Isolating and Screening of Effective Decomposing Strain of Silkworm Chrysalis Protein and Study on Parts of Its Enzymatic Properties

[Objective] The research aimed to obtain an effectively-decomposing strain of silkworm chrysalis protein and discuss its enzymatic properties.[Method] The effectively-decomposing bacteria of protein was isolated from the decayed silkworm chrysalis by using dilution plate and its enzymatic properties were tested after primary screening and second screening.The enzyme activity was determined and the intermediate and small molecule protein content in silkworm chrysalis was measured after solid-state fermentati...

Studies on the Isolation and Screening of α-amylase Producing Strain and Their Enzymatic Properties

[Objective] The aim was to obtain α-amylase producing strains with some excellent properties like high temperature resistance,strong acid resistance,strong alkali resistance,etc..[Method] α-amylase producing strains were isolated and screened; furthermore their enzymatic properties were studied.[Result] 10 strains with an obvious starch hydrolysis cycle were screened out from starch screening plate coated by diluted sample,from which 3 strains with higher α-amylase activity were screened out,that was X6,X8 and X10.As for X6,X8 and X10,their optimum pH values all belonged to neutral,and their optimum temperatures were all 60 ℃.Meanwhile,Ca^2＋ could increase their enzyme thermal stability.When the concentration of Ca^2＋ was 0.02-0.04 mol/L,the enzyme thermal stability of X6 and X8 reached the highest; When the concentration of Ca^2＋ was 0.03-0.04 mol/L,that of X10 reached the highest; When the concentration of Ca^2＋ was increased continuously,those of the 3 strains all decreased.[Conclusion] The research provides theoretical basis for satisfying the demands of different industries for α-amylase with different characteristics.

Characterization of a Novel Esterase Belonging to Family V from Marinobacter flavimaris

Lipolytic enzymes have attracted enormous attentions because of their ability in ester hydrolysis,ester synthesis,transesterification and other biochemical reactions.Bacteria are important sources of lipolytic enzymes applied in industry.Here,a novel lipolytic enzyme encoded by esterase gene est1347 was identified in Marinobacter flavimaris WLL162,and was purified and characterized.The lipolytic enzyme Est1347 consisted of 312 amino acid residues and a 21-amino-acids N-terminal signal peptide with a predicted molecular weight of 34.2 kDa.It belongs to family V of bacterial lipolytic enzymes based on the amino acid sequence homology analysis.Est1347 is a mesophilic and alkali-resistant enzyme with the highest activity at 45℃and pH 8.5;it is stable at temperatures below 50℃and pH 7.5–11.0.Est1347 showed a preference for middle-length chain substrate p-NPC10 and a wide range of other substrates.The Km,Vmax,Kcat and Kcat/Km values of Est1347 for p-NPC10 in pH 8.5 at 45℃were 0.9411 mmol L^(−1),1285μmol min^(−1)mg^(−1),698.91 s^(−1)and 743.65 s^(−1)(mmol L^(−1))^(−1),respectively.It is also tolerant to the metal ions,organic solvents and detergents.In conclusion,the esterase Est1347 laid a foundation for further study of bacterial lipolytic enzyme family V.

Cloning,expression in Escherichia coli,and enzymatic properties of laccase from Aeromonas hydrophila WL-11

A strain WL-11 with high laccase activity was isolated from activated sludge collected from the effluent treatment plant of a textile and dyeing industry. It was identified as Aeromonas hydrophila by physiological test and 16S rDNA sequence analysis. A gene encoding of laccase from a newly isolated Aeromonas hydrophila WL-11 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 1605 bp encoding a polypeptide comprised of 534 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other laccases, including the conserved regions of four histidine-rich copper-binding sites. The predicted amino acid sequence showed a high homology （more than 60%） with bacterial laccases in the genome and protein databases and the highest degree of similarity （61% identity） was observed with the multicopper oxidase of KlebsieUa sp. 601. When expressed in Escherichia coli, the recombinant enzyme was overproduced in the cytoplasm as soluble and active form. The purified enzyme had an optimum pH of 2.6 and 8.0 for ABTS （2,2＇-azino-bis（3-ethylbenzthiazolinesulfonic acid） and DMP （2,6-dimethoxyphenol）, respectively. The kinetic study on ABTS revealed a higher affinity of this enzyme to this substrate than DMP.

Molecular identification and enzymatic properties of laccase2 from the diamondback moth Plutella xylostella (Lepidoptera:Plutellidae)

Laccase（EC 1.10.3.2）is known to oxidize various aromatic and nonaromatic compounds via a radical-catalyzed reaction,which generally includes two types of laccase,Lac1 and Lac2.Lac1 oxidizes toxic compounds in the diet,and Lac2 is known to play an important role in melanizing the insect exoskeleton.In this study,we cloned and sequenced the cDNA of the diamondback moth,Plutella xylostella Lac2（PxLac2）,from the third instar larvae using polymerase chain reaction（PCR）and rapid amplification of cDNA ends techniques.The results showed that the full-length PxLac2 cDNA was 1 944 bp long and had an open reading frame of 1 794 bp.PxLac2 encoded a protein with 597 amino acids and had a molecular weight of 66.09 kDa.Moreover,we determined the expression levels of PxLac2 in different stages by quantitative PCR（qPCR）.The results indicated that PxLac2 was expressed differently in different stages.We observed the highest expression level in pupae and the lowest expression level in fourth instar larvae.We also investigated the enzymatic properties of laccase,which had optimal activity at pH 3.0 and at 35°C.Under these optimal conditions,laccase had a Michaelis constant（Km）of 0.97 mmol L-1,maximal reaction speed（Vm）of 56.82 U mL-1,and activation energy（Ea）of 17.36 kJ mol-1 to oxidize2,2＇-azino-bis（3-ethylbenzothiazoline-6-sulfonic acid ammonium salt）.Type II copper enhanced laccase activity below 0.8mmol L-1 and reduced enzyme activity above 0.8 mmol L-1 with an IC50 concentration of 1.26 mmol L-1.This study provides insights into the biological function of laccase.

Mutagenesis of Alkaline Protease-Producing Marine Strains and Enzymatic Properties

[ Objective] The aim of the research was to select alkaline pmtease-producing strains and provide basis for the application in production. [ Method ] An alkaline protease-preducing strain was isolated from the sea water and sea mud collected from Qingdao coastal area. After ultraviolet mutagenesis and microwave mutagenesis, a target strain ZR-58-3-1-3 was obtained. The protease activity and enzymatic properties of the strain were determined preliminarily. [ Result ] Prote- ase activity of the selected strain reached 145 U/ml. After the compound mutagenesis, protease activity of the fermentation liquid of strain ZR-58-3-1-3 reached 775 U/m｜, which was about 4.3 times higher than that of the original strain. The optimal temperature for alkaline protease produced by strain ZR-58-3-1-3 was 45℃, and the optimal pH was 8.5, suggesting it belongs to moderate temperature protease with relatively high thermal stabihty. [ Conclusion] Strain ZR-58-3-1-3 has stable alkaline protease production performance and can be used as an alkaline protease-producing mutant strain.

Characterization and Optimization of Amylase Production in Strains LZ-10 and LZ-11 Belonging to Bacillus subtilis

In order to obtain pure enzyme with high activity,two amylase producing strains were isolated from soil samples,and named as the strains LZ-10 and LZ-11.According to morphologic observation,physiology and biochemistry experiments,16S rRNA and gyrB gene analysis,the strains LZ-10 and LZ-11 were identified as Bacillus subtilis.Adopted the method of ammonium sulfate,DEAE-52 anion purify enzyme,finally used polyacrylamide gel electrophoresis(SDS-PAGE)to detect molecular weight.The strain LZ-10 had an amylase activity of 123.3 U·mL^(-1),a purification factor of 6.8,a recovery rate of 69.5% and an optimal temperature of 50℃.The amylase activity of the strain LZ-11 was 59.91 U·mL^(-1),the purification factor was 4.5,the recovery rate was 60.5%,and the optimum temperature was 55℃.The commodity enzyme derived from Bacillus subtilis was 37.5 U·mL^(-1).The relative molecular weight of amylase activity from the two strains was 55 ku.Both thermal stability and pH stability were higher than those of commercialized amylase.

Purification and Characterization of a Versatile Peroxidase from Edible Mushroom Pleurotus eryngii

A versatile peroxidase (VP-Peco60-7 ) was generated and purified from the liquid culture of Pleurotus eryngii. The purification procedure included ammonium sulfate precipitation, ion exchange chromatography, and gel chromatography. The molecular weight and isoelectric point (pI) of VP-Peco60-7 were determined to be approxi-mately 40 kDa and 4.1, respectively. By N-terminal sequence determination and peptide mapping analysis, VP-Peco60-7 was found to be similar to the versatile peroxidase isoenzyme VPL1, which was previously isolated from liquid cultures of the same species. However, the molecular weight and pI of VP-Peco60-7 were different from those of versatile peroxidases of liquid cultures, implying that the VP-Peco60-7 in this study is of a novel type. With 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as a substrate, the maximal enzyme activity was obtained at 50 °C and pH 3.0. The catalysis of ABTS by VP-Peco60-7 was expressed by the Michaelis-Menten equa-tion. At 50 °C and pH 3.0, the maximum velocity (V max ) was 188.68 U·mg-1 and the michaelis constant (K m ) was 203.09 μmol·L-1 .

Isolation,Mutagenesis,Identification and Cellulase Properties of a Mulberry Branches Decomposing Fungus from Insect

In order to utilize mulberry branches efficiently and reduce environment pollution, a strain named A1 was isolated from the intestinal tract of Acr/da Chinensis using Congo red staining method on the plate of carboxymethyl cellulose sodium （CMC-Na） medium and mulberry branch powder culture medium. The strain A1 was mutagenized by uhraviolet radiation and the strain Z3 with a significantly improved specific activity of filter paper enzyme was screened. Enzymatic characteristics, degradation ability of cellulose and taxonomic status of A1 and Z3 were also studied in this paper. The results showed that filter paper enzyme specific activity of Z3 increased 74.47% than that of A1 and the property of enzyme production was steady. The optimum pH values of filter paper enzyme produced by A1 and Z3 were 4. 6 and 3.8 respectively and the optimum temperatures were 45 and 50 ℃, respectively. When CMC-Na was used as substrate, Vmax of the two strains was similar while Km of Z3 was lower than that of A1 , which indicated that Z3 had a strong affinity for the substrate. Enzymes produced by Z3 were more resistant to high temperature. Strains A1 and Z3 could grow well on the culture mediums with mulberry branch powder as carbon source, but Z3 grew better than A1. A1 and Z3 were preliminarily identified as AspergiUus according to the morphological identification.

Immobilization and Properties of Polyphenol Oxidase

[Objectives]This study was conducted to investigate the effects of embedding conditions on activity and catalytic properties of immobilized polyphenol oxidase.[Methods]Polyphenol oxidase was immobilized in a polymer material by the embedding method,and the optimal immobilization conditions were obtained by single factor tests:CaCl_(2) concentration 2.0%,sodium alginate concentration 2.0%,immobilization time 2 h and mass ratio of enzyme to carrier 10 mg/100 g,under which the immobilized enzyme activity was 93.33 U/g.Under the above conditions,the properties of polyphenol oxidase immobilized by sodium alginate(A-PPO)and free polyphenol oxidase were studied.[Results]The thermostability of A-PPO was better than that of the free enzyme,but the pH stability of A-PPO was inhibited.The Michaelis constant K_(m) values of free polyphenol oxidase and A-PPO were 0.37 and 0.48 mmol/L,respectively,and the maximum reaction rate V_(max) values were 0.38 and 0.51 mmol/(L·g),respectively.[Conclusions]This study provides a theoretical basis for the study of the properties of polyphenol oxidase.

High-level soluble expression of the hemA gene from Rhodobacter capsulatus and comparative study of its enzymatic properties

The Rhodobacter capsulatus hemA gene, which encodes 5-aminolevulinic acid synthase （ALAS）, was expressed in Escherichia coil Rosetta （DE3） and the enzymatic properties of the purified recombinant ALAS （RC-ALAS） were studied. Compared with ALASs encoded by hemA genes from Agrobacterium radiobacter（AR-ALAS） and Rhodobacter sphaeroides （RS-ALAS）, the specific activity of RC-ALAS reached 198.2 U/mg, which was about 31.2% and 69.5% higher than those of AR-ALAS （151.1 U/mg） and RS-ALAS （116.9 U/mg）, respectively. The optimum pH values and temperatures of the three above mentioned enzymes were all pH 7.5 and 37 ℃, respectively. Moreover, RC-ALAS was more sensitive to pH, while the other two were sensitive to temperature. The effects of metals, ethylene diamine tetraacetic acid （EDTA）, and sodium dodecyl sulfate （SDS） on the three ALASs were also investigated. The results indicate that they had the same effects on the activities of the three ALASs. SDS and metal ions such as Co^2＋, Zn^2＋, and Cu^2＋ strongly inhibited the activities of the ALASs, while Mn^2＋ exerted slight inhibition, and K^＋, Ca^2＋, Ba^2＋, Mg^2＋, or EDTA had no significant effect. The specificity constant of succinyl coenzyme A [（kcatlKm）^S-CoA] of RC-ALAS was 1.4989, which was higher than those of AR-ALAS （0.7456） and RS-ALAS （1.1699）, showing its high catalytic efficiency. The fed-batch fermentation was conducted using the recombinant strain containing the R. capsulatus hemA gene, and the yield of 5-aminolevulinic acid （ALA） achieved was 8.8 g/L （67 mmol/L） under the appropriate conditions.

Characterization of putative mannoprotein in Kluyveromyces lactis for lactase production

Lactase is a member of theβ-galactosidase family of enzymes that can hydrolyze lactose into galactose and glucose.However,extracellular lactase production was still restricted to the process of cell lysis.In this study,lactase-producing Kluyveromyces lactis JNXR-2101 was obtained using a rapid and sensitive method based on the fluorescent substrate 4-methylumbelliferyl-β-D-galactopyranoside.The purified enzyme was identified as a neutral lactase with an optimum pH of 9.To facilitate extracellular production of lactase,a putative mannoprotein KLLA0_E01057g of K.lactis was knocked out.It could effectively promote cell wall degradation and lactase production after lyticase treatment,which showed potential on other extracellular enzyme preparation.After optimizing the fermentation conditions,the lactase yield from mannoprotein-deficient K.lactis JNXR-2101ΔE01057g reached 159.62 U/mL in a 5-L fed-batch bioreactor.

Human lactate dehydrogenase and malate dehydrogenase possess the catalytic properties to produce aromatic α‑hydroxy acid

Lactate dehydrogenase and malate dehydrogenase are the two main alpha-hydroxy acid dehydrogenases in the human body.We investigated the catalytic properties of human lactate dehydrogenase LDHC,LDHL6A and malate dehydrogenase MDH1 on aromatic α-keto acids phenylpyruvic acid,p-hydroxyphenylpyruvic acid and 3,4-dihydroxyphenylpyruvic acid.The optimum temperatures for LDHC,LDHL6A,and MDH1 are 37℃,35℃,and 45℃,respectively;and the optimum pH is 6.5,6.5,and 5.5,respectively.The K_(m)of LDHC for phenylpyruvic acid and 3,4-dihydroxyphenylpyruvic acid were 0.90 mM and 0.92 mM,respectively.LDHL6A has a high affinity for phenylpyruvic acid and 3,4-dihydroxyphenylpyruvic acid with K_(m)of 0.77 mM and 0.80 mM,respectively;MDH1 has an extremely high affinity(K_(m)=0.46 mM)and catalytic efficiency(k_(cat)/K_(m)=23.87 s^(-1)·mM^(-1))for p-hydroxyphenylpyruvic acid.It also has a high affinity for 3,4-dihydroxyphenylpyruvic acid with a K_(m)of 0.90 mM,but with a low affinity for phenylpyruvic acid(K_(m)=3.76 mM).The catalytic properties of human LDHC,LDHL6A,and MDH1 for the abovementioned aromatic α-keto acids may be one of the sources of L-phenyllactic acid,L-p-hydroxyphenyllactic acid,and L-3,4-dihydroxyphenyllactic acid in humans.