Bacterial succession on raw buffalo hide and their degradative activities during ambient storage

Inception of microbiology in leather industry was with reference to tanning processes where major concern was finished product bacteriology. Present investigation aims to study the bacteriology of unprocessed raw material, buffalo hide in particular using culture based and culture independent approach. Denaturing gradient gel electrophoresis (DGGE) analysis of the stored hide at different time intervals was done to study temporal successional shifts in the bacterial community. Considering the protein and fat content of hide, proteolytic and lipolytic bacteria were isolated and identified as major degradative flora. The degradative nature of individual isolate was confirmed through in vitro and in vivo investigations as collagenase production and ability to deteriorate raw hide to release amino acids and free fatty acids, respectively. Furthermore, the histological analysis of bacterially spiked raw hides was done as aid to the conclusions drawn. It was found that the temporal bacterial successional shifts occurring on ambient stored raw buffalo hide could be better understood using DGGE. Isolated and characterized degradative flora could be used as test organisms to discover new preservatives and to assess the process of preservation. Even though the process of degradation is very complex, the culture based and culture independent analysis of degradative microflora is comprehensive.     

Co-composting of hair waste from the tanning industry with de-inking and municipal wastewater sludges

Production of waste hair in the leather manufacturing industry is increasing every year due to the adoption of hair-save unhairing techniques, leaving the tanners with the problem of coping with yet another solid by-product. Numerous potential strategies for hair utilisation have been proposed. However, the use of hair waste as agricultural fertiliser is one of its most promising applications due to the high nitrogen content of hair. Agricultural value of hair can be increased by composting. This paper deals with the composting of hair from the unhairing of bovine hide. Results indicated that hair cannot be either composted on its own or co-composted with de-inking sludge, a chemical complementary co-substrate. However, good results were obtained when co-composted with raw sludge from a municipal wastewater treatment plant at hair:raw sludge weight ratios 1:1, 1:2 and, 1:4 in lab scale and pilot plant scale composters. In all cases, a more stable product was achieved at the end of the process. Composting in the pilot plant composter was effectively monitored using Static Respiration Indices determined at process temperature at sampling (SRI _T ) and at 37°C (SRI₃₇). Notably, SRI _T values were more sensitive to changes in the biological activity. In contrast, Respiratory Quotient (RQ) values were not adequate to follow the development of the process.     

Characterization of thermo- and detergent stable serine protease from isolated Bacillus circulans and evaluation of eco-friendly applications

Alkaline protease from Bacillus circulans has been purified and characterized in detail for its robustness and its eco-friendly application potential at leather processing and detergent industries. The molecular weight of the purified enzyme was estimated to be 39.5 kDa on SDS-PAGE. It exhibited optimum activity at broad temperature range and maximum at 70 °C under alkaline pH environment, in the presence of surfactants and oxidizing agents. It has revealed stain removal property and dehairing activity for animal hide without chemical assistance and without hydrolyzing fibrous proteins. This enzyme showed application potential in leather processing industry for production of better quality product in eco-friendly process. In addition, the stability (pH, temperature and surfactants) and hydrolysis of blood stain data also revealed its application in detergent industries.     

Short Term Preservation of Hide Using Vacuum: Influence on Properties of Hide and of Processed Leather

The objective of this work was to investigate vacuum influence on hide preservation time and how it affects hide structure. It was established that vacuum prolongs the storage time without hide tissue putrefaction up to 21 days when the storage temperature is 4°C. The microorganisms act for all storage times, but the action is weak and has no observable influence on the quality of hide during the time period mentioned. The hide shrinkage temperature decrease is negligible, which shows that breaking of intermolecular bonds does not occur. Optical microscopy, infrared spectroscopy and differential scanning calorimetry also did not show any structural changes which can influence the quality of leather produced from such hide. The qualitative indexes of wet blue processed under laboratory conditions and of leather produced during industrial trials are presented. Indexes such as chromium compounds exhaustion, content of chromium in leather, content of soluble matter in dichloromethane, strength properties, and shrinkage temperature were determined. Properties of the leather produced from vacuumed hide under industrial conditions conformed to the requirements of shoe upper leather.     

A Life Cycle Assessment on the Dehairing of Rawhides: Chemical Treatment versus Enzymatic Recovery through Solid State Fermentation

The leather industry needs to switch from the traditional chemically based dehairing process to an environmentally friendly one so that the overall burdens to the environment are reduced. The primary goal of the work was thus to compare the chemical leather dehairing process to an enzymatically based one using the enzymes that are extracted after the application of solid state fermentation (SSF) on hair wastes generated after dehairing. The environmental burdens of the dehairing stage were determined using a life cycle assessment (LCA) approach by comparing the two aforementioned management scenarios. The first scenario was the commonly used technology in which hair is removed via a chemical process and then composted in open piles. This scenario included two subscenarios where hair waste is either incinerated or landfilled. In the second scenario, the proteolytic enzymes extracted during the SSF of the residual hair are used to dehair the new rawhides instead of chemicals. Industrial and laboratory data were combined with international databases using the SimaPro 8.0 LCA software to make comparisons. The environmental impacts associated with the enzymatic dehairing were significantly lower than the ones associated to the conventional chemical dehairing process. This difference is attributed to the impacts associated with the original production of the chemicals and to the electricity consumed in the conventional method. A sensitivity analysis revealed that the results are affected by the amounts of chemicals used during dehairing.     

Hide depilation and feather disintegration studies with keratinolytic serine protease from a novel Bacillus subtilis isolate

Keratinases play an important role in biotechnological applications such as improvement of feather meal, enzymatic dehairing and production of amino acids or peptides from high molecular weight substrates. Bacillus subtilis P13, isolated from Vajreshwari hot spring (45–50°C) near Mumbai, India, produces a neutral serine protease and has an optimum temperature of 65°C. This enzyme preparation was keratinolytic in nature and could disintegrate whole chicken feathers, except for the remnants of shafts. The enzyme preparation also exhibited depilation of goat hides with the recovery of intact animal hair. The enzyme preparation could release peptides from ground feathers and bring about their weight reduction; however, similar action on hair was relatively weak. A single major PMSF-sensitive protease band could be detected upon zymogram analysis, indicating that a single enzyme may be responsible for feather degradation and hide depilation. The importance of these findings in the biotechnological application for feather and leather industries is discussed.     

Production and partial characterization of extracellular proteinases from <Emphasis Type="Italic">Streptomyces malaysiensis</Emphasis>, isolated from a Brazilian cerrado soil

Streptomyces malaysiensis AMT-3, isolated from a Brazilian cerrado soil, showed proteolytic activities detected by gelatin–sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum proteinase production was obtained when using 2.5% wheat bran and 0.1% yeast extract in the culture medium, after 5 days incubation at 30°C. The enzymatic complex degraded gelatin optimally at pH 7.0, and under these conditions eight proteolytic bands (four serine-proteinases and four metaloproteinases), ranging from 20 to 212 kDa, were detected on the culture supernatant filtrates. In addition, a 35-kDa proteinase was thermostable at 60°C for 120 min. These results point out to the applicability of gelatin zymograms in the characterization of crude enzymatic complexes. According to our results, this enzymatic complex could be used for biotechnological applications.     

The effects of treating bovine hide with steam at subatmospheric pressure on bacterial numbers and leather quality

AIMS: To examine the effect of subatmospheric steam treatment on total viable counts (TVCs) on bovine hide and on the quality of derived leather. METHODS AND RESULTS: Pieces of bovine hide were heated to 75 degrees C (+/-2 degrees C) (n = 3) or 80 degrees C (+/-2 degrees C) (n = 3) for periods of 1, 10 or 20 s by the application of steam at subatmospheric pressure in a laboratory scale apparatus. Treated hide pieces and untreated controls were tanned and the quality of leather was assessed. Treatment at 80 degrees C (T80) reduced the TVC on hide pieces by 2.95 (1 s), 3.33 (10 s) and 3.99 (20 s) log10 CFU cm-2 (P > 0.05). Treatment at 75 degrees C (T75) reduced the TVC on hide pieces by 1.87 (1 s), 2.51 (10 s) and 2.56 (20 s) log10 CFU cm-2 (P > 0.05). The grain on all treated hides was damaged resulting in sueding on derived leather. Sueding was observed on 100% of surfaces from T80-treated samples and on 18 (1 s) to 84% (20 s) of the surfaces of T75 samples. CONCLUSIONS: The magnitude of TVC reductions achieved using T75 and T80 could limit the impact and scale of contamination transfer to the carcass during dehiding. However, because of the sueding observed on derived leather, it is unlikely that either T75 or T80 would be a commercially valid operation during routine slaughter operations. SIGNIFICANCE AND IMPACT OF THE STUDY: Hide decontamination would provide an important critical control point for beef processing, however there are currently no commercially available treatments.     

Isolation and process parameter optimization of Aspergillus sp. for removal of chromium from tannery effluent

Five morphologically different fungi were isolated from leather tanning effluent in which Aspergillus sp. and Hirsutella sp. had higher potential to remove chromium. The potential of Aspergillus sp. for removal of chromium was evaluated in shake flask culture in different pH, temperature, inoculums size, carbon and nitrogen source. The maximum chromium was removed at pH 6, temperature 30 degrees C, sodium acetate (0.2%) and yeast extract (0.1%). Aspergillus sp. was applied in 2l bioreactor for removal of chromium, and it was observed that 70% chromium was removed after 3 days.     

Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326

Bacillus cereus MCM B-326, isolated from buffalo hide, produced an extracellular protease. Maximum protease production occurred (126.87+/-1.32 U ml(-1)) in starch soybean meal medium of pH 9.0, at 30 degrees C, under shake culture condition, with 2.8 x 10(8) cells ml(-1) as initial inoculum density, at 36 h. Ammonium sulphate precipitate of the enzyme was stable over a temperature range of 25-65 degrees C and pH 6-12, with maximum activity at 55 degrees C and pH 9.0. The enzyme required Ca(2+) ions for its production but not for activity and/or stability. The partially purified enzyme exhibited multiple proteases of molecular weight 45 kDa and 36 kDa. The enzyme could be effectively used to remove hair from buffalo hide indicating its potential in leather processing industry.     

The effect of acid proteinases on the activity and stability of glucoamylase preparations

It was demonstrated that the presence of acid proteinases in the preparation isolated from Aspergillus awamori decreased the activity and stability of glucoamylase. Patterns of changes in the enzymatic activity and stability of glucoamylase at increased temperature and various pH values were studied over a long-term storage. A biospecific sorbent for removal of acid proteinases was synthesized, and glucoamylase preparations free of proteolytic activity were produced.     

Production of an alkaline protease by Bacillus cereus MCM B-326 and its application as a dehairing agent

The present investigation describes microbial production of an alkaline protease and its use in dehairing of buffalo hide. Bacillus cereus produced extracellular protease when grown on a medium containing starch, wheat bran and soya flour (SWS). The ammonium sulphate precipitated (ASP) enzyme was applied for dehairing of buffalo hide. Microscopic observation of longitudinal section of buffalo hide revealed that the epidermis was completely removed and hair was uprooted leaving empty follicles in the hide. The ASP enzyme was stable for one month at ambient temperature between 25–35 °C. Enzymatic dehairing may be a promising shift towards an environment-friendly leather processing method.     

Enzymatic lysis of sulfated glycosaminoglycans reduces the electrophoretic mobility of vascular endothelial cells

The main purpose of this work was to identify the macromolecules carrying the surface charge of endothelial cells. This was done by measuring changes in cell electrophoretic mobility caused by enzymatic removal of glycocalyx components. Endothelial cells were removed from the bovine pulmonary artery using nonenzymatic procedures, plated, and identified by immunocytochemical methods and electron microscopy. Cultured cells were suspended in saline and placed in the lumen of a capillary in a Rank Brothers electrophoresis instrument. Voltage was applied between the ends of the capillary, and the velocity acquired by the cells was measured with a microscope. Preincubating the cells in protein-free saline for 1 h reduced the mobility by 25%. This reflects the loss of proteoheparan sulfate from the cell surface. Cell mobility was totally suppressed by exposing the entire cell surface to chondroitin sulfate lyase, but it was only slightly diminished when the enzyme was applied only to the cell side facing the culture medium. A partial decrease in mobility was obtained after enzymatic removal of either heparin, heparan sulfate, or collagen. The results indicate that sulfated glycosaminoglycans are the main carriers of the surface change in vascular endothelial cells. The asymmetrical effect of chondroitinase on the two sides of the cell indicates a distribution polarization for glycosaminoglycans in endothelial cells.     

Effects of heparan sulfate removal on attachment and reattachment of fibroblasts and endothelial cells

Human skin fibroblasts and calf aorta endothelial cells were grown as tissue culture monolayers in the presence of [35S]sulfate in order to label the glycosaminoglycan portions of proteoglycans for investigation of their role in cell attachment. The [35S]glycosaminoglycans were then selectively removed from the cell monolayers by the addition of various glycosaminoglycan-degrading enzymes. As previously described, in contrast to trypsin treatment none of these enzymes removed any cells from the culture plates. Incubation with a preparation from Flavobacterium heparinum left only small stubs of [35S]glycosaminoglycans on the cell monolayers, indicating that all the cell-surface proteoheparan [35S]sulfate and proteochondroitin [35S]sulfate was accessible to this enzyme preparation. The treatment did not change the amount or time of incubation with trypsin necessary for release of the cells from the monolayers. Thus, cell attachment was not weakened by removal of heparan sulfate or chondroitin sulfate. In contrast, neither fibroblasts nor endothelial cells in suspension would reattach in the presence of the F. heparinum preparation while reattachment occurred readily in the presence of chondroitin ABC lyase. This provides evidence that heparan sulfate, but not chondroitin sulfate, is involved in the process of cell attachment even though neither is necessary for maintaining attachment.     

Isolation, characterization and optimization of culture parameters for production of an alkaline protease isolated from Aspergillus tamarii

Aspergillus tamarii expresses an extracellular alkaline protease that we show to be effective in removing hair from cattle hide. Large quantities of the enzyme will be required for the optimization of the enzymatic dehairing process so the growth conditions for maximum protease expression by A. tamarii were optimized for both solid-state culture on wheat bran and for broth culture. Optimal protease expression occurred, for both cultural media, at initial pH 9; the culture was incubated at 30 °C for 96 h using a 5% inoculum. The crude enzyme was isolated, purified and characterized using MALDI TOF TOF. The alkaline protease was homologous to the alkaline protease expressed by Aspergillus viridinutans. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

The effect of acid proteinases on the activity and stability of glucoamylase preparations

It was demonstrated that the presence of acid proteinases in the preparation isolated from Aspergillus awamori decreased the activity and stability of glucoamylase. Patterns of changes in the enzymatic activity and stability of glucoamylase at increased temperature and various pH values were studied over a long-term storage. A biospecific sorbent for removal of acid proteinases was synthesized and glucoamylase preparations free of proteolytic activity were produced.     

Integrated reactor concepts for the enzymatic kinetic synthesis of cephalexin

Integrated process concepts for enzymatic cephalexin synthesis were investigated by our group, and this article focuses on the integration of reactions and product removal during the reactions. The last step in cephalexin production is the enzymatic kinetic coupling of activated phenylglycine (phenylglycine amide or phenylglycine methyl ester) and 7-aminodeacetoxycephalosporanic acid (7-ADCA). The traditional production of 7-ADCA takes place via a chemical ring expansion step and an enzymatic hydrolysis step starting from penicillin G. However, 7-ADCA can also be produced by the enzymatic hydrolysis of adipyl-7-ADCA. In this work, this reaction was combined with the enzymatic synthesis reaction and performed simultaneously (i.e., one-pot synthesis). Furthermore, in situ product removal by adsorption and complexation were investigated as means of preventing enzymatic hydrolysis of cephalexin. We found that adipyl-7-ADCA hydrolysis and cephalexin synthesis could be performed simultaneously. The maximum yield on conversion (reaction) of the combined process was very similar to the yield of the separate processes performed under the same reaction conditions with the enzyme concentrations adjusted correctly. This implied that the number of reaction steps in the cephalexin process could be reduced significantly. The removal of cephalexin by adsorption was not specific enough to be applied in situ. The adsorbents also bound the substrates and therewith caused lower yields. Complexation with beta-naphthol proved to be an effective removal technique; however, it also showed a drawback in that the activity of the cephalexin-synthesizing enzyme was influenced negatively. Complexation with beta-naphthol rendered a 50% higher cephalexin yield and considerably less byproduct formation (reduction of 40%) as compared to cephalexin synthesis only. If adipyl-7-ADCA hydrolysis and cephalexin synthesis were performed simultaneously and in combination with complexation with beta-naphthol, higher cephalexin concentrations also were found. In conclusion, a highly integrated process (two reactions simultaneously combined with in situ product removal) was shown possible, although further optimization is necessary.     

Production of Extracellular Acid Proteases by Aspergillus Clavatus

The production of extracellular acid proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on haemoglobin pH 5.0 at 37 °C. The highest acid proteolytic activity (80 U/ml) was observed in culture medium containing glucose and gelatin at 1%(w/v) at 30 °C at the third day of incubation. Cultures developed in Vogel medium with glucose at 2%(w/v) showed at about 45% of proteolytic activity when compared to the cultures with 1% of the same sugar. The optimum pH of enzymatic activity was 2.0 and the enzyme was stable at pH values ranging from 2.0 to 4.0. The optimum temperature was 40 °C and the half-lives at 40, 45 and 50 °C were 30, 10 and 5 min, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of the proteases involved in the N-terminal clipping of glucagon-like-peptide-1-antibody fusion proteins

In an attempt to develop high producing mammalian cell lines expressing glucagon-like-peptide-1-antibody fusion proteins (GLP-1), we have noted that the N-terminal GLP-1 portion of the fusion protein was susceptible to proteolytic degradation during cell culture, which resulted in an inactive product. The majority of the N-terminal clipped product appeared to be due to the removal of the entire biologically active peptide (30 amino acids) from the intact molecule. A number of parameters that influenced the degradative process were investigated. Additionally, protease inhibitors specific for each class of protease were tested. Results suggested that one or more serine-threonine class of protease(s) were involved in this process and inhibitors that are specific for this class of protease, including benzamidine hydrochloride could significantly inhibit the proteolytic degradation of the fusion proteins. Identification of the specific proteases involved in this process by shotgun proteomics methodology will pave the way for engineering the CHOK1SV cell line which will serve as a superior host for the production of future fusion protein products.     

Production of extracellular alkaline proteases by <Emphasis Type="Italic">Aspergillus clavatus</Emphasis>

Summary The production of extracellular alkaline proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on casein pH 9.5 at 37 °C. The highest alkaline proteolytic activity (38 U/ml) was verified for culture medium containing glucose and casein at 1% (w/v) as substrates, obtained from cultures developed at 25 °C for 6 days. Cultures developed in Vogel medium with glucose at 2% (w/v) and 0.2% (w/v) NH₄NO₃ showed higher proteolytic activity (27 U/ml) when compared to the cultures with 1% of the same sugar. Optimum temperature was 40 °C and the half-lives at 40, 45 and 50 °C were 90, 25 and 18 min, respectively. Optimum pH of enzymatic activity was 9.5 and the enzyme was stable from pH 6.0 to 12.0.