培养过程参数对霉菌Rhizopus oryzae IFO细胞催化植物油脂合成生物柴油的影响研究

采用脂肪酶催化可再生动植物油脂合成生物柴油已经成为目前研究的热点,其中利用全细胞催化剂是一个重要的研究方向。文中直接利用霉菌R.oryzaeIFO细胞催化植物油脂甲醇解反应合成生物柴油,系统研究了培养过程参数对细胞生长和该细胞催化剂催化甲醇解反应活性的影响。研究表明,细胞培养过程中所添加的油脂不同,细胞在后续反应中催化特定油脂进行生物柴油制备时所表现出的催化活性也会有所差别;由某种油脂作为碳源得到的细胞催化剂催化对应油脂与甲醇转酯化反应制备生物柴油时,表现出比催化其他油脂和甲醇反应制备生物柴油更高的催化活性。在优化的操作参数(大豆精制油20g/L,蛋白胨70g/L,NaNO31.2g/L,KH2PO41.2g/L,MgSO4·7H2O0.5g/L,培养温度35℃,摇床转速130r/min)下,培养得到的细胞催化剂能有效催化大豆油与甲醇三步转酯化反应,生物柴油(脂肪酸甲酯)最终得率可达到86%。

海洋真菌Rhizopus sp.菌丝体中化学成分的研究

目的对海洋真菌Rhizopussp.菌丝体的丙酮和甲醇萃取部分进行化学成分的研究。方法采用硅胶柱色谱、凝胶柱色谱、高效液相色谱等手段,利用理化和波谱分析方法,对海洋真菌Rhizopussp.菌丝体中的化合物进行分离鉴定。结果与结论从该真菌菌丝体中分离得到6个化合物。经光谱数据分析,鉴定其结构为5α,8α-过氧麦角甾-6,22-二烯-3β-醇(1)、1-O-9(Z)-十八碳烯酰甘油酯(2)、麦角甾-7,22-二烯-3β,5α,6β-三醇(3)、麦角甾-8(9),22-二烯-3β,5α,6β,7α-四醇(4)、戊五醇(5)、尿素(6)。这些化合物均为首次从该真菌菌丝体中分离得到。

Rhizopus sp.052凝乳酶的纯化

通过乙醇的分级沉淀,60%和71%浓度的乙醇沉淀凝乳酶分别获得14.55、18.44U/mg蛋白两种组分。通过SephadexG-100和DEAE-52-cellulose纯化蛋白,使凝乳酶活力提高2461倍,迭到2813.74U/mg蛋白。SDS-PAGE测定分子量是36.6kDa。

响应面法优化Rhizopus microsporus var.chinensis产生淀粉酶的条件

对Rhizopus microsporus var.chinensis固态发酵产生淀粉酶的条件进行了优化。首先采用单因子实验确定最适固态发酵基质为小麦麸皮,最适碳源和氮源分别为可溶性淀粉和硫酸铵,通过控制培养基初始pH为3.0、初始湿度为70%时,使其生淀粉酶产量提高3.5倍。在此基础上,利用响应面中心组合设计对显著因素进行优化,得出每500mL三角摇瓶中含小麦麸皮13.7g、可溶性淀粉0.063g、硫酸铵0.052g时,生淀粉酶产量达到48.50U/mL,比初始产量提高了8倍。

匍枝根霉(Rhizopus stolonifer)As3.38△6-脂肪酸脱氢酶基因的克隆与酵母表达

通过气相色谱法(GC)快速分析8种真菌的脂肪酸成分,发现匍枝根霉(Rhizopus stolonifer)具有较高的γ-亚麻酸含量,利用RT-PCR和RACE方法获得了全长为1475bp的匍枝根霉△6-脂肪酸脱氢酶基因的cDNA序列,其中开放阅读框为1380bp,编码459个氨基酸。生物信息学分析所克隆的基因具有△6-脂肪酸脱氢酶的典型结构:N端具有细胞色素b5结构、具有3个保守的组氨酸区序列和跨膜结构;把该基因的开放阅读框序列连接到表达载体pYES2.0上,构建重组表达载体pYRnD6D,并将其转入缺陷型酿酒酵母INVScl中进行表达。GC分析表明,该序列在酵母中获得了表达,表达产物表现出△6-脂肪酸脱氢酶的酶学活性,能将底物亚油酸转化为γ-亚麻酸。新生成的γ-亚麻酸占酵母细胞总脂肪酸的12.25%。

利用Native制备电泳分离来源于Rhizopus microsporus var. chinensis的葡萄糖淀粉酶同工酶

将Native制备电泳应用于Rhizopus microsporus var. chinensis中2个葡萄糖淀粉酶同工酶的分离,考察了电泳缓冲系统、凝胶浓度和凝胶长度对分离效果的影响。结果表明:先使用Ornstein-Davis系统分离同工酶,再使用0.02mol/L,pH6.2的NaAc.CH3COOH缓冲液洗脱,在长度为6cm的7%的Native电泳胶上,2个性质相近仅电泳迁移率略有差别的同工酶能有效分离。分离回收的蛋白质可保持其活力,便于后续研究。

Antagonistical Mode of Pichia membranefaciens to Rhizopus stolonifer in Wounds of Peach Fruit

膜醭毕赤酵母 (PichiamembranefaciensHansen)是本实验室从果实上分离获得的一种能有效防治桃果实采后软腐病的拮抗菌。本文将P .membranefaciens与葡枝根霉 (Rhizopusstolonifer)在桃果实伤口部位共培养 2 4h后 ,用扫描电子显微镜观测了它们的拮抗作用。结果表明 ,在有病原菌的地方聚集了大量的酵母拮抗菌 ,而且拮抗菌紧密地吸附在病原菌的菌丝体上。结合以前的研究结果可以推断 ,P .membranefaciens主要通过与病原菌进行营养和空间的竞争 ,紧密地吸附在病原菌菌丝体上分泌能降解病原菌细胞壁的水解酶 (如几丁质酶和 β_1,3_葡聚糖酶 ) ,并可能诱导寄主产生抗性 ,从而抑制桃软腐病的发生。

Polyphenol Oxidase, Peroxidase and PhenylalanineAmmonium Lyase Induced in Postharvest Peach Fruitsby Inoculation with Pichia membranefaciensor Rhizopus stolonifer

Rhizopus rot of peach fruits could be significantly suppressed by Pichia membranefaciens. Polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonium-lyase (PAL) activities induced by inoculation with P. membrane faciens or R. stolonifer were studied in postharvest peach fruits. The activities of PPO and PAL in peaches increased significantly after being inoculated with P. membrane faciens + R. stolonifer by 24 h, the activities maintained at a high level throughout the experiment. Under the condition of infected with R. stolonifer alone, activity of PPO and PAL could also increased, but the levels were lower than those treated with P. membrane faciens+ R. stolonifer. However, fruits inoculaed with P. membrane-faciens + R. stolonifer or R. stolonifer alone did not stimulated POD activity. The results suggest that the activation of these defense enzymes is involved in the action of P. membrane faciens against R. stolonifer.

Biosorption of cadmium(Ⅱ) from aqueous solutions by industrial fungus Rhizopus cohnii

An important filamentous industrial fungus,Rhizopus cohnii(R.cohnii),was used as an efficient biosorbent for removing cadmium from wastewater.The sorption conditions,such as pH,the dose of biomass and the initial concentration of cadmium were examined.Two kinds of adsorption models were applied to simulate the biosorption data.The uptake of cadmium was higher in weak acid condition than in strong acid condition.Nearly no sorption of cadmium occurred when the pH value was lower than 2.0. Biosorption isothermal data could be well simulated by both Langmuir and Freundlich models.Langmuir simulation of the biosorption showed that the maximum uptake of cadmium was 40.5 mg/g(0.36 mmol/g)in the optimal conditions,which was higher than many other adsorbents,including biosorbents and activated carbon.In addition,the reusability results showed that after five times of sorption and desorption process,the sorption capacity of R.cohnii could still maintain nearly 80%,confirming its practical application in cadmium treatment.Fourier transform infrared spectrum revealed that carboxyl,amino and hydroxyl groups on biosorbent R.cohnii surface were responsible for the biosorption of cadmium.

Production of Ammonium Lactate by Fed-batch Fermentation of Rhizopus oryzaefrom Corncob Hydrolysate

L-(+)-Lactic acid production from corncob hydrolysate as a cheap carbohydrate source by fed-batch fermentation of Rhizopus oryzaeHZS6 was studied. After 96 h of fermentation in a 5 L fermentor, the final concentration of ammonium L-(+)-lactate, average productivity(based on initial xylose concentration) and maximum dry cell weight were 132.4 g/L, 1.38 g/(L·h), and 8.9 g/L respectively. The optical purity of L-(+)-lactate was 98.8%.

Removal of lead in wastewater by immobilized inactivated cells of Rhizopus oligosporus

A novel technology for lead removal with nonliving Rhizopus oligosporus immobilized in calcium alginate was studied. The results show that the main influencing factors include pH value and interfering cations. pH value has different effects on biosorption of various heavy metals and lead adsorption can be proceeded by controlling pH value in a range of 2–5; interfering cations especially Cu(II) can make the adsorption amount of Pb(II) decrease by immobilized Rhizopus oligosporus. Desorption efficiency of different eluants and kinetics were investigated. Citrate acid with concentration of 0.3 mol ? L?1 is the best for the elution of Pb(II) and desorption rate is over 98%when the reaction equilibrium reaches 3 h. Immobilized biomass keeps high lead biosorption capacity after five cycles of regeneration.

Optimization of L-lactic Acid Production of Rhizopus Oryzae Mutant RLC41-6 by Ion Beam Implantation at Low-Energy

In order to obtain an industrial strain with a higher L（＋）-lactic acid yield, the strain Rhizopus oryzae RF3608 was mutated by means of nitrogen ion beam implantation and the mutant strain RLC41-6 was isolated. Under optimal conditions the yield of L（＋）-lactic acid produced in a shake-flask reached 133 g/L - 137 g/L after 36 h cultivation, indicating that the conversion rate based on glucose was as high as 88% - 91% and the productivity was 3.75 g/L.h. It was almost a 115% increase in lactic acid production compared with the original strain RF3608.

Optimization of L(+)-Lactic Acid Production from Xylose with Rhizopus Oryzae Mutant RLC41-6 Breeding by Low-Energy Ion Implantation

In order to obtain an industrial strain with a higher L（＋）-lactic acid yield, the strain Rhizopus oryzae PW352 was mutated by means of nitrogen ion beam implantation and the mutant strain Rhizopus oryzae RLC41-6 was obtained. An experimental finding was made in surprise that Rhizopus oryzae mutant RLC41-6 is not only an L（＋）-lactic acid producer from corn starch but also an efficient producer of L（＋）-lactic acid from xylose. Under optimal conditions, the production of L（＋）-lactic acid from 100 g/L xylose reached 77.39 g/L after 144 h fed-batch fermentation, A high mutation rate and a wide mutation spectrum of low-energy ion implantation were observed in the experiment.

Improvement of L(+)-Lactic Acid Production of Rhizopus Oryzae by Low-Energy Ions and Analysis of Its Mechanism

The wild type strain Rhizopus oryzae PW352 was mutated by means of nitrogen ion implantation （15 keV, 7.8×10^14 ～ 2.08 ×10^15 ions/cm^2） to find an industrial strain with a higher L（＋）-lactic acid yield, and two mutants RE3303 and RF9052 were isolated. In order to discuss the mechanism primarily, Lactate Dehydrogenase of Rhizopus oryzae was studied. While the two mutants produced L（＋）-lactic acid by 75% more than the wild strain did, their specific activity of Lactate Dehydrogenase was found to be higher than that in the wild strain. The optimum temperature of Lactate Dehydrogenase in Rhizopus oryzae RF9052 was higher. Compared to the wild strain, the Michaelis constant （Km） value of Lactate Dehydrogenase in the mutants was Changed. All these changes show that L（＋）-lactic acid production has a correlation with the specific activity of Lactate Dehydrogenase. The low-energy ions, implanted into the strain, may improve the specific activity of Lactate Dehydrogenase by influencing its gene structure and protein structure.

Free Amino Acid Profiles of Fermented Soybean Products Prepared by Bacillus subtilis and Rhizopus oligosporus

Fermented soybean is one of the traditional foods widely consumed in many Asian countries. Most products（i.e., Natto, Jang, Kinema and Thua nao） are produced mainly by the bacterial activity. This study was performed as a part of the program to improve the soybean fermentation, and a use of the co-culture between the bacterium Bacillus subtilis and the fungus Rhizopus oligosporus was selected. For fermentation, the raw soybeans were washed, sterilized by autoclaving, and inoculated with a mixed microbial starter culture of B. subtilis（102 CFU · g-1） and R. oligosporus（102 conidia · g-1）. The Free Amino Acid（FAA） contents were then investigated by using High Performance Liquid Chromatography（HPLC）. The results showed that the amounts of FAAs and essential amino acids in the fermented soybean samples were found at significantly higher concentration when compared to those of the cooked, non-fermented soybean samples（p〈0.05）. These data indicated that an involvement of microbial fermentation helped to accelerate an increase of FAA. The food compositional data were useful as a reference（and/or guidance） for the nutritional value, when compared to other fermented soybeans and related products.

Optimization of L(+)-Lactic Acid Fermentation Without Neutralisation of Rhizopus Oryzae Mutant RK02 by Low-Energy Ion Implantation

In order to get an industrial strain which can yield a high concentration of lactic acid for ISPR （in situ product removal）, the original strain Rhizopus oryzae RE3303 was mutated by low-energy ion beam implantation. A mutant RK02 was screened, and the factors such as the substrate concentration, nitrogen source concentration, inoculum size, seed age, aeration and temperature that affect the production of lactic acid were studied in detail. Under optimal con- ditions, the maximum concentration of L（＋）-lactic acid reached 34.85 g/L after 30 h shake-flask cultivation without adding any neutralisation （5% Glucose added）, which was a 146% increase in lactic acid production after ion implantation compared with the original strain. It was also shown that RK02 can be used in ISPR to reduce the number of times of separation.

Rhizopus microsporus lung infection in an immunocompetent patient successfully treated with amphotericin B:A case report

BACKGROUND Rhizopus microsporus(R.microsporus)lung infection is an invasive fungal disease with high mortality that is increasingly common in immunocompromised patients.However,it is very rare in immunocompetent patients.Here,we present the case of a 19-year-old girl who developed R.microsporus lung infection without any known immunodeficiency.CASE SUMMARY The patient presented to our hospital because of hemoptysis and irritative cough without expectoration.She was first treated for community-acquired pneumonia until the detection of R.microsporus in bronchoalveolar lavage fluid by metagenomics next-generation sequencing(mNGS).After a combination therapy of intravenous inhalation and local airway perfusion of amphotericin B,she eventually recovered,with significant absorption of lung infections.CONCLUSION Early diagnosis and treatment are very important for pulmonary mucormycosis.Compared to fungal culture,mNGS is a relatively precise and convenient method to obtain pathogenic results.A combination therapy of intravenous inhalation and local airway perfusion of amphotericin B may be a promising strategy for the treatment of pulmonary mucormycosis in the future.

Chronic pulmonary mucormycosis caused by rhizopus microsporus mimics lung carcinoma in an immunocompetent adult:A case report

BACKGROUND Pulmonary mucormycosis is a rare but life-threatening invasive fungal infection that mostly affects immunocompromised patients.This disease usually develops acutely and progresses rapidly,often leading to a poor clinical prognosis.Chronic pulmonary mucormycosis is highly unusual in immunocompetent patients.CASE SUMMARY A 43-year-old man,who was a house improvement worker with a long history of occupational dust exposure,presented with an irritating cough that had lasted for two months.The patient was previously in good health,without dysglycemia or any known immunodeficiencies.Chest computed tomography revealed a mass in the left lower lobe,measuring approximately 6 cm in diameter,which was suspected to be primary lung carcinoma complicated with obstructive pneumonia.Thoracoscopic-assisted left lower lobectomy was performed,and metagenomic next-generation sequencing detection,along with special pathological staining of surgical specimens,suggested Rhizopus microsporus infection.Postoperatively,the patient’s respiratory symptoms were relieved,and no signs of recurrence were found during the six-month follow-up.CONCLUSION This article reports a rare case of chronic pulmonary mucormycosis caused by Rhizopus microsporus in a middle-aged male without dysglycemia or immunodeficiency.The patient’s surgical outcome was excellent,reaffirming that surgery remains the cornerstone of pulmonary mucormycosis treatment.

脂肪酶Rhizopus chinensis催化水解大豆粉末磷脂制备L-α-甘油磷脂酰乙醇胺

为考察脂肪酶Rhizopus chinensis催化水解大豆粉末磷脂制备L-α-甘油磷脂酰乙醇胺(L-α-GPE)的可行性,采用脂肪酶Rhizopus chinensis催化水解大豆粉末磷脂制备L-α-GPE,通过单因素实验考察了pH、大豆粉末磷脂质量浓度、反应温度、CaCl_(2)质量浓度、酶浓度对L-α-GPE得率的影响,并对纯化产品进行了红外光谱和超高效液相色谱电喷雾四极杆飞行时间质谱(UPLC-Q-TOF-MS)结构验证。结果表明:在pH 7,大豆粉末磷脂质量浓度5 mg/mL,大豆粉末磷脂磷酸盐缓冲溶液中添加终质量浓度为2 mg/mL的CaCl_(2)、终浓度为30 U/mL的脂肪酶Rhizopus chinensis,于45℃下反应15 h后,L-α-GPE得率为93.8%。产品经分离纯化后得到纯度为99.2%的浅黄色黏稠液体,经结构验证确为L-α-GPE。

Rhinocerebral mucormycosis caused by Rhizopus oryzae in a patient with acute myeloid leukemia: A case report

BACKGROUND Rhinocerebral mucormycosis(RCM)is a rare fatal fungal infection which is on the increase among immunocompromised hosts such as patients who have had hematological cancers,or have received immunosuppressive drugs,corticosteroids,or other T cell suppressing agents.CASE SUMMARY We report a case of RCM caused by Rhizopus oryzae,one of the most common opportunistic pathogens,in a patient suffering from a fourth relapse of acute myeloid leukemia.The patient developed RCM after he had received long-term antibiotic agents and corticosteroids.The pathogen was isolated three times from nasal secretions collected from the deep parts of the nasal cavity and was identified by morphology and internal transcribed spacer sequencing.Blood infection was excluded by droplet digital polymerase chain reaction and blood culture.The patient was empirically treated with caspofungin and voriconazole for several days while the lesions continued to progress.The patient was given amphotericin B in combination with caspofungin after RCM was suspected,and the lesions improved over the course of treatment,which lasted several days.However,the patient eventually died of the primary disease.CONCLUSION This case indicates that immunosuppressive drugs,including corticosteroids and antimetabolites in hematological tumor,do increase the risk of infections of this type.Early diagnosis,prompt and frequent surgical debridement,and treatment with amphotericin B without delay are all essential in combatting RCM.