An antifungal chitinase produced by Bacillus subtilis using chitin waste as a carbon source

The production of inexpensive chitinolytic enzymes is an element in the utilization of shellfish-processing waste. In this study, shrimp and crab shell powder, prepared by treating shrimp- and crab-processing waste by boiling and crushing, was used as a substrate for the isolation of an antifungal chitinase-producing microorganism. Bacillus subtilis NPU 001, a strain isolated from soil samples, excreted a chitinase when cultured in a medium containing 2% (w/v) shrimp and crab shell powder as the major carbon source. The chitinase, which was purified by sequential chromatography, had a Mw of 31 kDa and a pI of 5.4. The purified chitinase (2 mg ml⁻¹) inhibited hyphal extension of the fungus Fusarium oxysporum. Compared with other known bacterial chitinases, the unique characteristics of NPU 001 chitinase include antifungal activity against plant-pathogenic fungi and the production of chitotriose as the major enzymatic hydrolysate from colloidal chitin.     

Isolation and Identification of a Novel Antioxidant with Antitumour Activity from <Emphasis Type="Italic">Serratia ureilytica</Emphasis> Using Squid Pen as Fermentation Substrate

The antioxidant activity of the culture supernatant of Serratia ureilytica TKU013 with squid pen as the sole carbon/nitrogen source was assessed by three methods, and the phenolic contents were assayed. The supernatant with the highest antioxidant activity was further purified by liquid–liquid partition, revealing the ethyl acetate extract exhibited the strongest antioxidant activity and the highest total phenolic content. Eight fractions were retrieved from silica gel column chromatography of this extract, designated F1–F8. F4 was found to possess the strong antioxidative activity and the highest total phenolic content and also exhibited strong cytotoxic activities against two different tumoural cell lines. A new compound (Serranticin) with antioxidant and antitumor activity was obtained from F4. The structure of Serranticin is analogous to that of siderophores (hexacoordinated catecholamine), which are iron chelators. As such, Serranticin has the potential for use as a deferration agent in various iron overload diseases.     

Production and characterization of exopolysaccharides and antioxidant from Paenibacillus sp. TKU023

Using squid pen powder (SPP) as the sole C/N source, Paenibacillus sp. TKU023 produced exopolysaccharides (EPS) and antioxidant. With medium containing 1.5% SPP, 0.1% K(2)HPO(4), and 0.05% MgSO(4)·7H(2)O, pH 7.23, the culture was incubated at 37°C in liquid (50 mL/250 mL) for five days. The resultant culture supernatant had higher EPS productivity (4.55 g/L). The crude EPS were isolated by centrifugation, methanol precipitation and deproteinization. The characterization of the EPS demonstrated that it was mainly composed of glucose and maltose. In addition, the culture supernatant incubated for four days by using baffled base flask showed the strongest antioxidant activities and the highest total phenolic content, but maximum EPS production was found at the fifth day by using flat base flask. The production of two invaluable environmental-friendly biomaterials (EPS and antioxidant) is unprecedented. Besides, the use of SPP (waste) is green that made the whole process more valuable and attractive.     

Conversion of squid pen by a novel strain Lactobacillus paracasei subsp. paracasei TKU010, and its application in antimicrobial and antioxidants activity

TKU010 was isolated from infant vomited milk and identified as Lactobacillus paracasei subsp. paracasei. TKU010 had desirable properties concerning its ability to withstand adverse conditions in the gastrointestinal tract. The hydrolysate of casein enhanced the growth of TKU010 most obviously (17.20-18.25 OD(660)), followed by the hydrolysate of SPP (16.00-15.06 OD(660)). Incubating with SPP, both the culture supernatant of TKU010 on the first day and the fourth day showed inhibitory activities on E. coli BCRC13086, F. oxysporum BCRC32121 and A. fumigatus BCRC30099. TKU010 culture supernatant (1% SPP) incubated for 3 days has high antioxidant activity; the DPPH scavenging ability was 75% per ml. Thus, TKU010 could be preferably used as a starter to produce fermented milk with possibly interesting organoleptic properties. Besides, we have shown that squid pen wastes can be utilized to generate a high value-added product, and have revealed its hidden potential in the production of biocontrol agents and functional foods.     

Bioconversion of shellfish chitin wastes for the production of Bacillus subtilis W-118 chitinase

Bacillus subtilis W-118, a strain that produces antifungal materials, excreted a chitinase when cultured in a medium containing shrimp- and crab-shell powder as the major carbon source. This chitinase, purified by sequential chromatography, had a molecular mass of 20,600 Da and a pI of 6. The optimum pH, optimum temperature, and pH stability of the chitinase were pH 6, 37 degrees C, and pH 5-7, respectively. The unique characteristics of the purified chitinase include low molecular mass and acidic pI. In the investigation of the inhibitory activity, it was found that the growth of Fusarium oxysporum was 100% inhibited after incubation for 1 day with sterilized W-118 chitinase solution (5.6 units/mL). The chitinase hydrolyzates of chitin with low degrees of polymerization (DP 1-6) were analyzed by HPLC. Longer reaction times led to the generation of chitin oligosaccharides with lower DP. The chitin oligosaccharides were examined for their inhibitory effects on F. oxysporum and human leukemia cell lines.     

Fermented and enzymatic production of chitin/chitosan oligosaccharides by extracellular chitinases from Bacillus cereus TKU027

Two chitinases, Chi I and Chi II, were purified from the culture supernatant of Bacillus cereus TKU027 with shrimp head powder (SHP) as the sole carbon/nitrogen source. The molecular masses of Chi I and Chi II determined using SDS-PAGE were approximately 65kDa and 63kDa, respectively. Chi I toward various surfactants showed high stability, such as SDS, Tween 20, Tween 40 and Triton X-100, and these surfactants were stimulator of Chi I chitinase activity. Concomitant with the production of Chi I and Chi II, chitin oligosaccharides were also observed in the culture supernatant, including chitobiose, chitotriose, chitotetrose and chitopentose at concentrations of 0.44mg/mL, 0.08mg/mL, 0.09mg/mL and 0.43mg/mL, respectively. Chitosan with 60% deacetylation was degraded by TKU027 crude enzyme to prepare chitooligosaccharides. MALDI-TOF MS analysis of the enzymatic hydrolyzates indicated that the products were mainly chitooligosaccharides with degree of polymerization (DP) in the 4-9 range.     

Purification and characterization of chitinases and chitosanases from a new species strain Pseudomonas sp. TKU015 using shrimp shells as a substrate

A chitinase (CHT1) and a chitosanase (CHS1) were purified from the culture supernatant of Pseudomonas sp. TKU015 with shrimp shell wastes as the sole carbon and nitrogen source. The optimized conditions of this new species strain (Gen Bank Accession Number EU103629) for the production of chitinases were found to be when the culture was shaken at 30 degrees C for 3 days in 100 mL of medium (pH 8) containing 0.5% shrimp shell powder (SSP) (w/v), 0.1% K2HPO4, and 0.05% MgSO(4).7H2O. The molecular weights of CHT1 and CHS1 determined by SDS-PAGE were approximately 68 kDa and 30 kDa, respectively. The optimum pH, optimum temperature, pH stability, and the thermal stability of CHT1 and CHS1 were pH 6, 50 degrees C, pH 5-7, <50 degrees C and pH 4, 50 degrees C, pH 3-9, <50 degrees C, respectively. CHT1 was inhibited completely by Mn2+ and Fe2+, and CHS1 was inhibited by Mn2+, Cu2+, and PMSF. CHT1 was only specific to chitin substrates, whereas the relative activity of CHS1 increased when the degree of deacetylation of soluble chitosan increased.     

Production of xylanases from rice bran by Streptomyces actuosus A-151

In this study, the agricultural waste was used to screen for an organism that is capable of producing enzymes for degrading xylan and cellulose. Results showed that Streptomyces actuosus A-151, isolated from northern Taiwan, produced β-xylanase when rice bran was used as the sole carbon source. Four xylanases, designated as FI-A, FI-B, FII-A, FII-B, were identified and purified from the culture filtrate of S. actuosus A-151. Their specific activities after purification were 41.3, 86.2, 20.4, 85.2 U/mg, respectively. The pH stability of the four enzymes was: FI-A, 5–8; FI-B, 3–8; FII-A, 5–9; and FII-B, 3–9. The optimum pH for FII-B was 4, and the others were near 5–6. The optimum temperatures for enzyme activities were 60 °C for FII-B, and 70 °C for the others. The thermal stability for all four enzymes were up to 60 °C. The molecular weights of FI-A, FI-B, FII-A, and FII-B xylanases were 30,000, 45,000, 26,000, and 20,000, respectively, by sodium dodecylsulfate–polyacrylamide gel electrophoresis and 30,000, 43,000, 25,000, and 21,000, respectively, by gel filtration. Addition of xylan, shrimp and crab shell powder, and orange peel to the culture medium was found to enhance the production of xylanase.     

Conversion of squid pen by Serratia ureilytica for the production of enzymes and antioxidants

Two proteases (P1 and P2) and a chitinase (C1) were purified from the culture supernatant of Serratia ureilytica TKU013 with squid pen as the sole carbon/nitrogen source. The molecular masses of P1, P2 and C1 determined by SDS-PAGE were approximately 50 kDa, 50 kDa and 60 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of P1, P2 and C1 were (pH 10, 40 degrees C, pH 7-11, and <50 degrees C), (pH 10, 40 degrees C, pH 8-11, and <40 degrees C) and (pH 6, 50 degrees C, pH 5-8, and <50 degrees C), respectively. P1 and P2 were inhibited by Mg(2+), EDTA and C1 was inhibited completely by Cu(2+). The antioxidant activity of TKU013 culture supernatant was 72% per mL (DPPH scavenging ability). With this method, we have shown that squid pen wastes can be utilized and have revealed its hidden potential in the production of functional foods.     

Purification and characterization of extracellular lipases from <Emphasis Type="Italic">Pseudomonas monteilii</Emphasis> TKU009 by the use of soybeans as the substrate

A lipase-producing bacterium was isolated and identified as Pseudomonas monteilii TKU009. A lipase (F2) and lipase-like materials (F1) were purified from the culture supernatant of P. monteilii TKU009 with soybean powder as the sole carbon/nitrogen source. The molecular mass of F1 and F2 was estimated to be 44 kDa by SDS-PAGE and gel filtration. The optimum pH, optimum temperature, and pH and thermal stabilities of F2 were 7, 40°C, 8–11, and 50°C; and of F1 were 6, 40°C, 6–7, and 50°C, respectively. F2 was completely inhibited by EDTA and slightly by Mg²⁺, Fe²⁺, Mn²⁺, and SDS. F1 was completely inhibited by EDTA and Fe²⁺ and strongly by Zn²⁺, Mn²⁺, Ca²⁺, Mg²⁺, and SDS. The activities of both the enzymes were enhanced by the addition of non-ionic surfactants Triton X–100 and Tween 40, especially for F1. F2 preferably acted on substrates with a long chain (C10–C18) of fatty acids, while F1 showed a broad spectrum on those with chain length of C4–C18. The marked activity of F2 in organic solvents makes it an ideal choice for application in a water-restricted medium including organic synthesis. Li-June Ming is a visiting Professor at the National Cheng Kung University.