816 U/mg protein, recovery of 25% and molecular weight of 25 kDa. The purified lipase had the optimal pH and temperature at 7 and 35°C and was stable for 36–72 h under those conditions. Ca^{2 +} significantly increased the enzyme activity and NaCl decreased it at all the tested concentrations. In addition, Mn^{2 +} had no effect on enzyme activity but Mg^{2 +} and Zn^{2 +} increased it only at the highest concentration used. Three out of the four inhibitors used, significantly decreased the purified lipase activity so that most inhibition and changes in the enzyme kinetic parameters were obtained by using different concentrations of EDTA. Knowledge of enzymology provides important information for the development of fungi as microbial pest control agents opening new avenues for study of the role of enzymes in virulence.     

Biodiesel synthesis in a solvent-free system by recombinant Rhizopus oryzae: comparative study between a stirred tank and a packed-bed batch reactor

A simultaneous synthesis of biodiesel, as fatty acid methyl esters, and monoacylglycerols catalysed by the recombinant Rhizopus oryzae lipase immobilized by adsorption on Relizyme OD/403M is presented. The use of this 1(3)-positional specific lipase prevents the formation of glycerol as a by-product, thus avoiding its drawbacks. The synthesis was carried out in a solvent-free system and it has been studied in two different reactor systems: stirred tank and packed-bed reactor. Stirred tank reactor presented a high-initial reaction rate and achieved a 33.6% yield, which corresponds to a value of 50.4% of the maximum yield that can be achieved with a 1(3)-positional specific lipase. In packed-bed reactor there was a smaller initial reaction rate, but it was achieved a 49.1% yield, which corresponds to a 73.6% of the maximum yield. When a second batch is performed, the yield decreased only 4% when packed-bed reactor is employed whereas a drastic decrease is observed in a stirred tank operation. Therefore, packed-bed reactor showed a best performance and minor damage to the biocatalyst.     

共表达伴侣蛋白对重组毕赤酵母产米根霉(Rhizopus oryzae)脂肪酶发酵过程的影响

[目的]通过共表达伴侣蛋白Erolp和PDI获得米根霉(Rhizopus oryzae)脂肪酶在毕赤酵母中的高效表达.[方法]利用毕赤酵母基因重组菌高密度发酵的方法,在7L发酵罐水平上分析共表达伴侣蛋白菌株(BH128)与非共表达伴侣蛋白菌株(H238)对脂肪酶表达的差异.[结果]在相同条件下,BH128发酵产酶能力高于H238,最高酶活可达到2 338.7U/mL,最大比生长速率达到0.02 h-1,最大产物比形成速率达到944.5 U/(gDCW·h),最大底物比消耗速率也能达到0.15 gmethanol/(gDCW·h),分别是H238的1.7、0.5、4.1和1.3倍,且发酵周期缩短了20h.[结论]毕赤酵母基因重组菌BH128通过共表达伴侣蛋白Ero1p和PDI,提高了米根霉脂肪酶的产量,而且缩短了发酵周期,为工业化生产奠定了基础.     

Immobilization and stability of a Rhizopus oryzae lipase expressed in Pichia pastoris: comparison between native and recombinant variants

The stability of a soluble extract containing a recombinant lipase from Rhizopus oryzae (Cursive) lipase (rROL) produced by Pichia pastoris (Cursive), as well as that for the commercial extract containing the lipase produced by the native organism (nROL), was investigated. The results showed higher residual activity values of the commercial protein compared with the recombinant one. Moreover, two different kinds of support, the polypropylene powder EP100 and Eupergit®C, were tested to immobilize the enzymes. The residual activity of the immobilizated derivatives was also tested to determine whether their stability was enhanced. The results showed a slight improvement in rROL using both supports but a decrease in nROL using Eupergit®C. The study of the residual activity of soluble and immobilized enzymes was performed by means of a central composite rotatable experiment design. In addition, EP100 adsorption isotherms were determined. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

米根霉发酵生产L-乳酸

报道了Ｌ－乳酸菌株的分离与筛选,探讨了不同碳源、氮源、通气量、温度等发酵条件对产Ｌ－乳酸的影响,从７８株米根霉中筛选出１３株产Ｌ－乳酸较高的菌株,其中米根霉（Ｒｈｉｚｏｐｕｓ ｏｒｙｚａｅ）Ｒｓ９２８产Ｌ－乳酸最高,产酸最稳定。试验结果表明,该菌株最适发酵培养组成（％）：淀粉水解糖１６,ＭｇＳＯ４ ０．０８,ＫＨ２ＰＯ４ ０．０５,ＺｎＳＯ４ ０．０１,ＣａＣＯ３ ７,ｐＨ自然。在６０ｔ发酵罐中,     

Expression and characterization of recombinant Rhizopus oryzae lipase for enzymatic biodiesel production

The Rhizopus oryzae lipase containing prosequence was expressed in Pichia pastoris. Recombinant lipase subunit showed a molecular mass of 32 kDa. The maximum activity of recombinant lipase obtained from Mut(s) recombinant was 90 IU/ml. The enzyme was stable in broad ranges of temperatures and pH, with the optimal temperature at 35 °C and pH 7.0. The crude recombinant R. oryzae lipase can be directly used for the transesterification of plant oils at high-water content of 60-100% (w/w) based on oil weight. The addition of 80% water to the transesterification systems resulted in the yield of methyl ester of 95%, 94% and 92% after 72 h using soybean oil, Jatropha curcas seed raw oil and Pistacia chinensis seed raw oil as raw material, respectively. These results indicate that the recombinant lipase is an effective biocatalyst for enzymatic biodiesel production.     

少根根霉(Rhizopus arrhizus)脂肪酶基因在毕赤酵母中的分泌表达

利用PCR技术从少根根霉中扩增出脂肪酶基因(包括前导序列和成熟肽),并将其连接到酵母分泌表达载体pPIC9K中,转化毕赤酵母GS115。利用抗生素G418从重组阳性克隆中筛选得到高拷贝的转化子。在5 L的发酵罐中,当碳源耗尽后开始流加甲醇诱导脂肪酶的表达,经过96 h培养后发酵液上清液中重组脂肪酶(rRAL)的表达量约为90 mg/L。rRAL经过超滤,SP-Sepharose离子交换层析和Butyl-Sepharose疏水层析纯化。纯化后的蛋白在SDS-PAGE上为单一条带,表观分子量为32 kDa,比酶活为1 543 U/mg。N-端序列分析表明rRAL是经过加工后的产物。同时没有发现全长的Rhizopus arrhizus脂肪酶(RAL)被分泌表达。     

米根霉利用纯糖和不同预处理玉米秸秆酶解糖生产L-乳酸

通过单因素实验设计,优化米根霉摇瓶发酵产L-乳酸。在此基础上,以蒸气爆破和碱处理玉米秸秆酶解液为混合C源,与纯糖对比,研究不同预处理玉米秸秆混合C源对米根霉发酵产L-乳酸的影响。结果显示:在初始葡萄糖质量浓度100g/L、(NH4)2SO4质量浓度2g/L、接种量6%(体积分数)、转速170r/min、发酵12h后添加30g/LCaCO3的条件下,米根霉发酵产L-乳酸质量浓度为69.15g/L。米根霉发酵不同预处理玉米秸秆酶解混合C源,木糖的存在影响了米根霉的C代谢网络,降低L乳酸的产量。     

One-pot conjugated linoleic acid production from castor oil by Rhizopus oryzae lipase and resting cells of Lactobacillus plantarum

Conjugated linoleic acid (CLA) has attracted as novel type of fatty acids having unusual health-promoting properties such as anticarcinogenic and antiobesitic effects. The present work employed castor oil as substrate for one-pot production of CLA using washed cells of Lactobacillus plantarum (L. plantarum) and lipases as catalysts. Among the screened lipases, the lipase Rhizopus oryzae (ROL) greatly assisted resting cells to produce CLA. Mass spectral analysis of the product showed that two major isomers of CLA were produced in the reaction mixture i.e. cis-9, trans-11 56.55% and trans-10, cis-12 43.45%. Optimum factors for CLA synthesis were found as substrate concentration (8 mg/mL), pH (6.5), washed cell concentration (12% w/v), and incubation time of 20 h. Hence, the combination of ROL with L. plantarum offers one pot production of CLA selectively using castor oil as a cost-effective substrate.     

Enhanced esterification activity through interfacial activation and cross‐linked immobilization mechanism of Rhizopus oryzae lipase in a nonaqueous medium

Interfacial activation via surfactant (Tween 80, Triton X‐100) treatment was conducted to improve the esterification activity of Rhizopus oryzae lipase that had undergone immobilization through cross‐linked enzyme aggregates (CLEA®) technique. Surfactant pretreated immobilized enzymes exhibited better esterification activity compared to free and non‐pretreated immobilized enzyme (Control CLEAs) since higher conversion rates were obtained within shorter times. The superiority of surfactant pretreated CLEAs, especially Tween 80 pretreated CLEAs (T 80 PT CLEAs), were clearly pronounced when longer alcohols were used as substrates. Conversion values exceeded 90% for octyl octanoate, oleyl octanoate and oleyl oleate synthesis with T 80 PT CLEAs whereas Control CLEAs and free enzyme showed no activity. Maximum conversions were achieved in the case equal molars of the substrates or in the case excess of the alcohol to acid in cyclohexane. In solvent free medium containing equal molars of substrates the conversion rates were 85% and 87% with T 80 PT CLEAs respectively for octyl octanoate and oleyl oleate within 2 hours. T 80 PT CLEAs showed 59% of its original activity after 7 consecutive usage for oleyl oleate synthesis. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:899–904, 2016     

Comparison of Sucrose-Hydrolyzing Enzymes Produced by Rhizopus oryzae and Amylomyces rouxii

Rhizopus oryzae produces lactic acid from glucose but not efficiently from sucrose, while Amylomyces rouxii, a species closely related to R. oryzae, ferments these sugars equally. The properties of two sucrose-hydrolyzing enzymes purified from culture filtrates of R. oryzae NBRC 4785 and A. rouxii CBS 438.76 were compared to assess lactic acid fermentation by the two fungi. The substrate specificity of the enzymes showed that the enzymes from strains NBRC 4785 and CBS 438.76 are to be classified as glucoamylase and invertase respectively. The entity of the enzyme from strain NBRC 4785 might be a glucoamylase, because eight residues of the N-terminal amino acid sequence coincided with those of the deduced protein from the amyB gene of R. oryzae. The enzyme from NBRC 4785 was more unstable than that from strain CBS 438.76 under conditions of lower pH and higher temperature. These observations mean that the culture conditions of R. oryzae for lactic acid production from sucrose should be strictly controlled to prevent inactivation of the glucoamylase hydrolyzing sucrose.     

根霉TC1653产纤维素酶提纯及酶学性质研究

对根霉所产纤维素酶酶系进行了分析并研究了部分酶学性质。实验选择超滤和凝胶柱分离相结合的方式提纯纤维素酶,结果显示根霉TC1653纤维素酶系是一个完全酶系,具有一个较为明显的内切葡聚糖酶组分。β-葡萄糖苷酶组分的最适反应温度为70℃,温度高于70℃时,活性迅速下降,但在这种高温下具有最高反应活性的酶很少见,很可能又是一种新的β-葡萄糖苷酶。     

N-terminal peptide of Rhizopus oryzae lipase is important for its catalytic properties

In a culture medium, the Rhizopus oryzae strain produces only one form of lipase, ROL32. When the concentrated culture medium was stored at 0 degrees C during several months or kept at 6 degrees C during a few days, we noticed the appearance of a second shorter form of ROL32 lacking its N-terminal 28 amino acid (ROL29). ROL29 was purified to homogeneity and its 21 N-terminal amino acid residues were found to be identical to the 29-49 sequence of ROL32. The cleavage of the N-terminal peptide reduced the specific activity of ROL29 by 50% using either triolein or tributyrin as substrates. In order to explain this decrease of the specific activity of ROL29, we measured its critical surface pressure of penetration into phosphatidyl choline from egg yolk films which was found to be 10 mN/m, in contrast to a value of 23 mN/m found in ROL32. A kinetic study on the surface pressure dependency, stereoselectivity and regioselectivity of ROL29 was performed using the three dicaprin isomers spread as monomolecular films at the air-water interface. Our results showed that in contrast to ROL32, ROL29 presented a preference for the distal ester groups of one diglyceride isomer (1,3-sn-dicaprin). Furthermore, ROL32 was markedly more stereoselective than ROL29 for the sn-3 position of the 2,3-sn-enantiomer of dicaprin. A structural explanation of the enhanced penetration capacity as well as the catalytic activity of ROL32 was proposed by molecular modeling. We concluded that the N-terminal peptide of ROL32 can play an important role in the specific activity, the regioselectivity, the stereoselectivity and the binding of the enzyme to its substrate.     

无载体固定化米根霉重复间歇发酵生产L-乳酸

通过研究影响米根霉菌丝体形态的培养基因素,初步构建了无载体固定化米根霉重复间歇发酵生产L-乳酸的工艺条件.研究结果表明,首批次发酵培养基采用120 g/L葡萄糖,3 g/L硝酸铵,K 和Na 浓度比为1:1,发酵72 h后,米根霉菌体形态为均匀的茵丝体小球,直径为1.0 mm～2.0 mm,此时L-乳酸产量可达100.8 g/L,葡萄糖转化率为84%.在此基础上,利用米根霉菌丝体小球重复间歇发酵16批次,每批次发酵24h,此时葡萄糖转化率均高于75%,L-乳酸产量保持在60.0 g/L以上,米根霉菌丝体小球形态保持稳定.     

壳聚糖固定化德氏根霉脂肪酶的研究

研究了壳聚糖吸附和戊二醛交联对脂肪酶固定化条件,在室温条件下将0．4g酶粉溶于pH6．0缓冲液中,加入10g壳聚糖,摇匀,再加入浓度为0．6％戊二醛交联6h,得到固定化酶,酶活力回收率约为54．2％。固定化酶的半失活温度比游离酶的高,半失活温度由游离酶的47℃提高到100℃,最适反应温度由40℃上升至80℃,最适pH由6下降到5．5,固定化酶K’m值由游离酶的Km 50mg／mL增加到56mg／mL。该固定化脂肪酶用于酯的合成;在80℃条件下经过10批次连续水解植物油反应,固定化酶的活力仍保持在82．6％以上。     

离子注入选育高产L-乳酸的米根霉突变株及其发酵特性研究

通过氮离子注入获得米根霉突变株RQ4012,其利用木糖的能力比出发菌株提高了1.6倍;通过多次传代,证明其具有良好的遗传稳定性。试验测定菌株RQ4012发酵木糖生产L-乳酸的最佳发酵条件:木糖10%,生理盐水浸泡孢子9 h,(NH4)2SO43 g/L,接种量4%,CaCO3添加量6%,装液量20%,温度37℃,转速200 r/min,在此条件下,乳酸产量达到79.51 g/L。对混合糖的发酵进行了探索,结果表明该菌能高效利用混合糖生产L-乳酸,在利用植物纤维素水解液生产L-乳酸上有良好的应用前景。     

Unlocking the mystery behind the activation phenomenon of T1 lipase: a molecular dynamics simulations approach

The activation of lipases has been postulated to proceed by interfacial activation, temperature switch activation, or aqueous activation. Recently, based on molecular dynamics (MD) simulation experiments, the T1 lipase activation mechanism was proposed to involve aqueous activation in addition to a double-flap mechanism. Because the open conformation structure is still unavailable, it is difficult to validate the proposed theory unambiguously to understand the behavior of the enzyme. In this study, we try to validate the previous reports and uncover the mystery behind the activation process using structural analysis and MD simulations. To investigate the effects of temperature and environmental conditions on the activation process, MD simulations in different solvent environments (water and water-octane interface) and temperatures (20, 50, 70, 80, and 100°C) were performed. Based on the structural analysis of the lipases in the same family of T1 lipase (I.5 lipase family), we proposed that the lid domain comprises α6 and α7 helices connected by a loop, thus forming a helix-loop-helix motif involved in interfacial activation. Throughout the MD simulations experiments, lid displacements were only observed in the water-octane interface, not in the aqueous environment with respect to the temperature effect, suggesting that the activation process is governed by interfacial activation coupled with temperature switch activation. Examining the activation process in detail revealed that the large structural rearrangement of the lid domain was caused by the interaction between the hydrophobic residues of the lid with octane, a nonpolar solvent, and this conformation was found to be thermodynamically favorable.     

里氏木霉与米根霉混合固态发酵产纤维素酶

以里氏木霉及米根霉单菌固态发酵为对象,考察不同混合发酵形式对里氏木霉与米根霉混合固态发酵产纤维素酶的影响。结果表明:同时接种里氏木霉与米根霉,试验考察的两菌种接种量比1∶1(以孢子个数计)及5∶1条件下,两菌未产生明显协同产酶作用。米根霉延时(24 h)接种且菌种量比5∶1以及米根霉延时(48 h)接种且菌种量比1∶1,2种发酵形式产酶情况类似,滤纸酶活(FPA)及羧甲基纤维素酶(CMCase)酶活相对米根霉单菌发酵有所提高,而β-葡萄糖苷酶(β-GA)酶活相对里氏木霉单菌固态发酵结束时分别增加4.66及4.40倍,可以发现两菌产生一定协同作用。在米根霉延时(48 h)接种且菌种量比5∶1的发酵形式下,FPA及CMCase在发酵第7天酶活分别达到44.04 IU/g、627.14 U/g(以1 g干曲计),分别是里氏木霉固态单菌发酵产酶达到稳定期时酶活的1.36和1.63倍,两菌产生了有效的协同作用。     

Preparation and testing of Sardinella protein hydrolysates as nitrogen source for extracellular lipase production by Rhizopus oryzae

Various fish protein hydrolysates (FPH) from sardinella (Sardinella aurita) were used as nitrogen sources for the production of extracellular lipase by the filamentous fungus Rhizopus oryzae. The best results were obtained with defatted meat–fish protein hydrolysates (DMFPH), indicating the presence in the lipid fraction of some constituents which may repress lipase synthesis. Furthermore, it was found that the extensive hydrolysis of fish proteins resulted in a higher lipase production. The use of 40 g DMFPH l^–1 for the growth of Rhizopus oryzae in medium R1 resulted in a lipase production of 394 U ml^–1, higher than the yield obtained with standard soy peptone as nitrogen source (373 U ml^–1). The most appropriate medium for the growth and the production of lipase is composed only of 24 g DMFPH l^–1 and 10 g glucose l^–1, indicating that the strain can obtain its nitrogen and salts requirements directly from fish substrate.     

利用木霉与根霉两步发酵秸秆制备L-乳酸研究

以秸秆为原料进行生物转化大量制备有机酸意义重大.在秸秆汽爆法预处理的基础上,以绿色木霉为菌种转化制备秸秆糖,对降解单糖接种米根霉进行二次发酵制备L-乳酸.试验结果表明,第一步绿色木霉固态培养制备纤维素酶时,控温30℃、通气0.12L/(L.min)、发酵40h后制备干曲,后按10g干曲/L汽爆液的配比进行55℃酶解36h,五、六碳糖累积浓度达到86g/L.第二步米根霉发酵时,控制温度32℃、通气0.4L/(L·min)、转速450r/min,发酵48h,最终产L-乳酸累积浓度为81.6g/L.秸秆制备L-乳酸的两步发酵法发酵工艺具有推广价值.